Proceedings Biofloc Technology and Shrimp Disease Workshop December 9-10, 2013 Saigon Exhibition and Convention Center, Ho Chi Minh City, Vietnam Edited by Craig Browdy, John Hargreaves, Tung Hoang and Yoram Avnimelech Proceedings of the Biofloc Technology and Shrimp Disease Workshop December 9-10, 2013 Saigon Exhibition and Convention Center, Ho Chi Minh City, Vietnam Chairs Yoram Avnimlech and Hoang Tung Organizing Committee Yoram Avnimlech, Hoang Tung and Craig L. Browdy Proceedings Editors Craig L. Browdy, John Hargreaves, Hoang Tung and Yoram Avnimelech How to cite this volume: Browdy. C.L., J. Hargreaves, H. Tung and Y. Avnimelech. 2014. Proceedings of the Biofloc Technology and Shrimp Disease Workshop. December 9-10, 2013, Ho Chi Minh City, Vietnam. The Aquaculture Engineering Society, Copper Hill, VA USA. www.aesweb.org/shrimp_health.php. 2 PREFACE Disease continues to be one of the greatest challenges facing commercial marine shrimp aquaculture around the world. Over the past thirty years, improved shrimp farming technologies have enabled production intensification, better shrimp growth and more sustainable farming practices. Despite these advances, catastrophic disease outbreaks have periodically plagued growers in major shrimp producing countries worldwide, most recently exemplified by the ongoing epidemic of Acute Hepatopancreatic Necrosis Disease, formerly named Early Mortality Syndrome. A growing number of anecdotal observations from producers and results from a limited number of controlled research studies suggest that biofloc systems can reduce the incidence and severity of shrimp and fish disease outbreaks. This may be related to more-stable and diverse microbial communities, improved biosecurity related to low water exchange, and stable water quality from high aeration rates in biofloc systems. Although intriguing, the reliability, scope and confidence in the existing data is limited and contradictory evidence has also been reported; uncertainty about the effect of biofloc technology on fish or shrimp disease is high. Objective reporting and balanced consideration of the value of the information available by members of the professional aquaculture community is needed. This can provide a foundation for the establishment of research priorities and practical recommendations that can be applied towards holistic solutions and best practices to manage the grave disease problems currently facing commercial shrimp aquaculture. The goal of this two-day workshop was to gather a group of interested, knowledgeable and experienced professionals and practitioners to discuss the issue, summarize what is known with an estimation of certainty, and identify and prioritize the major information gaps that can lead to practical management approaches. Based on workshop presentations and discussions, this white paper identifies attributes of biofloc systems that limit disease and highlights recommended research priorities and potential practical measures to reduce the incidence and severity of disease outbreaks. The workshop was organized and supported by the World Aquaculture Society (WAS), the Aquacultural Engineering Society (AES), the International University VNUHCM and Novus. INVE, Tomboy Skretting, AES, Blue Archipelago, Intron LifeSciences and Blue Aqua International sponsored different activities associated with the workshop. Yoram Avnimelech, Tung Hoang, Craig Browdy and John Hargreaves 3 CONTENTS PREFACE ………………………………………………………………………..………..…... 3 WORKSHOP EXECUTIVE SUMMARY ……………………………………………….….. 8 Biofloc Technology ………..……………………………………………………….…... 8 Early Mortality Syndrome ………………………...……………………………….…… 8 Biofloc and Shrimp Immunity ……….…………………………………………………. 8 Effects of the Microbial Ecology of Biofloc Systems on Shrimp Disease …………... 9 Biofloc Technology and Gut Health ..…………………………………………………. 10 Co-culture of Fish and Shrimp and Shrimp Disease …………………………………… 11 Attributes of Biofloc Technology System Management that Reduce Disease Risk…. 11 Practical Recommendations………………...…………………………………….…….. 12 Priority research areas ……………………………...……………….……….…. 12 Short-term research topics ………….…..………..……………………. 12 Longer-term research topics …………………………..……………...... 13 EARLY MORTALITY SYNDROME ………………………...…………………………….. 14 CONFIRMATION OF THE INFECTIOUS NATURE OF THE AGENT OF EARLY MORTALITY SYNDROME (EMS) AFFECTING FARMED PENAEID SHRIMP IN MEXICO Donald V. Lightner, Linda Nunan, Rita M. Redman, Leone L. Mohney, Carlos R. Pantoja, and Loc Tran Extended abstract ………………………………………….…………………… 15 Presentation …………………………………………….………………………. 18 EARLY MORTALITY SYNDROME: OBSERVATIONS AND DOUBTS Victoria Alday-Sanz Extended abstract …………………………….………………………………… 24 Presentation ………………………………………………………….…………. 26 CO-INFECTION IN SHRIMP AND ITS MEANING IN BIOFLOC SYSTEMS Phuoc L. H., Corteel M., Nauwynck H. J., Pensaert M. B., Alday-Sanz V., Van den Broeck W., Sorgeloos P., Bossier P. Extended abstract ………………………………………………………………. 32 Presentation ……………………………………………………………….……. 35 A REVIEW OF ACUTE HEPATOPANCREATIC NECROSIS SYNDROME RESEARCH IN VIETNAM Dang Thi Hoang Oanh Extended abstract ………………………………………………………………. 40 Presentation ……………………………………………………………….……. 42 4 BIOFLOCS IMMUNITY AND SHRIMP DISEASE ……………………………………… 51 IMMUNE MECHANISMS IN CRUSTACEANS Kenneth Söderhäll Extended abstract ………………………………………………………………. 52 Presentation ……………………………………………………………….……. 54 EVALUATION OF IMMUNE ENHANCEMENT OF SHRIMP GROWN IN BIOFLOC SYSTEMS In-Kwon Jang and Su-Kyoung Kim Extended abstract ………………………………………………………………. 63 Presentation ……………………………………………………………….……. 65 THE EFFECTS OF BIOFLOCS GROWN ON DIFFERENT CARBON SOURCES ON SHRIMP IMMUNE RESPONSE AND DISEASE RESISTANCE Julie Ekasari, Muhammad Hanif Azhar, Enang Harris Surawidjaja, Peter De Schryver and Peter Bossier Extended abstract ………………………………………………………………. 76 Presentation ……………………………………………………………….……. 80 BIOFLOC SYSTEMS MANAGEMENT ………………………………..………………….. 83 SHRIMP BIOFLOC TECHNOLOGIES, FEEDS AND GUT HEALTH Craig L. Browdy Extended abstract ………………………………………………………………. 84 Presentation ……………………………………………………………….……. 87 IS IT POSSIBLE TO CONTROL THE BACTERIAL COMPOSITION IN SHRIMP AND FISH PONDS? Stephen G. Newman Extended abstract ………………………………………………………………. 93 Presentation ……………………………………………………………….……. 97 HIGH PERFORMING BIOFLOC SYSTEMS USING PROBIOTICS: THE VIEW FROM ASIA AND LATIN AMERICA Olivier Decamp, Marcos Santos, Hoa Nguyen Duy, Fauzan Bahri and Jaime Munoz Medina Extended abstract …………………………………………….……………… 103 Presentation …………………………………………………………….……. 104 THE USE OF A BIOFLOC TECHNOLOGY SYSTEM WITH PROBIOTICS TO LIMIT SHRIMP VIBRIOSES Dariano Krummenauer, Luis Poersch, Luiz A. Romano, Gabriele R. Lara, Bárbara Hostins and Wilson Wasielesky Jr. Extended abstract …………………………………………….……………… 109 Presentation …………………………………………………………….……. 111 5 PROBIOTIC EFFECTS OF BIOFLOC TECHNOLOGY: DEPRESSION OF TILAPIA INFECTION BY Streptococcus Yoram Avnimelech Extended abstract …………………………………………….……………… 119 Presentation …………………………………………………………….……. 122 ROLE OF SELECTIVE BREEDING IN BIOFLOC SHRIMP PRODUCTION AND DISEASE MITIGATION Shaun M. Moss, Dustin R. Moss, Clete A. Otoshi, Steve M. Arce, and Donald V. Lightner Extended abstract …………………………………………….……………… 124 Presentation …………………………………………………………….……. 130 CO-CULTURE OF FISH AND SHRIMP ………………………………………………… 135 EFFECTS OF TILAPIA IN CONTROLLING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) Loc H. Tran, Kevin M. Fitzsimmons and Donald V. Lightner Extended abstract …………………………………………….……………… 136 Presentation …………………………………………………………….……. 138 EXPERIENCE ON Penaeus monodon/RED TILAPIA CO-CULTURE USING A BIOFLOC SYSTEM Boonsirm Withyachumnarnkul, Compoonut Gerdmusic, Teerapong Jutipongraksa, Padmaja J. Pradeep and Sage Chaiyapechara Extended abstract …………………………………………….……………… 143 Presentation …………………………………………………………….……. 148 GREENWATER TECHNOLOGY FOR SHRIMP FARMING: MODES OF ACTION Marc C.J. Verdegem and Eleonor A. Tendencia Extended abstract …………………………………………….……………… 151 Presentation …………………………………………………………….……. 153 COMMERCIAL APPLICATIONS ……………………………………………………….. 157 BIOFLOC: PAST, PRESENT AND FUTURE Robins McIntosh Extended abstract …………………………………………….……………… 158 Presentation …………………………………………………………….……. 162 PRACTICAL MEASURES FOR SHRIMP FARMING DURING AN EMS OUTBREAK Tung Hoang and Marc Le Poul Extended abstract …………………………………………….……………… 168 Presentation …………………………………………………………….……. 171 6 SHRIMP FARMING: BIOFLOC AS BIOSECURITY? Nyan Taw Extended abstract …………………………………………….……………… 175 Presentation …………………………………………………………….……. 177 INTRODUCTION TO THE MIXOTROPHIC SYSTEM: AN INTENSIVE SHRIMP FARMING MANAGEMENT TECHNOLOGY Farshad Shishehchian Extended abstract …………………………………………….……………… 184 Presentation …………………………………………………………….……. 185 LIST OF PARTICIPANTS…………………………………………………………………. 192 7 WORKSHOP EXECUTIVE SUMMARY A two-day workshop to explore the interaction between biofloc technology systems for aquaculture and shrimp health management was held in Ho Chi Minh City, Viet Nam. The workshop was motivated by the large economic losses (e.g. in Vietnam alone, about US$ 1 billion per year over the last 3 years) suffered by the commercial shrimp aquaculture sector caused by the occurrence of acute hepatopancreatic necrosis disease (AHPND), formerly known as early mortality syndrome (EMS). The workshop specifically focused on characteristics of biofloc technology systems that reduce the risk of the incidence and severity of this disease. There were over 200 participants (including at least 15 producers) and 21 presentations were made. Workshop presentations are included in this proceedings and can be found through a link on the web page of the Biofloc Technology Working Group of the Aquacultural Engineering Society (www.aesweb.org). Biofloc technology improves shrimp health by stimulating the nonspecific immune system of shrimp, by providing excellent biosecurity because water exchange rates are typically low, and by providing stable water quality from high rates of aeration. Biofloc Technology Biofloc technology is an intensive approach to aquaculture production that relies on elevated suspended solids concentration to provide water treatment and supplementary food for cultured animals. Biofloc technology has been applied in commercial shrimp farming since the 1990s. Anecdotal reports over recent years have suggested that aspects of shrimp production in biofloc systems provide an improved level of disease protection in comparison to conventional pond production. Early Mortality Syndrome The outbreak and epidemic disease called Early Mortality Syndrome (EMS), now known more formally as acute hepatopancreatic necrosis disease (AHPND), has caused tremendous losses in commercial shrimp aquaculture in Asia (China, Vietnam, Malaysia, Thailand) and Mexico since it first appeared in China in 2009. The disease affects both Litopenaeus vannamei and Penaeus monodon. White and atrophied hepatopancreas, soft shells, and minimal or no gut contents are common clinical signs. Mortality typically occurs in the first 30-45 days of culture and can begin as soon as 10 days after stocking. The causative organism has been identified as the bacterial pathogen, Vibrio parahaemolyticus. Very high concentrations of bacteria (108 CFU/mL) are required to cause disease. Pathogenesis appears to be regulated through quorum sensing. Vibriosis often occurs as a co-infection with viral diseases. Risk factors for EMS outbreaks include elevated salinity, especially greater than 10 ppt, and high temperature. Bioflocs and Shrimp Immunity Evidence is accumulating that exposure to bioflocs stimulates the non-specific immune system in shrimp. Constituents of bacterial cell walls (lipopolysaccharides, peptidoglycans and β -1, 3glucans) activate the non-specific immune system in shrimp. Specifically these components activate a proteolytic cascade of reactions leading to the production of prophenoloxidase (proPO), leading ultimately to melanization. Activation of proPO occurs more quickly than 8 immune responses that require altered gene expression. Other biochemical pathways that are part of the shrimp immune system are likewise stimulated by contact with or consumption of biofloc. In Kwon Jang and Su-Kyoung Kim measured mRNA expression of six genes that are involved in the innate immune response of shrimp [proPO1 (prophenoloxidase 1), proPO2 (prophenoloxidase 2), PPAE (prophenoloxidase activating enzyme), SP1 (serine protease), mas (masquerade-like serine proteinase), ran (ras-related nuclear)]. Gene expression measured in mysid, post-larvae and adult Letopenaeus vannamei is enhanced in the presence of bioflocs. Gene expression is greater in L. vannamei than in other shrimp species (F. chinensis, Metapenaeus japonicus), possibly related to differences in morphology of the third maxilliped. The number, type, length and distance between setae and setules on the third maxilliped affect the ability of different shrimp species to capture and use bioflocs as food. Julie Ekasari and co-workers reported that phenoloxidase activity increases in response to organic carbon loading from different sources (molasses, tapioca, tapioca by-product and rice bran). Biofloc systems contribute to the enhancement of immune response and survival of L. vannamei after IMNV challenge, regardless of organic carbon source. Biofloc technology can be viewed as a mechanism that provides shrimp with pattern recognition and other molecules that lead to stimulation of the non-specific immune system. These molecules are provided to shrimp constantly. There is an energetic cost associated with constant immunostimulation although it is difficult to conclude whether or not this effect is deleterious. Biofloc “primes” the immune system but it is not fully activated until a pathogen is encountered. Avnimelech presented results indicating significantly lower infection of tilapia by Streptococcus iniae released to the water from challenged fish in biofloc systems as compared to clear water. This may be related to antagonism between the pathogen and other bacteria that limits the pathogen. It is possible that a similar antagonism occurs between dense heterotrophic bacteria and Vibrio parahaemolyticus, the causative agent of AHPND Effects of the Microbial Ecology of Biofloc Systems on Shrimp Disease Water in biofloc technology systems contains a large number of bacterial species. Jang found 351-773 operational taxonomic units (essentially equivalent to ‘species’) in water from biofloc systems. Others reported as many as 2000 species. The most dominant group is Bacteroidetes, a common constituent of wastewater in treatment plants, responsible for organic matter degradation. Vibrio is an opportunistic “early successional” species that quickly reaches a very high population density, dominating the microbial community of new mixed bacterial cultures, but is controlled in “mature” or aged water with a more diverse assemblage of bacteria. Characteristics of “mature” and stable water that confer control of Vibrio parahaemolyticus are not known with certainty. Better characterization of the microbial composition of flocs, especially the bacteria that have protective effects, is needed. 9 Additional evidence on the capacity of biofloc systems to control Vibrio was provided by Oliver Decamp, citing the Ph.D. dissertation of R. Crab (2010). Shrimp were fed either artificial diet or artificial diet partially replaced with biofloc. Bioflocs were grown on different carbon sources with or without the addition of a Bacillus-based probiotic. Treatments were feed only, feed + sucrose, feed + sucrose + Bacillus, feed + glycerol, feed +glycerol + Bacillus. With both types of carbon source, with or without Bacillus addition, the density of Vibrio was less than the feedonly control. Within either type of organic carbon source added to develop bioflocs, adding Bacillus reduced Vibrio cell density further. Numerous questions about microbial community management were raised: • What is the optimal biofloc concentration? • How to manage/control biofloc community composition for optimal shrimp health? • How to measure functionality of system in terms of disease control? • Biofloc systems are quite unstable at the species level; what is the optimum balance of species? • What is the best way to “feed” the biofloc? o manipulate C:N ratio? o continuous or intermittent inputs? • How to quickly establish a functional diverse biofloc community? Regarding this last question, information was presented regarding normal practice and methods to accelerate aging or maturity of water with respect to microbial community composition. AHPND occurs early in the culture period, within the first 30-45 days of grow-out. In newly started systems, 30-40 days are normally required before flocs fully develop and the system is considered acclimated. Thus, there is a pressing need to develop flocs quickly before AHPND occurs. Development of bioflocs that are target oriented, considering feed composition, immune effects, shrimp growth rates and other desirable properties is presently taking place in academic institutions and by commercial operations. Biofloc Technology and Gut Health The anatomy of the shrimp digestive system includes barriers to protect against pathogen infection, including a gastric sieve, the cuticular lining of the stomach and a periotrophic membrane in the midgut. As mentioned previously, Pacific white shrimp have the capacity to harvest bioflocs from culture water. This attribute allows this species to take advantage of biofloc as a food resource and as a mechanism to stimulate one part of the non-specific immune system. Consumption of biofloc can influence the microbial community of the shrimp gut, thereby affecting nutrient digestibility and shrimp health. Various feed supplements, as alternatives to antibiotics, can improve shrimp health by influencing the structure and function of the gut microbial community. These additives include immunostimulants, prebiotics, probiotics, organic acids and essential oils. 10 Co-culture of Fish and Shrimp and Shrimp Disease Data on the effect of co-culturing shrimp and tilapia and the effect on decreasing the incidence of shrimp diseases was presented, based on experiences in the Philippines and Thailand. Including tilapia or other fish in a shrimp production system appears to confer some protection to AHPND in shrimp, although the mechanism is not clearly understood. The possibilities for the mechanism include: 1) Some zooplankton may serve as concentrators of Vibrio in ponds. Consumption of these zooplankton by shrimp may lead to infection. Grazing by filter-feeding fish may reduce the density of concentrator zooplankton. 2) The green alga Chlorella inhibits Vibrio harveyi. If present at sufficient biomass, grazing of blue-green algae by filter-feeding fish may shift algal community composition towards green algae. 3) Fish mucus contains antimicrobial compounds. There may be a link between these compounds and suppression of Vibrio. 4) The fish gut contains bacteria with antibacterial effects that are constantly shed into the water. More research is needed to elucidate these mechanisms. Co-culture of shrimp with fish is practiced by shrimp farmers in the Philippines, Vietnam, and China, although a farmer from China reported that results were not very good. In Thailand, an equal biomass of shrimp and tilapia are produced in co-culture ponds. Fish used in co-culture systems include tilapia and silver carp. Attributes of Biofloc Technology System Management that Reduce Disease Risk 1) Biofloc technology systems are characteristically operated with very low rates of water exchange. Inherently this improves biosecurity because exclusion of pathogens is enhanced by limiting contact with water from external aquatic ecosystems adjacent to farms. Low rates of water exchange is only one aspect of farm biosecurity, which also includes using post-larvae that have been evaluated and certified as disease-free, filtering incoming water (250 microns), erecting barriers to crustacean carriers (crab fences), and maintaining a clean pond bottom. 2) Biofloc technology systems are typically operated with high levels of aeration and mixing. This characteristic creates a stable water quality environment with respect to dissolved oxygen concentration and pH, conditions that are favorable for good shrimp growth and elevated immunocompetence. 3) Many biofloc systems provide mechanisms for removal of settled solids or control of suspended solids concentration. Removal of accumulated sludge is seen as essential to reduce the risk of AHPND outbreaks. Pond areas with accumulated sludge deposits are areas of impaired water quality. These areas are the location of active production of sulfide, a potent toxicant of shrimp, and other growth-inhibiting chemicals. Furthermore, bacterial population densities in the fluid sediment layer near the pond bottom are very high. Grazing by shrimp in this area increases exposure to high bacterial density. It appears that the lethal dose of V. parahaemolyticus is quite high (108 CFU/mL), a density that could be encountered in the fluid 11 sediment layer. Thus, regular removal of waste solids is viewed as essential. Tung and coworkers recommend solids removal 2 hours after every feeding. To summarize this discussion, the main attributes of biofloc systems that reduce the risk of shrimp disease are: • Low rates of water exchange improves pathogen exclusion (biosecurity). • Continuous aeration provides stable water quality (DO and pH). • Diverse and stable microbial community stimulates the non-specific immune system and limits development of opportunistic species (e.g. Vibrio). • Regular removal of accumulated sludge crops solids and controls biofloc concentration to moderate levels. Practical Recommendations Biofloc systems can limit the development of shrimp or fish diseases. This conclusion is based on the results of controlled research and a significant number of field observations. Due to the severity of the shrimp disease problem, the use of biofloc technology can be recommended as an effective approach to shrimp health management. • • • • Efforts should be taken to grow shrimp in “mature” water with a diverse and active micro-biota in the pond. As a practical matter, this calls for the development of mature water prior to shrimp stocking, through inoculation protocols including probiotics, and minimizing water exchange during the production cycle. Probiotics and certain feed additives can increase shrimp resistance to diseases. Only additives that have been properly tested with demonstrated efficacy should be used. Prevent bottom sludge accumulation to reduce shrimp stress and possibly disease. Plan ponds in a way to enable drainage and washout of sludge when it accumulates. Biosecurity, selection of healthy stock and other best management practices and technologies deployed on modern commercial shrimp farms should be implemented. Priority Research Areas The economic losses caused by shrimp diseases are huge and the probability that implementation of biofloc technology improves the situation is quite high. Research costs are relatively low compared to economic losses. Research oriented to develop, optimize and ascertain means to minimize disease outbreaks and severity is essential. Thus, investment in research oriented toward further development of the biofloc technology approach is recommended. Short-term research topics • • • Objective and controlled evaluation of the effects of biofloc systems and co-culture with tilapia on the infection of shrimp by viral and microbial diseases. Objective and controlled evaluation of the effects probiotic products and feed additives on the infection of shrimp by viral and microbial diseases. Effects of dense heterotrophic population on Vibrio parahaemolyticus survival in water and infecting shrimp. 12 • • • Effects of anaerobic bottom sludge on AHPND infections. Means to treat the bottom sediment to prevent negative effects. Effects of inhibiting quorum sensing on the infection of shrimp by Vibrio parahaemolyticus. Establishing the essential parameters to define biofloc systems. Longer-term research topics • • • • Methods to establish a diverse and stable microbial community. Defining the optimal microbial community composition in biofloc systems. o for competitive exclusion of pathogens o for target crop growth o for water quality management Methods for manipulation of the microbial community to maintain an optimal composition. o fertilization o filtration o sterilization o inoculation, probiotics o habitat o environment Identification of the best tools for measuring and describing the complex microbial floc community 13 EARLY MORTALITY SYNDROME 14 CONFIRMATION OF THE INFECTIOUS NATURE OF THE AGENT OF EARLY MORTALITY SYNDROME (EMS) AFFECTING FARMED PENAEID SHRIMP IN MEXICO Donald V. Lightner1*, Linda Nunan1, Rita M. Redman1, Leone L. Mohney1, Carlos R. Pantoja1, and Loc Tran1,2 1 Aquaculture Pathology Laboratory School of Animal and Comparative Biomedical Sciences University of Arizona, Tucson, AZ 85721, USA 2 Department of Soil, Water and Environmental Science University of Arizona, Tucson, AZ 85721, USA dvl@u.arizona.edu A new emerging disease in shrimp, first reported in 2009, was initially named Early Mortality Syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as Acute Hepatopancreatic Necrosis Syndrome/Disease (AHPNS/AHPND). Affecting both Pacific white shrimp Litopenaeus vannamei and black tiger shrimp Penaeus monodon, the disease has caused significant losses in Southeast Asian shrimp farms. Most recently the disease has crossed the Pacific Ocean and is affecting shrimp farms in western Mexico. AHPND was first classified as idiopathic because no specific causative agent had been identified. However, since early 2013, the Aquaculture Pathology Laboratory at the University of Arizona (UAZ-APL) isolated the causative agent of AHPND in pure culture. More recently, the UAZ-APL has obtained a number of isolates that have subsequently been confirmed to cause AHPND. Immersion challenge tests have been employed for infectivity studies with isolates from Southeast Asia and Mexico, which induced complete mortality with typical AHPND pathology to experimental shrimp exposed to the pathogenic agent (Table 1, Figures 1-2). Subsequent histological analyses indicated that AHPNS lesions could be experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas of Southeast Asia and western Mexico. Bacterial isolation from experimentally infected shrimp enabled recovery of the same bacteria colony type found in field samples. In all challenge studies run to date with the unique strain of Vibrio parahaemolyticus (VP) found to cause AHPND, the same pathology was reproduced in experimental shrimp 15 regardless of whether isolates were derived from Vietnam or Mexico. Hence, AHPND has a bacterial etiology and Koch’s Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus. TABLE 1. Biochemical comparison of Southeast Asian and Mexican Vibrio parahemolyticus isolates that cause AHPND. API 20 NE Test Result NO3 => NO2 Indole Glucose fermentation Arginine dihydrolase Urease Esculin hydrolysis Gelatin liquefaction B-Galactodiase Assimilation of: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosameine, maltose, L-malate D-gluconate, caprate, citrate, phenyl acetate Oxidase 16 VP A/3 SE Asia + + - (usually +) + + VP from Mexico + + - (usually +) + + + + + + FIGURE 1. Number of surviving shrimp after exposure. Positive for EMS: 13-306C, 13-306D and 13-306F, Negative for EMS: 13-306A, 13-306B and 13-306E. FIGURE 2. Litopenaeus vannamei after challenge with a strain of VP from Mexico. 17 Spread of “New Disease” in w estern Mexico Documentation of an Emerging Disease (Early Mortality Syndrome or Acute Hepatopancreatic Necrosis disease) in Mexico Mid May, 2013 Early May, 2013 D.V. Lightner 1, R.M. Redman1, C.R. Pantoja1, B.L. Noble1, L.M. Nunan 1, Loc Tran 1 and Silvia Gomez J. 2 OI E Reference Laboratory for Shrimp Diseases, School of Animal and Comparative Biomedical Sciences, The University of Arizona, Tucson, AZ, USA 2 CI AD, Hermosillo, Sonora, Mexico April, 2013 1 2 1 The hepatopancreas is the target organ for EMS (= AHPND) Normal histology of the hepatopancreas EMS in Sonora, Mexico – Photo by Ms. Silvia Gomez Al-Mohana & Nott. 1989. Functional cytology of the hepatopancreas of Penaeus semisulcatus (Crustacea: Decapoda) during the moulting cycle. Marine Biology (101) 535-544. 3 4 Normal hepatopancreas 5 18 6 Case 11-041. Normal hepatopancreas (HP) Normal hepatopancreas histology 7 Comparison of EMS in Asia vs. EMS in Mexico Gross Signs of EMS/ AHPNS     8 EMS/ AHPND shrimp in from Vietnam EMS/ AHPND shrimp from a shrimp pond near Mazatlan, Sinaloa, Mexico Significant atrophy of the hepatopancreas (HP). Often pale, yellowish or white within the HP connective tissue capsule. Black spots or streaks sometimes visible. HP does not squash easily between thumb & finger. 10 Acute Hepatopancreatic Necrosis Disease Has Two Distinct Phases:  An acute phase      Histopathology showing acute phase HP dysfunction  Samples from Mexico Acute Hepatopancreatic Necrosis Disease or AHPND. HP tubule cells (R, B, F & later E-cells) show acute loss of function. Significant acute sloughing of HP tubule epithelial cells. collected in May 2013 Abundant bacteria in the hepatopancreas at this stage are not easily demonstrated. Terminal phase ends with destruction of the HP by opportunistic Vibrio spp. 11 12 19 Mexico - Acute Phase of AHPND; UAZ-APL 13-218; 4x Mexico - Acute Phase of AHPND; UAZ-APL 13-220; 10x 13 14 Mexico - Acute Phase of AHPND; UAZ-APL 13-220; 20x Histopathology showing terminal phase of HP destruction due to Vibriosis Samples from Mexico collected in May 2013 16 15 Mexico - Terminal Phase of AHPND; UAZ-APL 13-220A-3; 4x Mexico - Terminal Phase of AHPND; UAZ-APL 13-220A-3; 20x 17 18 20 Proposed Case Definition for EMS/ AHPND  I diopathic – no specific disease causing agent (infectious or toxic) was identified until March 2013.  Pathology:  acute progressive degeneration of hepatopancreas (HP) from medial to distal with dysfunction of all HP cells, prominent necrosis & sloughing of these tubule epithelial cells. The agent found to induce EMS/ AHPND pathology was identified as a strain of  terminal stage shows marked inter- & intra-tubular Vibrio parahaemolyticus. hemocytic inflammation & development of massive secondary bacterial infections that occur in association with necrotic & sloughed HP tubule cells. 20 Recent work on the Agent of AHPND/ EMS Discussion    EMS/ AHPNS is caused by Vibrio parahaemolyticus that can be found in infected shrimp’s stomachs.  Biochemical characterization.  Molecular characterization of extra The agent did not show pathogenicity when grown on solid media, but when liquid media is used, pathogenicity does occur. chromosomal elements of the strains of VP that cause AHPND/ EMS. Studies using the bacterial agent of EMS/ AHPNS satisfied the four points of Koch’s Postulates. 21 22 Biochemical comparison of a SE Asian & a Mexican VP isolate that cause AHPND API 20 NE Test Result VP A/ 3 SE Asia Comparison of VP isolates from Mexico for ability to cause EMS/ AHPND VP from Mexico NO3= > NO2 + + I ndole + + Glucose fermentation - (usually + ) - (usually + ) Arginine dihydrolase - - Urease - - Esculin hydrolysis - - Gelatin liquefaction + + B-Galactodiase + + Assimilation of: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosameine, maltose, L-malate + + D-gluconate, caprate, citrate, phenyl acetate - - Oxidase + +   23 A mixed culture & a pure culture of Vibrio parahaemolyticus from Mexico were tested by immersion exposure to determine if the mixed & pure culture can cause EMS/ AHPND. The mixed culture & the pure culture of VP caused EMS/ AHPND. 24 21 I mmersion test using a pure bacterial culture obtained from Mexico P. vannamei (UAZ 13-334A&E) after challenge with VP from Mexico Histological confirmation: 13-306 A, 13-306 B, and 13-306E: negative for EMS/ AHPND 13-306 C, 13-306D, and 13-306F(= VP A/ 3) : positive for EMS/ AHPND 25 Two VP isolates underwent metagenomic sequencing Four Vibrio parahaemolyticus isolates from Vietnam & one from Mexico Causes AHPND  Sequenced were VP A/ 2 & VP A/ 3. 13-028A/ 2 NO  VP A/ 2 does not cause AHPND/ EMS. 13-028A/ 3 YES  VP A/ 3 does cause AHPND/ EMS. 12-297B YES 1335 YES 13-306D/ 4 (Mexico) YES Designation 26   Primers were designed from the metagenomic sequencing data for the extra-chromosomal genetic material that was found. These primers gave the following results: 28 Screening for mobile genetic elements Vibrio parahaemolyticus isolates 13- 028 A/ 3 123 4 56 1. 2. 3. 4. 5. 6. 12- 297B 1234 56 Samples: Tox R Samples: 1335 1234 56 1 2 3 4 5 6 1. 2. 3. 4. 5. 6. 1 Kb marker 13-028 A/ 3- Vietnam 1335- Vietnam 12-297B- Vietnam 13-306D/ 4- Mexico 13-028 A/ 2- Vietnam All of the isolates are Vibrio parahaemolyticus 1 Kb marker Phage Contig 32 Contig 52 Contig 73 Contig 89 13- 306 D/ 4 13- 028 A/ 2 1 234 5 6 1234 56 I NFECTI OUS VP STRAI NS NONI NFECTI OUS 22 Contigs 52 & 89 Acknowledgements  Contigs 52 & 89 are consistent  OI E (World Organization for Animal Health)  Department of Animal Health, MARD, Vietnam for local arrangements in Vietnam. amplicons present among the four AHPND-causing isolates.  Uni-President feed company in Vietnam for funding toxicity  A PCR kit is being developed for      the VP agent of AHPND / EMS based on Contig 89. & infectivity studies. CP Food, Thailand for funding recent work on EMS. World Bank & Global Aquaculture Alliance for travel. FAO project for partial support. Minh Phu Seafood for diagnostic services. Grobest for molecular biology work. 32 Thank you for your attention! Reference Lab for Crustacean (Shrimp) Diseases 23 EARLY MORTALITY SYNDROME: OBSERVATIONS AND DOUBTS Victoria Alday-Sanz Pescanova Rua Antonio Fernandez s/n, Chapela, Pontevedra, Spain valday@pescanova.es The disease called Early Mortality Syndrome has been affecting Asian shrimp production at least since 2009 and Mexico in 2013. Only recently, an infectious etiological agent has been identified as Vibrio parahaemolyticus. Despite the different culture systems, with intensive to superintensive production with high biosecurity in Asia and extensive to semi-intensive production with low biosecurity in Mexico, the presentation of the disease in both locations has been very similar. In both regions, the main EMS risk factor appears to be the pond bottom, whether lined or earthen. There was no mortality of shrimp held in suspended cages in EMS-affected ponds but mortality occurred when shrimp were released and established contact with the pond bottom. Although the agent may enter the production system with incoming water, its presence is not sufficient to elicit disease because there is no mortality of wild shrimp present in the reservoir and canals or in the estuaries that collect effluents from EMS ponds. However, in Mexico, mortality was associated with water exchange a few days before. Inbreeding or loss of heterozygosity associated with domesticated stocks does not seem to be a risk factor because shrimp collected from the wild and stocked in an EMS area had the same pattern of mortality. Additionally Litopenaeus vannamei, Penaeus monodon and Penaeus chinensis are susceptible to EMS. Environmental factors that influence the severity of EMS are temperature and salinity. EMS is most active as temperature increases and in ponds with salinity greater than 10 ppt. In ponds with salinities less than 10 ppt, EMS is much less prevalent. EMS has been described in ponds with a range in pH from 7.5 to greater than 8.6 in Asia and Mexico. Shrimp culture with well water, regardless of salinity, did not result in EMS in Asia and Mexico. It is possible that filtration either removes the bacteria or a needed concentrator of the bacteria, such as filter feeders. The identification of some EMS isolates at biochemical and molecular levels presents certain inconsistencies. Vibrio can conjugate and be altered by phage and plasmid inclusions. Two 24 extrachromosomal genes are consistently present on highly virulent EMS bacteria. Similar histopathological lesions to EMS have been described before; however, EMS lesions appear to be more severe and acute in their presentation. Experimental infections with purified agent indicate that a very high load of bacteria is required to reproduce EMS lesions, in comparison with reports of other Vibrio pathogens. Experimental infections via what could be the natural route have been achieved by feeding mollusks and copepods (?) from EMS-affected ponds. The mortality pattern of experimental infections can be modified using tilapia culture water with Chlorella and other green algae. Quorum sensing may play a role in the pathogenicity of this Vibrio and might suggest a strategy to control the disease. In order to understand this new disease, a new vision needs to be developed. The term “biocomplexity” was used to characterize biological system interactions that represent a network that relates weather patterns, aquatic reservoirs, phages/plasmids, zooplankton, cell attachment behavior and an adaptable genome. Learning to manage EMS will require understanding the ecological influence of bacterial populations. 25 Different culture system APHNS: OBSERVATIONS AND DOUBTS Mexico Asia • • • • • Victoria Alday‐Sanz, D.V.M., M.Sc., Ph.D. Director Animal Health Pescanova Disease pattern Superintensive Treated water Linned and soil ponds High biosecurity Most severe mortality up to 1gr • • • • • Semi‐intensive No water treatment Soild pond bottom Low biosecurity Most severe mortality up to 4gr Pond bottom • Both in Mexico (soil) and Asia (soil and linned): • Acute: – High mortality in early stages (40‐90%) – Shrimp suspended in cages: no mortality • Chronic: – Moderate or low mortality with slow growth • Trial in Mexico: removal of 15cm of soil: NO mortality up to 10gr and growing • Trial in Thailand: plastic buckets: No mortality It is a risk factor • Not all farms in an area affected • Not all ponds affected in a farm • Variable mortality between ponds Water Industria acuicola Viven pescadores del Sur temporada de “Ensueño” Son varios los factores que se han conjugado, para que los pescadores del Sur de Sonora vivan una temporada de ensueño, entre otros el alto precio del camarón. La Oficina Federal de Pesca y Acuacultura en Ciudad Obregón, informó que también la captura del crustáceo ha sido buena pues a la fecha se han obtenido un total de 569 toneladas, un 10% más que en la misma fecha del año pasado. “Se tiene reportada una captura mayor a la del año pasado en esta misma fecha, sin embargo, les puedo decir que ya superamos lo que se capturó en toda la temporada del 2012, que fue de 551 toneladas”, comentó. Aunque la captura ha sido alta, dijo, actualmente es poca la cantidad de camarón que se extrae del mar, por lo que difícilmente se superará la cifra récord de capturas del año 2011, cuando se extrajeron 638 toneladas del crustáceo. • Possibly bacteria enters the pond with the water but it is not a sufficient cause of disease: – No mortality of wild shrimp in the reservoir – No mortality of wild shrimp in the estuaries that collect effluent of EMS farms 26 Water exchange Well water • Not a link in Asia (low water exchange) • Mexico: mortality is often linked to water exchange (a few days after) • Asia and Mexico: – No EMS in well water farms independently of their salinity (up to 20ppt) – Stirring of pond bottom?? Stress?? Loss of estability ?? Molt induction?? • Does the well filter the bacteria? Does it filter the conditions for proliferation?? Does it filter ¨concentrators¨ of the bacteria??? • Mexico, nowadays: 20%exchange/day better survival Protective factor LARVAE Genetics • Comments about inbreeding or loss of heterozygosity associated with domesticated stocks • Not likely to be the related: ASOCI ACI ÓN NACI ONAL DE PRODUCTORES DE LARVA DE CAMARON, A.C. Participación de Laboratorios Productores de Postlarvas al 30 de Septiembre, 2013. LABORATORIOS / ESTADOS – P. vannamei, P. monodon and P. chinensis – Different genetic programs from different countries SINALOA SONORA • Trial in Mexico stocking wild shrimp in EMS affected farms: mortality Not a risk factor SINALOA SONORA NAYARIT 4.143.157.831 958.181.389 472.001.000 40.100.000 2.127.927.738 NAYARIT 154.820.000 BCS 263.324.250 526.505.251 COLIMA TOTAL 10.600.000 0 0 GOLFO (Tamaulipas COLIMA Y , Campeche CHIAPAS y Yucatán) BCS % TOTAL 0 330.000.000 400.000.000 63,36% 0 0 0 0 21,79% 112.406.000 0 0 0 29.409.000 377.948.420 0 0 12,03% 0 0 1.800.000 0 4.612.002.0813.612.614.378 615.616.000 377.948.420 330.000.000 400.000.000 2,69% 0,12% 100,00% ‐Could be coming with the larvae but the environmental conditions are not present? EMS is seasonal cases increase with temperature More frequently reported in ponds with salinity >10ppt 80 Fujian, China Failure Rate: 2010 70 60 50 40 30 20 10 Thailand: Jan to May 2013 0 Mar Apr May Jun July Aug Sept Oct 27 in vitro culture: <2ppt: no growth 2‐8ppt: slow growth pH Affected stages • pH: reported EMS cases between 7.0 and 9.5 Experimental infections: ‐All stages might be susceptible (up to 15grs at least) Feeding Natural infections: most common early stages ‐Related to feeding behaviour ?? ‐Proportionally higher dose of toxins?? • Stop feeding improves pond population symptoms Disease process in the shrimp Colonization of the stomach cuticle Production of toxins Damage of the hepatopancreas epithelium Secondary bacterial infection • • • • How to explain these observations? Hypothesis How to manage the disease? Antibiotics will not solve the disease Disease process in a population Survival rate Treat 1 Why is the pond the unit of risk? • Are we promoting EMS bacteria? – Fertilization of water – Pellet feed Procedure Sea water 2 Sea water + 3% TSB media 3 Sea water + 60 ppm Molass + 3 ppm NH4 4 Sea water + 60 ppm Molass + 3 ppm NH4 Rep. 1 2 1 2 1 2 1 2 Shrimp Stage PL PL PL PL PL PL PL PL MBW. , , , , , , , , ###### ###### ###### ###### ### ### ### ### DOE1 DOE2 DOE3 DOE4 Bacteria need nutrients to cause disease 28 A ¨high dose¨ is required to cause disease Oral experimental infections • Experimental challenges • EMS: high dose needed: – If fed biofilm from infected ponds: no disease – If fed mussel from an infected pond: disease – High mortality in a short term challenge: 108 cfu – No mortality: 104 cfu with majority of isolates Where are the concentrated forms in a pond? Filterfeeders? • Other vibrio publications: 102 to 106 cfu Difference in virulence Ez Taxon • Both in Thailand and Mexico, isolates of different degree of virulence have been found: • time to cause mortality and • dose • Might the bacteria require certain chromosomic capacity to express maxium virulence? • Vibrio nigripulchritudo (plasmid): acute, semi‐acute and chronic • Might this virulence gene ¨jump¨ between strains and species?? Owens 2013 Conditions for APHNS ocurrence • Transmission of VHML bacteriophage isolated from Vibrio harveyi: • Pathogen – V. harveyi – V. campbelli – Vibrio parahaemolyticus with specific virulence gen/genes • Transmission of VOB bacteriophage isolated from Vibrio owensii • Suitable environmental conditions for the pathogenic bacteria to proliferate • Mechanisms to concentrate the bacteria to threshold level – V. owensii – V. harveyi – V. campbellii Transfer of virulence 29 Validation of treatments and desinfections Can we avoid the pathogen? • VBNC: ¨Viable but not culturable¨ • Stock APHNS free PL • Use of well water or fresh water • Treating water to eliminate it? – – – – – Chlorine: loss of diversity Freezing Ozono Chlorine UV • ¨Resurrection¨ protocol • V. parahaemolyticus Generation time (8‐12min) • Negative effect! 1. Alcaline 3% peptone water at 37 ± 1 ºC for frozen or refrigerated samples for 20 ± 2 hours (from surface, no growth) 2. Alcaline 2% peptone water at 41.5 ± 1 ºC for 18 ± 1 hours (from surface) ‐TCBS (blueish‐green, round, flat, smooth and shinny) ‐CHROMagar Can we avoid proliferation of the pathogen? Is dry out effective? • Asia: ponds are dried for up to 5 months: EMS reappears • Ploughing and liming does not seem effective • Why? • Promote diversity: mature waters • Promote QSi (quorum sensing inhibitors) – Tilapia • Avoid ¨concentrators¨ (filter feeders) – V. parahaemolyticus develop resting forms Thongchankaew Can. J. Microbiol. 57: 867–873 (2011) – V. cholera resting forms may survive 60 years – Drum filters (50microns)? – Cupper sulphate? • Keep high water exchange: avoid ¨fermentation¨ in the pond: recirculation • Remove first layer of soil? Management strategies How it can be moved around? APHNS is a new disease, and requieres a new way of management • • • • • • Current biosecurity measures have been developed for viruses 30 Life infected animals Fresh feed for broodstock Currents Frozen shrimp (remains virulent?) Ballast water Careful with microalgae and probiotics Environmental indicator Cargo vessel traffic densities based on AMVER (2001) (Source: DNV). Vibrio parahaemolyticus culture RESULTADO Años 2011 2012 Total general FECHA DE ENVÍO ene feb may jun jul ago sep oct nov dic ene feb mar may jun jul ago sep RESULTADO 2 Ausencia Ausencia 21 5 73 64 73 76 52 39 16 55 28 8 9 51 80 74 108 45 877 RESULTADO <3 6 1 2 1 6 7 2 2 (3 ‐ 100) (101 ‐ 1000) > 1000 Total general 7 2 4 4 8 5 15 1 1 2 3 1 1 5 4 43 1 16 12 10 25 111 1 7 11 8 27 1 4 4 8 17 34 6 75 67 83 87 54 49 21 70 29 11 9 55 99 98 138 90 1075 http://www.issg.org/database/species/search.asp?st=sss&sn=&rn=&hci=8&ei=‐1&x=22&y=5 84species reportadas Colwell 2002 • The disease cholera can no longer be considered a simple equation of bacteria and human host, but represents a complex network that includes global weather patterns, aquatic reservoirs, phages, zooplankton, collective behaviour of surface attached cells, an adaptable genome and the deep sea inter alia. This approach to view biological systems as a whole was termed biocomplexity. Sniezco Colwell 2005 31 CO-INFECTION IN SHRIMP AND ITS MEANING IN BIOFLOC SYSTEMS Phuoc L. H., Corteel M., Nauwynck H. J., Pensaert M. B., AldaySanz V., Van den Broeck W., Sorgeloos P., Bossier P.* Department of Animal Production Laboratory of Aquaculture and Artemia Reference Center University of Ghent, Ghent, Belgium Peter.Bossier@UGent.be Species of Vibrio (mainly Vibrio anguillarum, V. alginolyticus, V. parahaemolyticus, V. harveyi, V. penaeicida and V. campbellii) are well known in penaeid shrimp culture as causative agents of vibriosis. This important disease occurs in hatcheries and grow-out systems (Lightner 1988, Lavilla-Pitogo et al. 1990). Infections with luminescent V. harveyi strains cause major losses in shrimp larviculture in Australia (Pizzutto and Hirst 1995), South America (Álvarez et al. 1998, Robertson et al. 1998) and Mexico (Vandenberghe et al. 1999). Vibriosis usually occurs during the first month of culture and can cause more than 50% mortality. So far, it is not clear whether Vibrio spp. are opportunistic or primary pathogens. According to Saulnier and colleagues (2000a), Vibrio spp. may act as opportunistic agents in secondary infections or be true pathogens. Only a few cases of polymicrobial disease have been described in shrimp aquaculture. In 20012002, retardation of the Penaeus monodon growth rate was noted in shrimp ponds in Thailand. This problem was named Monodon Slow Growth Syndrome. Samples of affected shrimp were screened by histopathology, polymerase chain reaction (PCR) and transmission electron microscopy for a wide range of pathogens. Several causative agents were involved. Many shrimp specimens had dual or multiple infections with monodon baculovirus (MBV), heptopancreatic parvovirus (HPV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) (Chayaburakul et al. 2004). After screening shrimp samples from 18 ponds in India, Umesha and colleagues (2006) found that shrimp in seven ponds had dual infections with WSSV and MBV and in 10 ponds had triple infections with HPV, WSSV and MBV. Selvin and Lipton (2003) demonstrated the presence of a virulent strain of V. alginolyticus in shrimp from a pond hit by a 32 WSSV outbreak. Although both pathogens could not be isolated from all sampled shrimp, shrimp weakened by WSSV would succumb to a secondary infection by V. alginolyticus. The objective of this study was to reproduce a co-infection of shrimp with WSSV and Vibrio under laboratory conditions using standardized challenge protocols and to investigate the existence of any synergistic effects. More specifically, the study aimed to determine whether a WSSV infection already present in specific pathogen-free (SPF) Litopenaeus vannamei would allow Vibrio to cause faster and greater mortality than the virus or bacteria administered separately. Shrimp started to die 108 hours after injection with WSSV and all shrimp died by 336 hours (see figure). Animals co-infected by WSSV and V. campbellii died quickly after the challenge with V. campbellii, with mortality reaching 66.7% by 48 hours after injection and all shrimp died by 96 hours. Dual infections did not cause any change in the number of WSSV-infected cells. Infected cells were found in all organs, but there was no significant difference (P < 0.01) between groups of shrimp administered pathogens together or WSSV alone. The number of WSSV-infected cells in haematopoietic tissue (15–568 cells/mm2) was greater than that in gills (55–241 cells/mm2) and 33 lymphoid organs (3–165 cells/mm2) (Table 1). In the stomach epithelium, 2–29% of cells were infected. The number of V. campbellii isolated from bacteria-only-injected shrimp was less than 100 cfu/mL. In contrast, a very high density of V. campbellii (1.8 × 106 cfu/mL) was observed in the haemolymph of shrimp co-infected with WSSV and Vibrio. TABLE 1. Cumulative shrimp mortality after challenge with WSSV and V. campbellii. Quantification of WSSV-infected cells (mean ± SD) and V. campbellii (cfu/mL of haemolymph) in gills (G), stomach epithelium (SE), lymphoid organ (LO) and haematopoietic tissue (HP) of shrimp collected 10 h after V. campbellii injection (experiment 6; shrimp in dual treatment were moribund). Numbers between brackets are minimum and maximum values of six shrimp. Numbers of infected cells in the same tissue or cfu/mL with different superscripts were significantly different between the two treatments (P < 0.01). WSSV-infected cells in organs Treatments G (cells/mm2) SE (%) HP (cells/mm2) VC (cfu/mL) WSSV 167 ± 58a 17 ± 10a 83 ± 56a 315 ± 194a – VC WSSV + VC (55–221) – 184 ± 40a (130–241) (2–29) – 14 ± 5a (4–19) (70–568) – 208 ± 168a (15–503) 43 ± 61a (0–157) 183,721 ± 73,177b (113,000–314,600) LO (cells/mm2) (12–138) – 90 ± 67a (3–165) The importance of these findings in relation to biofloc systems might be dual. First, WSSVinfected shrimp become more vulnerable to bacterial infection and can die because of Vibrio infection much faster than from WSSV infection alone. It remains to be established if this would be the case with other virus-Vibrio combinations. Hence contamination of shrimp ponds with WSSV should be avoided at all times to prevent subsequent bacterial infections. Second, it remains to be established if a biofloc system allows steering of the microbial community composition away from infectious vibrio species, acknowledging that the complete elimination of Vibrio is unattainable. 34 Outline Co-infection in shrimp and • On the importance of viral – bacterial co-infections its meaning in bioflocs systems • Microbial management in bioflocs L.H. Phuoc, M. Corteel, H.J. Nauwynck, M.B. Pensaert, V. Alday-Sanz, W. Van den Broeck, P. Sorgeloos, P. Bossier • Vibrio management in bioflocs Ugent Aquaculture R&D consortium Ghent University Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 1 of 26 Injection challenge with different doses of V. campbellii Slide 2 of 26 Co-challenge of SPF L. vannamei Cumulative SPF shrimp mortality (%) after challenge with different doses of V. campbellii with WSSV and V. campbellii 100 Cumulative mortality (%) 90 Treatments 80 70 WSSV WSSV 60 50 30 SID50 VC - WSSV + VC 20 10 Negative control 0 0 6 12 Control 105 CFU shrimp-1 24 36 48 103 CFU shrimp-1 106 CFU shrimp-1 Treatments Cumulative mortality (%) 80 70 60 WSSV 50 40 VC injection 10 WSSV + VC 0 Control 48 72 WSSV 96 10 CFU shrimp - - 1 5 extra 6 + 5 extra 6 + 5 extra 6 + 5 extra 120 144 168 192 216 240 264 288 312 336 Hpi VC (106 CFU shrimp-1) WSSV + VC (106 CFU shrimp-1) Co-infection in shrimp and its meaning in bioflocs systems Slide 4 of 26 Quantification of WSSV-infected cells in the moribund shrimp collected at different time-points HPoi AG Stomach -2 -2 (cells mm ) (cells mm ) (%) Shrimp Hpi Gill -2 (cells mm ) 1 237 32 168 90 24 30 SID50 - + Co-infection in shrimp and its meaning in bioflocs systems Slide 3 of 26 100 0 1 6 6 107 CFU shrimp-1 Cumulative SPF shrimp mortality (%) after challenge with WSSV and V. campbellii 20 10 CFU shrimp - 72 96 120 Hpi 104 CFU shrimp-1 Co-infection in shrimp and its meaning in bioflocs systems 30 6 40 30 Number of shrimp V. campbellii 20 12.7 CEC (%) 23.9 2 334 39 118 28 15.2 18.8 3 232 9 13 7 6.5 7.9 4 170 52 80 16 15.7 19.4 5 119 68 50 12 34.0 19.1 6 119 132 190 16 20.7 17.3 1 96 85 30 18 23.6 18.9 2 35 10 3 1 0.8 0.5 3 83 199 115 60 12.4 14.9 4 37 0 0 0 0 0 5 41 1 0 0 0 0 6 29 0 0 0 0 0 Hpoi = Haematopoietic tissue; AG = Antennal gland; CEC = Cuticular epithelial cells Co-infection in shrimp and its meaning in bioflocs systems Slide 5 of 26 35 Slide 6 of 26 Cumulative shrimp mortality (%) after challenge with WSSV and V. harveyi BB120 Quantification of WSSV and V. campbellii of the moribund shrimp collected at 10 h after V. campbellii injection 100 90 WSSV-infected cells in organs 80 VC in haemolymph (CFU mL-1) G (cells mm-2) SE (%) LO (cells mm-2) HPoi (cells mm-2) WSSV 167 ± 58a 17 ± 10a 83 ± 56a 315 ± 194a - VC - - - - 43 ± 61a Cumulative mortality (%) Treatments 70 60 50 40 30 20 10 0 WSSV + VC 184 ± 40a 14 ± 5a 90 ± 67a 208 ± 168a 183721 ± 73177b 0 G = Gills; SE = Stomach epithelium; LO = Lymphoid organ; Hpoi = Haematopoietic tissue Co-infection in shrimp and its meaning in bioflocs systems 24 48 Control 72 96 WSSV 120 144 168 192 216 240 264 288 312 336 360 Hpi BB120 (106 CFUshrimp-1) VC (106 CFUshrimp-1) WSSV+BB120 (106 CFUshrimp-1) WSSV+VC (106 CFUshrimp-1) Co-infection in shrimp and its meaning in bioflocs systems Slide 7 of 26 Slide 8 of 26 Dual infection: conclusions • WSSV is a lethal killer Vibrio’s in bioflocs • WSSV is inactivating the shrimp immune response • In a WSSV compromised shrimp, upon Vibrio infection, Vibrio is killing before WSSV can kill, pointing at a strong synergistic action • This phenomenon is Vibrio-strain dependent Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 9 of 26 Slide 10 of 26 Vibrio are opportunistic colonizers: Vibrio’s in bioflocs infection of seawater with bleach 100 • Vibrio’ s are omni-present CFU/ml • Prevalence in bioflocs should be studied within the concept of microbial community management (MCM) • Optimal biofloc concentration 80 60 1e+6 40 1e+5 20 • Optimal biofloc feeding strategies 1e+4 • Specific interference with Vibrio numbers and activity % Opportunists Vibrio Pseudom. Others 1e+7 0 0 1 2 3 4 5 7 9 13 17 20 25 Days Data: NTNU Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 11 of 26 36 Slide 12 of 26 Vibrio are opportunistic colonizers: suggestions for MCM Vibrio are opportunistic colonizers: suggestions for specific growth inhibition • Keep substrate/biofloc ratio low, either by • Keeping biofloc high • Negative operational consequences POLY-β-HYDROXYBUTYRATE (PHB) • Keeping substrate low • Biofloc feeding regimes • continuous or intermediant? • C/N ratio’s • Biofloc substrates Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 13 of 26 SHORT-CHAIN FATTY ACIDS POLY-β-HYDROXYBUTYRATE (PHB) • Known to inhibit growth of enteric bacteria (Salmonella, Klebsiella, Escherichia coli) • Linear polymer of β-hydroxybutyric acid O HO O O – Acidification of cytoplasm – Energy needed to keep internal pH optimal – Effect is pH-dependent (lower pH → higher effect) O O Slide 14 of 26 OH n 103-106 H+ fatty acid H+ H+ proton pump H+ bacterium Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 15 of 26 Slide 16 of 26 PHB UPTAKE BY ARTEMIA EFFECT in GART: Artemia survival • Starved nauplii without feed or with PHB particles • Artemia nauplii challenged with Vibrio campbellii • PHB particles added to culture water at start of test PHB 100 PH B T he PH B pa r t icle s a re inge st e d by t he na uplii Fluorescenc e microscopy (Nile Blue) Significantly 1000 mg/l PHB: increased 60 t ive e ffe c ly prot e c t s Art e m ia survival atprotection complete Artemia survival (%) No PHB Light microscopy 80 from lum inemg/l sc ePHB nt vibriosis 100 40 or more 20 0 ol C on tr Co-infection in shrimp and its meaning in bioflocs (bar = 250 Slideµm) 17 of 26 systems o 0) 00 ) 00 0) Vibri HB(1 H B(1 H B(1 o+P o+P o+P Vibri Vibri Vibri Co-infection in shrimp and its meaning in bioflocs systems 37 Slide 18 of 26 Vibrio are opportunistic colonizers: QU ORU M QU EN CH I N G (QQ) suggestions for specific inhibition of activity: quorum sensing = disruption of quorum sensing • Possible targets: HO OH - B O O H N O OH O H • • • O OH O OH AI-2 OH O CAI-1 HAI-1 Signal production Signal molecules Signal detection Receptor LuxP LuxQ LuxN LuxS CqsS Target genes CqsA LuxM LuxU Synthase LuxO σ54 sRNA’s + Hfq Signal Promoter of target genes LuxR Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 19 of 26 EN Z Y M AT I C AH L I NACT I VAT I ON EN Z Y M AT I C AH L I NACT I VAT I ON • AHL degradation by Bacillus strains isolated from shrimp (LT3, LT12) and sea bass (LCDR16) Use of signal-degrading bacteria as probionts, e.g. in Macrobrachium larvae: 6 60 5 50 4 Survival (%) Control 3 LT3 LT12 2 LCDR16 1 20 3 6 9 0 12 Time (h) O O NH O R NH lactonase O O R OH HO Co-infection in shrimp and its meaning in bioflocs Defoirdt et al. (2011) Aquaculture 311: Slide 21 of258-260 26 systems Co-infection in shrimp and its meaning in bioflocs Nhan et al. (2011) J. Appl. Microbiol. Slide 109:221007-1016 of 26 systems CI N NAM ALDEH Y DE CI N NAM ALDEH Y DE • Cinnamaldehyde Long time known for antibacterial properties Subinhibitory concentrations  QS disruption Effective in different host-microbe systems 100 100 100 80 80 80 Survival (%) Non-toxic synthetic flavouring substance that is widely used in food (GRAS) 60 40 60 40 40 20 20 0 0 0 Macrobrachium – V. harveyi Crustaceans: 10-100 µM 38 60 20 Brine shrimp – V. harveyi Brackman et al. (2008) BMC Microbiol. 8: 149 Survival (%) 0 • • 30 10 0 • 40 Survival (%) [HHL] (mg/L) • Slide 20 of 26 Burbot – A. hydrophila Fish: 0.01 µM Health management in biofloc system • Vibrio’ s are omni-present and can be especially problematic in virus-compromised shrimp • microbial community management (MCM); strategies for feeding floc to the benefit of the host • Specific interference with Vibrio numbers (PHB) and activity (QQ) • Holistic and integrative strategies need to be designed Co-infection in shrimp and its meaning in bioflocs systems Co-infection in shrimp and its meaning in bioflocs systems Slide 25 of 28 39 Slide 26 of 28 A REVIEW OF ACUTE HEPATOPANCREATIC NECROSIS SYNDROME RESEARCH IN VIETNAM Dang Thi Hoang Oanh Department of Aquatic Pathology College of Aquaculture and Fisheries Cantho University dthoanh@ctu.edu.vn Acute Hepatopancreatic Necrosis Syndrome (AHPNS) appeared and caused serious mortality in farmed penaeid shrimp in coastal provinces of the Mekong Delta of Vietnam in 2010 and then appeared on shrimp farms in some northern coastal provinces. The disease occurred year-round with greater severity from April to July. Disease signs at the pond level included pale to white and significant atrophy of hepatopancreas (HP), soft shells and guts with discontinuous or no contents. The onset of clinical signs and mortality start as early as 10 days post-stocking, with moribund shrimp drifting to pond embankments or sinking to the bottom. Gram-staining of a fresh smear of HP from affected shrimp clearly shows the presence of Gram-negative, rodshaped bacteria. In addition, gregarine-like entities were found in samples from AHPNS-infected and non-infected ponds alike. Vibrio spp. bacterial isolates were recovered from shrimp HP, the majority identified as V. parahaemolyticus. Rep-PCR analysis of these isolates resulted in different DNA profiles. Thermolabile hemolysin (TLH) encoded by tlh gene was detected from all tested isolates but neither tdh nor trh genes. Incidence of AHPNS seems to be greater on farms with high salinity and during the dry season with high temperature. Environmental studies demonstrated that water quality is not the main cause. The concentrations of water quality variables (i.e., salinity, pH, dissolved oxygen, unionized ammonia, nitrite, hydrogen sulfide) were within acceptable ranges as frequently in ponds with AHPNS-infected shrimp as in ponds with healthy shrimp. Moreover, there were no differences in average concentrations of pesticides between waters of ponds related to AHPNS status of shrimp. Environmental studies were carried out in laboratory conditions to determine whether AHPNS will develop in L. vannamei held in water-sediment systems containing V. parahaemolyticus and 40 pesticides (deltamethrin, fenitrothion and hexaconazole). Physical parameters were stable among the different treatments during the first (after 5 days) and second samplings (after 10 days). Chemical parameters increased during the second sampling, but remained suitable for shrimp culture, with the exception of total nitrogen and total phosphorus. Deltamethrin was not detected after five days; fenithrothion and hexaconazole were detected during the second sampling but decreased with time. The combination of pesticides and bacteria increased mortality of experimental shrimp. Shrimp exposed to pesticides without bacteria did not show typical AHPNS pathology. Transmission studies were carried out to determine if AHPNS can be transmitted via water and by co-habitation. Experimental hapas containing healthy L. vannamei were set up in AHPNSinfected ponds to study transmission of AHPNS via water. Hapas containing healthy and AHPNS-infected shrimp were set up in AHPNS-infected ponds to study transmission in EMS/AHPNS by co-habitation. Thirty shrimp per treatment were checked for AHPNS by histology after 10 days. Bacterial isolation was carried out to recover V. parahaemolyticus in AHPNS-affected shrimp and these were used for DNA profiling and detection of tdh and trh genes. Gross signs and histopathology revealed typical AHPNS clinical signs and lesions in experimental shrimp. Transmission of AHPNS occurred via water (16/60 tested shrimp) and cohabitation (27/60 tested shrimp) of healthy shrimp in AHPNS-infected ponds. V. parahaemolyticus isolates were recovered from the HP of shrimp samples. 41 AHPNS in Vietnam Officially reported as hepatopancreatic necrosis syndrome since April 2010, in Soc Trang province. A REVIEW OF ACUTE HEPATOPANCREATIC NECROSIS SYNDROME RESEARCH IN VIETNAM • Dead shrimp found at the bottom of cultured ponds • Mortality up to 60% after several days (3-7 days) • Behavior of the shrimps was different from other diseases, such as WSD, YHD,… • Abnormal hepatopancreas (atrophied, swollen, soften) Dang Thi Hoang Oanh, Pham Phong Vu, Dao Thi Thanh Hue, Le Van Khoa, Pham Anh Tuan, Don Lightner, Tim Flegel and Melba B. Reantaso (DAH, 2012) Occurrence of EMS/AHPNS in Vietnam (2012) (1) MD, VN Occurrence of EMS/AHPNS in Vietnam (2012)(2) Occurred in 19 coastal provinces 1. The disease occurred all year round with severity from April to July. 2. It affected on farms cultured of black tiger (P. monodon) or white shrimp (L. vannamei) 3. Mainly in areas of intensive and semi-intensive farming systems. 4. Incidences of AHPNS seem to be higher in farms with high salinity; during dry season with high temperature. 5. Results of environmental parameter tests showed that water quality is not the main cause Effected areas (September 2012): Country: 46,093 ha Soc Trang: 12,882 ha Bac Lieu: 11,554 ha Ca Mau: 9,188 ha Effected areas were reported by observation on gross sign of AHPNS without histopathology. Actual effected areas may be less than the reported areas. Source: DoF (2012) Source: DoF (2012) Disease card - AHPNS Collection and testing of samples in existing outbreak and non-outbreak areas in the Mekong Delta A disease card was prepared based on the outcomes of the Asia Pacific Emergency Regional Consultation on EMS/AHPNS held in Bangkok, Thailand on 9-10 August 2012. The workshop was co-organized by the Australian Government Department of Agriculture, Fisheries and Forestry and the Network of Aquaculture Centres in Asia-Pacific (NACA). 42 2.Locations and date of collecting samples Sampling times 18 ponds (9 infected) 23 ponds (16 infected) Sampling and samples analysis 19 ponds (11 infected) Sampling pond (~ 100-300 shrimp/pond ) Hepatopancreatic Fresh smear Bacterial isolation Phage assay Fungal isolation Davidson’s fixative EtOH PCR analysis Histopathology Infectivity 43 Liquid nitrogen, then -80C Collection of samples Testing of samples (Cont.) 1. Histologycal analysis: RAHO6, UAZ and CENTEX 1. 60 samples (each sample has more than 20 shrimp) were preserved in Davidson’ fixative 2. 60 samples (pooled gills and pleopods) were preserved in 95% ethanol 3. 175 bacterial isolates (~ Vibrio) were recovered from hepatopancreatic of shrimp specimens (- 80 °C at CTU) 4. 10 fungal isolates were recovered from hepatopancreatic of shrimp specimens (4C at CTU) 5. 60 frozen samples are kept at -80C at CTU 2. PCR analysis: RAHO7 3. Bacterial and fungal identification: CTU 4. Bacterial metagenomics: CENTEX 5. Infectivity: UAZ TCP/VIE/3304 (E) Disease signs at pond level    Diagnosis studies     Gross signs P.vanamei Abnormal HP Normal HP 44 Pale to white hepatopancreas (HP) Significant atrophy (shrinkage) of HP. Soft shells and guts with discontinuous contents or no content. Black spots or streaks sometimes visible within the HP. HP does not squash easily between thumb & finger. Onset of clinical signs and mortality starting as early as 10 days post stocking. Moribund shrimp came to the pond side or sink to bottom. Necrostic HP from inside to outside 45 Results of histopathology Laboratory UAZ CENTEX RAHO6 60 ponds were ST BL CM SUM ST BL CM SUM ST BL CM SUM examined EMS/AHPNS WSSV MBV HPV Gregarine 3 16 10 7 4 1 0 18 1 0 2 0 - - Gregarine like - - - 29 12 19 2 - 6 17 7 7 1 1 0 8 2 0 1 0 - - - 30 9 10 2 - 4 10 1 0 1 18 9 7 5 7 0 1 0 6 1 31 22 8 1 8 12 8 33 6 2 10 15 2 BL: Bac Lieu CM: Ca Mau ST: Soc Trang Bacterial isolation and identification (3) Results of PCR analysis Results Test WSSV YHV IMNV Positive Negative 23 37 0 60 0 60 V. parahemolyticus ( API20E, bioMerieux, Pháp) V. vulnificus (API20E, bioMerieux, Pháp) 46 Detection of TLH gene from V. parahaemolyticus M 1 2 3 4 5 6 7 8 9 , 10 11 12 13 14 15 16 17 18 18 isolates from Baclieu AHPNS samples: BL6.1; 02; 07; 01; 4.2; 5.3; 5.6; 5.8; 5.10; 6.1; 6.9 (1); 6.9 (2); 6.10; 8.2 (1); 8.2 (2); 8.3; 8.5; 8.7 Detection of TDH and TRH genes from V. parahaemolyticus M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 TCP/VIE/3304 (E) 17 18 Field study on transmission in AHPNS infected pond 18 isolates from Baclieu AHPNS samples: BL6.1; 02; 07; 01; 4.2; 5.3; 5.6; 5.8; 5.10; 6.1; 6.9 (1); 6.9 (2); 6.10; 8.2 (1); 8.2 (2); 8.3; 8.5; 8.7 Objectives TIME AND LOCATION • Time: April-May, 2013. To determine if AHPNS is tranmissible by water and by co-habitation of healthy shrimp in AHPNS infected ponds • Location: Cống Ấp 10, Tân Duyệt Commune, Đầm Dơi district, Cà Mau province. 47 Gross sign Experimental set up • Transmission in AHPNS via water (setting up hapas containing 100 healthy shrimp/hapas) • Transmission in AHPNS by co-habitation (hapas containing 100 healthy shrimp and 15 AHPNS infected shrimp) • After 10 days: check for AHPNS by histology and bacterial isolation 48 RESULTS Mortality Vibrio bacterial isolation and identification Detection of TLH gene from V. parahaemolyticus • 74 bacterial isolates (~ Vibrio) were recovered from hepatopancreatic of shrimp specimens • 31 isolates were identified as V. parahaemolyticus by API 20E 49 Detection of TDH and TRH genes from V. parahaemolyticus Conclusions AHPNS is tranmissible by water and by cohabitation of healthy shrimp in EMS/AHPNA infected ponds ACKNOWLEDGEMENTS Funding resources 1. FAO project: TCP/VIE/3304 Thank you for your attention! 2. Vietnam National task force for EMS Technical advisors 1. Prof. Don Lightner, University of Arizona, USA 2. Prof. Tim Flegal, Mahidol University, Thailan 50 BIOFLOCS IMMUNITY AND SHRIMP DISEASE 51 IMMUNE MECHANISMS IN CRUSTACEANS Kenneth Söderhäll Department of Comparative Physiology Uppsala University Sweden Kenneth.Soderhall@ebc.uu.se Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. The continuous renewal of hemocytes and their release from hematopoietic tissue is tightly regulated in crustaceans by two different astakines, members of a new family of cytokines sharing a prokineticin (PROK) domain. Astakine 1 is essential for the release of new hemocytes into the open circulatory system of Pacifastacus leniusculus, while astakine 2 plays a role in granular cell differentiation. These two astakines differ in their three-dimensional structure and receptor binding and, interestingly, show differences in the timing of expression. Hemocyte release from hematopoietic tissues is under circadian regulation and this phenomenon correlates with a rhythmic expression of astakine1 and astakine 2. Time variations in astakine1 and astakine 2 expression has an impact on hemocyte number and immunity, assessed as susceptibility to a pathogenic Pseudomonas species. These findings indicate the significance of comparing immune responses at predetermined times and not to ignore circadian regulation of immune responses. We are also studying the antiviral response, particularly the response to WSSV. The crossroad between cell death and proliferation is a general target for viral infections because viruses need to obstruct apoptosis to use cells for their own replication. Inducing immunogenic cell death in proliferating cells is also an important aim of anticancer chemotherapy. The C1q-binding proteins calreticulin (CRT) and gC1qR are highly conserved and ubiquitous proteins, which are putative targets for viral manipulation and are associated with cancer. These proteins form a complex in the cytoplasm as a response to viral infection, resulting in apoptosis prevention. The formation of a cytosolic CRT/gC1qR complex prevents cell death by reducing gC1qR translocation into the mitochondria. This mechanism is conserved from arthropods to human cancer cells. Furthermore, it is possible to prevent this complex from being formed in cancer cells. When peptides of the complex proteins are overexpressed in these cells, the cells undergo 52 apoptosis. This causal link between virus and cancer may be used to develop new tools in anticancer or antiviral therapy. Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is activation of prophenoloxidase (proPO) by a proteolytic cascade, finalized by the proPO activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly-regulated and limited proteolysis of downstream processes and are also involved in inflammatory responses to infections, such as activation of interleukin 1ß. We demonstrate a link between caspase cleavage of proPO, release of this protein and the biological function of these fragments in response to bacterial infection. 53 INVERTEBRATES How do they defend themselves? IMMUNE GENES AND THEIR FUNCTION IN A CRUSTACEAN Limulus polyphemus/ Tachypleus tridentatus Tenebrio molitor Pacifastacus leniusculus PACIFASTACUS LENIUSCULUS Professor Kenneth Söderhäll Department of Comparative Physiology, Uppsala University, Sweden Kenneth.Soderhall@ebc.uu.se Drosophila melanogaster CRUSTACEAN IMMUNITY Bombyx mori and Manduca sexta CLOTTING - COAGULATION • Clotting-Coagulation • Pattern recognition proteins •The ProPO-system • Blood cell synthesis • DSCAM Specific immunity • Viral immunity • Intestinal immunity • Antimicrobial peptides CP in plasma SS SS Ca2+ Ca2+ Ca2+ TGase Ca2+ TGase from hemocytes SS Ca2+ SS α2M SS SS SS SS SS Hemocyte Most recent review. Cerenius L and Söderhäll K. 2011 J. Innate Immunity Recognition Lipid A Lipopolysaccharide LPS Certain structures/molecules are conserved in microorganisms Gram-positive bacteria O-antigen Peptidoglycan Core Peptidoglycan DAP/lys =N-acetylmuramic acid Fungi Lipopolysaccharide, LPS Peptidoglycan =N-acteylglucosamine Glycoprotein ß1,3-glucan Chitin ß-1,3-glucan Gram-negative bacteria 54 Recognition ß-1,3-glucan-binding proteins Pattern recognition proteins Pattern recognition receptors LPS-binding proteins Faktor C (Limulus polyphemus), serine proteinase LGBP (Pacifastacus leniusculus), binds both LPS och ß-1,3-glucans GNBP (Anopheles gambiae) BmLBP (Bombyx mori) CCF-1 (Eisenia foetida), binds both LPS och ß-1,3-glucans PATTERN RECOGNITION PROTEINS Plasma THE PROPO SYSTEM Hepatopancreas Masquerade SPH1 SPH2 Bacteria Fungus Ficolin-like MIP βGBP Serpin Fungus MBL Bacteria Bacteria Bacteria C-type lectins Masquerade Hemocyte Most recent review. Cerenius L and Söderhäll K. 2008 Trends in Immunology C i L t l 2010 T d i Bi h i l S i MELANIZATION MELANIZATION is induced by bacteria and fungi No bacteria Increased number of bacteria Tyrosinase/Penoloxidase-activity 55 MELANIZATION MELANIZATION Tyrosinase/Penoloxidaseactivity MELANIZATION Plants Fungi ”Arctic” GMO which lacks PO Normal apple BLOOD CELL SYNTHESIS CHF MELANIZATION Clock BMAL1 RACK1 In nearly all organisms PCNA Ast1 PCNA TGase CHF RUNX RUNX PER CHF Ast1 ProPO RUNX PER ProPO RUNX KPISG CHF Ast2 ProPO RUNX PER SOD MBL Review Lin X and Söderhäll I. 2010 Blood 56 HPT cell culture HEMATOPOIETIC STEM CELL CULTURE Fresh isolated HPT cells HPT cells after 3 and 14 days in astakine or plasma Söderhäll I. et al. 2005 J. Immunology Jiravanichpaisal P. et al. 2006 J. Gen.Virol. Söderhäll. I. et al J. Immunology. 2005, 174: 6153‐6160. Virus replicates in HPT stem cell cultures Astakines in arthropods Astakine 1 Astakine 2 A new anti-viral factor identified in HPT Viral replication in HPT stem cells Crustacea Insecta Chelicerata New method for gene silencing in Stem cell cultures Lin X, Novvotny M, Söderhäll K & Söderhäll I.2010 J. Biol. Chem.285, 21467‐ 21477 Four new antibacterial peptides identified in HPT and hemocytes Down syndrome cell adhesion molecule Dscam DSCAM Specific immunity cDNA An immunoglobulin superfamily member, Dscam, in bacterial defense Protein 5’ 6009 bp 3’ 2002 aa Dscam protein structure and its variable regions PlDscam can generate more than 22,000 different unique isoforms 57 Specific Dscam isoforms in bacterial clearance Clustering of all Dscam isoforms E. coli S. aures Specific Dscam isoforms in phagocytosis Dscam and bacterial defense propsed model Dscam Coated E. coli E. coli E. coli induced isoforms Uptake of pathogen Coated S. aureus P. leniusculus hemocyte S. aureus S. aureus induced isoforms : rE. coli induced isoforms ; : rS. aureus induced isoforms VIRAL IMMUNE STRATEGIES crossroad between apoptosis and proliferation mRNA expression level gC1qR CRT/g C1qR Apoptosis PlgC1qR PlCRT 0 Watthanasurorot et al. 2010 J. Virology Watthanasuroroot A. et al. 2013 J. Mol. Cell Bi l 6 12 24 Hours after WSSV infection 58 A CRT-gC1qR complex is formed in virus-infected cells CRT and gC1qR interact in early virus infection gC1qR CRT The CRT-gC1qR complex CRT-gC1qR complex during WSSV infection Confirmation by IP (immunoprecipitation) In vitro: HPT cell DTT DTT CRT‐gC1qR complex CRT gC1qR CRT‐gC1qR complex CRT gC1qR WSSV replication cycle + reducing condition - non reducing condition The CRT-gC1qR complex is present in cancer cells N- and P-domains of CRT bind to gC1qR 19 1 170 285 C N 18 Signal peptide N-domain 169 P-domain 284 404 bp C-domain 59 Overexpression of CRT, gC1qR and their domains induce apopotosis CRT gC1qR CRT CRT CRT PLA (proximity ligation assay) shows CRTgC1qR in situ gC1qR C-domain of CRT overexpression and MTX reduce the complex Catalase (CAT) is used as internal control Hypothetical model VIRAL IMMUNITY ALF Antilipopolysaccharide factor TCTP (Fortilin) Translationally Controlled Tumor Protein ANTIMICROBIAL PEPTIDES Plasma Astakine 2 - links Melatonin to Circadian Regulation in Crustaceans Hepatopancreas Kazal –type PI Astacidine 1 FKVQNQHGQVVKIFHH‐COOH Astacidine 2 RPRPNYRPRPIYRP Crustins s-s s-s WAP Crustin 1&2 Gly-rich WAP Crustin 3 Liu H. et al. 2007 J. Virology Watthanasurorot et al. 2010 J. Virology Watthanasuroroot A. et al. 2013 J. Mol. Cell Bi l Hemocyte Cerenius L. et al. 2010 Dev. Comp. Immunol. Jiravanichpaisal P. et al. 2007 Dev. Comp. Immunol. Lee SY. et al. 2003 J. Biol. Chem. 60 mRNA expression level Circadian rhythmic variation of the number of blood cells have important implication in immune defense AST2 AST1 Day Wathanasurorot A, Jiravanichpaisal P Söderhäll K & Söderhäll I. 2011.Cell Molecular Life Sci 68,315‐323 Night Melatonin induces Astakine 2 transciption Melatonin induces Astakine 2 transciption In vitro, HPT cell culture In vivo, hemocytes In vivo, HPT In vivo, brain An Ast2-Bmal1-Rack1 complex at night only Melatonin induces Astakine 2 translation Day In vitro In vivo Night 61 Melatonin induces Ast2-Bmal1-Rack1 High expression Day Induce Induce High expression E‐box CLK-PlBMAL1 complex Inhibit High expression Night Induce Melatonin Induce AST2-PlBMAL1PlRACK1 complex In case dsAST2 Knock down Induce CLK-PlBMAL1 complex THANKS TO Comparative Immunology group, Uppsala University PI Dr Irene Söderhäll, Protein expression level E‐box Dep. Comparative Physiology Uppsala University Present postdocs Dr. Enen Guo, China Dr. Veronica Chico-Gras, Spain Dr. Pikul Jiravanichpaisal BIOTECH, Bangkok Thailan Now Fish Vet Group,Asia ltd Bangkok Present PhD students Chadanat Noonin Apiruck Wathanasurorot Kingkamon Junkunlo Gizem Korküt AST2 CLK PlBMAL1 Recent PhD students and their present employment Dr. Young-A Kim, PlRACK 1 Harvard Medical School Boston Dr. Haipeng Liu, State Key Laboratory of Marine Environmental Science, Xiamen University Dr. Xionghui Lin, Cedric Blanpain Lab IRIBHM, Université Libre de Bruxelles Dr. Chenglin Wu, Day Karolinska Institute Stockholm Sweden Night 62 Visiting PhD students Sirinit Tharntada, Bangkok Thailand Valaiporn Charoensapsri, Bangkok Thailand Suchao Donpudsa, Bangkok Thailand Netnapa Saelee, Sonkla Thailand Benjamas Nupan, Sonkla Thailand Shibata Toshiba, Fukuoka Japan Collaborators Dr. Marian Novotny, Prague, Check Republic Dr. Seiko Nakamura, Uppsala University, Prof.Chu Fang Lo Taiwan EVALUATION OF IMMUNE ENHANCEMENT OF SHRIMP GROWN IN BIOFLOC SYSTEMS In-Kwon Jang* and Su-Kyoung Kim West Sea Fisheries Research Institute National Fisheries Research & Development Institute Incheon 400-420, Republic of Korea jangik@korea.kr Biofloc technology (BFT) for shrimp production has been proposed as a sustainable practice capable of reducing environmental impacts and preventing viral introduction. The microbial community associated with bioflocs not only removes nutrients, but can improve feed utilization and animal growth. Biofloc systems contains abundant bacteria of which the cell wall consists of various components such as bacterial lipopolysaccharide, peptidoglycan, and β-1, 3-glucans. Bacterial cell wall components stimulate non-specific immune activity in crustaceans, including shrimp. Bioflocs, therefore, are assumed to enhance immunity of shrimp that consume them as a food source. Although there are many studies that show biofloc effects on shrimp growth and survival, more work is needed to obtain a better understanding of the roles of biofloc microorganisms, particularly at the molecular level. To examine biofloc effects on shrimp immunity, short-term culture trials were conducted with postlarvae of three farmed species (Litopenaeus vannamei, Marsupenaeus japonicus and Fenneropenaeus chinensis) in the presence and absence of bioflocs. Six genes (prophenoloxidase1, prophenoloxidase2, prophenoloxidase activation enzyme, serine proteinase1, masquerade-like proteinase, and ras-related nuclear protein) that are involved in a series of responses known as the prophenoloxidase (proPO) cascade, one of the major innate immune responses in crustaceans, were selected to determine mRNA expression levels using a TaqMan real-time PCR method. The mRNA expression of most selected genes was significantly different between clean seawater and biofloc treatment groups in the three species, except for a few genes, such as ras-related nuclear protein gene in M. japonicus. In addition, mRNA expression levels of the genes are strongly affected by total suspended solids (TSS) concentration of treated biofloc. This suggests that the presence of biofloc and its concentration may have effects on shrimp immunity, depending on species. 63 Using bioflocs as an additional food source that consequently may have a positive effect on shrimp immunity is well reported in L. vannamei, but little is known about this effect in other penaeid species. In crustaceans, efficiency of harvesting and consuming food particles such as bioflocs are largely affected by the morphological structure of the third maxilliped in the mouthparts. Based on microscopic observations, the type, length and distance of the setae and setules (secondary setae) on the third maxilliped are significantly different among the three species. This observation may well explain differences in survival and growth in biofloc systems among the three species evaluated. 64 65 No Discharge High Production High Food Safety High Immunity Low FCR 66 No antibiotics High Biosecurity Low CO2 emmission Final B.W. (g) Total Harvest (kg) Initial Stocking B.W. Density (g) (/m2) Days Mean 0.13 465.5 145 16.7 4.83 2,754 64.5 1.91 Min 0.038 404 112 13.6 3.37 2,020 33.7 1.19 Max 0.26 WT ( ) 500 DO (mg/L) 177 Sal (psu) 19.9 Yield (kg/m2) 6.40 3,280 Survival rate(%) 88.3 W.T. DO Sal (℃) (mg/L) FCR 2.91 pH TAN (mg/L) NO2 (mg/L) NO3 (mg/L) TSS (mg/L) VSS (mg/L) Alk (mg/L) Tur (NTU) phase 1 (2013.06.03 - 07.11) tank1 28.9 6.2 32.3 7.4 0.65 0.38 33.76 375.8 296.4 137.1 206.2 tank2 28.6 7.0 32.0 7.4 0.92 1.55 28.63 327.8 237.3 142.2 177.1 phase 2 (2013.07.11 - 08.22) tank1 29.6 5.9 30.2 7.2 0.18 0.13 99.50 457.67 343.17 143.0 308.3 tank2 29.4 6.0 29.7 7.2 0.16 0.14 92.50 350.33 258.00 145.8 243.5 phase 3 (2013.08.23 -10.07) tank1 28.8 6.2 31.3 7.3 0.28 0.16 147.69 638.00 404.00 143.4 328.0 tank2 28.8 6.4 31.1 7.3 0.29 0.16 154.23 610.33 392.33 145.4 287.0 67 pH TAN NO2-N NO3-N Chl-a TSS VSS (mg/L) (mg/L) (mg/L) (㎍/L) (mg/L) (mg/L) Alkal (mg/L) Tur. (NTU) Mean 28.5 5.6 32.9 7.3 0.45 3.35 57.7 206.9 821.4 312.2 157.6 152.8 Min 28.1 5.1 31.4 7.0 0.2 1.2 33.2 565 204.5 99 90.5 Max 29.2 6.2 34.3 7.7 0.8 5.9 94.4 402.6 1134 383 216.3 212.2 52.2 Bioflocs consist of bacteria, fungi, algae, detritus and other suspended particles (Hargreaves 2006, Avnimelech, 2011) mas Bacterial cell wall consists of LPS, PG and β -1, 3-glucans, activating the non-specific immune system (Johansson & Söderhäll 1985) Bioflocs can be consumed by cultured animals (Avnimelech et al. 1994; McIntosh, 2000) proPO PPAE Bioflocs may contribute to enhance the shrimp immunity Select six genes which are involved to innate immune response of shrimp GenBank accession No. & Remarks EF115296.1 EF115296.1 proPO1-RT- P EF115296.1 proPO2-RT- F EF565469.1  proPO1(prophenoloxidase 1), proPO2 (prophenoloxidase 2), PPAE proPO2-RT- R proPO2-RT- P EF565469.1 (prophenoloxidase activating enzyme), SP1 (serine protease), mas PPAE1-RT- F Jang et al., 2011 PPAE1-RT- R Jang et al., 2011 (masquerade-like serine proteinase), Ran (ras-related nuclear) PPAE1-RT- P Culture shrimp in biofloc media  Mysis to PL3, postlarvae (PL20) and adult (about 10g) Experimental groups (L. vannamei) BF100 (TSS 678 mg/L), BF 75 (TSS 508), BF 50 (TSS 340), BF 25 (TSS 170), BF0 (TSS 13) 68 EF565469.1 Jang et al., 2011 SP1- RT- F JX644456, present study SP1-RT- R JX644456, present study SP1-RT- P JX644456, present study mas-RT- F Measure mRNA expression of six genes using real-time RT PCR • Name proPO1-RT- F proPO1-RT- R JX644451, present study mas-RT- R JX644451, present study mas-RT- P JX644451, present study Ran-RT- F JX644455, present study Ran-RT- R JX644455, present study Ran-RT- P JX644455, present study β-actin-RT- F Jang et al., 2011 β-actin-RT- R Jang et al., 2011 β-actin-RT- P Jang et al., 2011 PO activity (Dopachrome formation/min/mg protein) 1.0 0.8 b b 0.6 a 0.4 a a 0.2 0.0 Exp.I (100% ) Exp.II (75%) Exp.III (50%) Exp.IV (25%) Control (0%) • In short-term culture (two weeks) of L. vannamei postlarvae (2.2 mg BW) • BF100 (TSS 678 mg/L), BF 75 (TSS 508), BF 50 (TSS 340), BF 25 (TSS 170), BF0 (TSS 13) • Gene expression level was normalized to β-actin. *: P<0.05, **: P<0.01. 0.6 proPO mRNA expression levels (Normalize to β-actin) PPAE mRNA expression levels (Normalize to β-actin) 4.5 proPO 0.5 biofloc seawater 0.3 0.2 • BF100% (TSS 820 mg/L), 75% (TSS 610), 50% (TSS 400), 25% (TSS 200) mRNA expression of selected genes and PO activity are significantly higher in L. vannamei larvae or adult cultured in biofloc water than control. 3 2.5 2 1.5 0.1 Long-term culture of adult L. vannamei (10 g) PPAE 4 3.5 0.4 1 0.5 0 mRNA expression levels and PO activity are different in animals cultured in different TSS concentrations 0 L. vannamei F. chinensis Species L. vannamei M. japonicus F. chinensis Species M. japonicus 7.00E+00 6.00E+00 Ran mRNA expression levels (Normalize to β-actin) Mas mRNA expression levels (Normalize to β-actin) 1.20E+00 mas 1.00E+00 Ran 5.00E+00 8.00E‐01 Effects of bioflocs on shrimp immunity are different in different species. It may depend on species ability to use bioflocs as food source. 4.00E+00 6.00E‐01 3.00E+00 4.00E‐01 2.00E+00 1.00E+00 2.00E‐01 0.00E+00 0.00E+00 L. vannamei • Bio-floc ratio • F. chinensis Species M. japonicus L. vannamei F. chinensis Species M. japonicus L. vannamei, F. chinensis and M. japonicus Fleshy shrimp, F. chinensis Kuruma shrimp, M. japonicus Pacific white shrimp, L. vannamei 69 Short-term culture BF0 BF25 BF50 BF75 BF100 Long-term culture BF0 BF25 BF50 BF75 BF100 Final B.W. (mg) 76.97a 104.19b 113.97b 114.86b 132.07 b Final B.W.(g) 8.72a 10.85b 10.87b 11.12b 9.21c Standard Error 6.66 9.06 8.53 9.52 9.76 FCR 1.70 1.38 1.37 1.65 1.45 Survival rate (%) 83a 89a 85a 92a 96a Survival rate (%) 95.0 95.5 96 88.8 98.8 • In short-term culture (two weeks) of postlarvae (2.2 mg BW) • BF100 (TSS 678 mg/L), BF 75 (TSS 508), BF 50 (TSS 340), BF 25 (TSS 170), BF0 (TSS 13) In long-term culture trial (two months) of juveniles (1.69 g BW) TSS conc. ranges from 200 mg/L (BF25) to 700 mg/L (BF100) The 3rd maxilliped performs multiple function Culture media Factor L. vannamei F. chinensis M. japonicus SGR5(% bw d-1) 15.93±0.91 a 3.98±0.57 b 5.10±0.43 b Survival rate (%) 91.50±2.88 a 32±1.41 b 75±1.57 c SGR5(% bw d-1) 11.88±0.98 a* 4.89±0.56 b 5.25±0.53 b Survival rate (%) 83±8.36 a 52±0.06 b 83±2.50 a including manipulation of food particles, preening, and moving as a response to chemical stimuli Biofloc Different types of setae play different roles (Garm 2004) Seawater Setae on 3rd mxp forming ‘mesh’ structure Specific growth rate (SGR)=(loge final B.W-loge initial B.W)/day*100 which potentially trap suspended particles (~10 μm) (Moss 1993, Kent et al 2011) Postlarvae (PL10-20) cultured for two weeks TSS 637 mg/L for L. vannamei; TSS 486 mg/L for F. chinensis and TSS 577 mg/L for M. japonicus Segments Ischium The morphology of the third maxilliped in three penaeid species.  A: Litopenaeus vannamei;  B: Fenneropenaeus chinensis; 70 M. japonicus mainly simple setae mainly simple setae a row of simple, serrulate setae Merus 2 rows of plumose setae a row of simple setae 2 rows of plumose setae a row of simple, serrulate setae 2 rows of simple 3 cuspidate setae (distally), 2 5 cuspidate setae (distally), 7 cuspidate setae (distally), Dactylus Scale bars: A-C = 0.5 mm, DG = 0.1 mm. of simple setae F. chinensis Carpus Propodus  C: Marsupenaeus japonicus L. vannamei a row of plumose setae, a row rows of plumose setae 0.7x propodus, 2 rows of plumose setae simple and serrulate setae simple long setae 0.3x propodus, simple and 0.4x propodus, simple and serrulate setae on entire serrulate setae on entire surface surface Table 3. Mean values of body length, third maxilliped length, total number of seta, seta 200 Number of seta (piece) N indicates the number of specimens examined. (±S.D, n=5, p<0.01) Factor Species L. vannamei F. chinensis M. japonicus Body length (mm) 23.23±1.25 22.07±3.31 21.18±2.81 Third maxilliped length (mm) 1.18±0.13a 0.84±0.22b 0.73±0.04b 250 F. chinensis b 100 a a a a b b c b 154±12b b a a 100 29.1±4.7 b 29.3±3.6 b Setule length (µm) 13.7±2.5 a 3.6±0.7 b 6.9±0.9 b Setule distance (µm) 7.58±0.66 a 2.26±0.22 b 9.64±4.23 a Filter area (cm2) 32.05±2.16 a 16.82±2.24 b 11.36±1.78 b a b b b b b 40 2nd seg. 3rd seg. 4th seg. 5th seg. 1st seg. total C a 12 a 10 8 b b b b b 2 0 1st seg. 2nd seg. 3rd seg. 4th seg. 4th seg. 5th seg. b b b b b a b b b a 30 14 4 3rd seg. 40 35 6 2nd seg. Each segment of the thrid maxilliped D a 16 5th seg. Each segment of the thrid maxilliped Distance of seta (㎛) 21.4±1.8 a b b 60 Each segment of the thrid maxilliped 71.1±13.1 b Seta distance (µm) b b 80 20 b a,b 20 187±24 c 79.0±19.7b b a 120 0 Length of setule (㎛) 123.1±23.0a a 140 a Seta Length (µm) a 160 0 1st seg. 226±10a a b 18 Total number of seta c M. japonicus 150 50 B a 180 a L. vannamei 200 Length of seta (㎛) A length, seta distance, setule length, setule distance and filter area of three penaeid species. a 25 a a a a 20 15 10 5 0 1st seg. 2nd seg. 3rd seg. 4th seg. 5th seg. Each segment of the thrid maxilliped . Mean values of the number (A), length (B) and distance (C) of setae, and length of setule (D) for each segment of the third maxilliped. 1st to 5th segments represents from the ischium to the dactylus. (±S.D, n=5, p<0.05) Growth and survival rate of L. vannamei are higher F. chinensis and M. japonicus, suggesting that L. vannamei may use bioflocs as food source better than the other two species. Setal structure of 3rd maxilliped in L. vannamei is well adapted for collecting fine particulates such as bioflocs, comparing with F. chinensis and M. japonicus. The results suggest that L. vannamei postlarvae may effectively collect and consume bioflocs using 3rd maxilliped structure Habitat Microscopic counts/mL Culturability Seawater 105-106 0.001-0.1% freshwater 106-107 0.01-0.1% Groundwater 104-105 <1% Sediments 106-109 <1% Bioflocs 107-108 3.9 x 108 Avnimelech 2012 Otish et al 2006 • 102-103 times more bacteria in seawater than can grow on plates • Pyrosequencing (GS-FLX-Titinium) 71 No. Sampling date 1 2013-07-31 Sources YA1 (outdoor pond1-SW) Culture conditions 2013.05.30-09.27, 550 m 2, density 161/m2 2 2013-07-31 YA3 (outdoor pond 3: low sal) 2013.06.03-09.27, 550 m 2, density 200/m2 3 2013-07-31 SQ1-1 (indoor tank1: low sal) 2013.06.04-9.27, 10 m3, density 1000/m3 4 2013-07-31 SQ2-1 (indoor tank2: SW) 2013.06.04-9.27, 10 m3, density 1000/m3 5 2013-09-31 SQ1-2 (tank 1-2: low sal) 2013.06.04- 9.27, 10 m3, density 1000/m3 6 2013-09-31 SQ2-2 (tank 2-2: SW) 2013.06.04-9.27, 10 m3, density 1000/m3 7 2013-07-31 GH (greenhouse: SW) 2012.10.26-2013.09.27, 300 m 2 raceway 8 2013-10-01 TA1 (private indoor farm: SW) 2013.06.22~present, 670 m 2 9 2013-08 INC1 (private earthen pond) 1 ha earthen pond: 30/m2 10 2013-08 INC2 (private BFT pond) 1,000 m2 HDPE pond: 100/m2 Biofloc Sample Samples YA1 (outdoor pond1-SW) Number of tre ated sequence s 8,143 Number of OTUs Chao1 richness estimator Shannon index Simpson div ersity index 576 909.35 4.61 0.02 YA1 YA3 GH TA1 INC1 INC2 Bacteroidetes 3.6 2.83 0.99 1.55 4.33 3.24 2.39 1.73 1.67 4.15 Alphaproteobacteria 3.37 2.35 2.84 0.69 3.77 2.12 0.6 0.47 2.88 Chloroflexi 1.46 0.49 0.58 0.05 0.1 0.46 3.85 4.28 0.08 0.32 Actinobacteria 0.45 1.21 1 0.33 0.43 1.59 0.15 0.41 3.95 0.34 Gammaproteobacteria 0.6 0.38 1.05 0.47 0.28 0.33 1.73 0.5 0.35 0.44 Planctomycetes 0.54 1.34 0.86 0.2 0.29 0.75 0.44 0.2 0.09 0.34 Deltaproteobacteria 0.3 0.85 0.79 0.19 0.39 0.95 0.68 0.07 0.17 0.28 0 0.01 0.12 0.03 0.13 0.47 0.07 0.06 0.67 1.41 TM7 0.12 0.36 1.08 0.4 0.09 0.12 0.34 0.19 0 0.07 Armatimonadetes 0.04 0 0.62 0 0 0.09 0.98 0.05 0 0 0 0.21 0.1 0 0 0.45 0.15 0.01 0.28 Acidobacteria 0.06 0.29 0.46 0.03 0.02 0.01 0.02 0.02 0 0.02 Betaproteobacteria 0.12 0.02 Gemmatimonadetes Cyanobacteria YA3 (outdoor pond 3: low sal) 8,932 605 821.92 5.05 0.01 SQ1-1 (indoor tank1: low sal) 8,772 449 604.04 3.89 0.05 SQ2-1 (indoor tank2: SW) 8,908 773 1,134.25 5.19 0.01 SQ1-2 (tank 1-2: low sal) 8,525 459 632.87 4.43 0.03 SQ2-2 (tank 2-2: SW) 3,990 351 595.61 3.96 0.06 GH (greenhouse: SW) 9,687 550 756.19 4.04 0.08 TA1 (private indoor farm: SW) 6,748 420 567.12 3.61 0.14 INC1 (private earthen pond) 8,247 497 884.65 4.64 0.01 INC2 (private BFT pond) 8,270 729 1,097.44 4.51 0.04 GN02 SQ2-2 1.93 0 0.17 0.06 0 0 0.42 0 0.04 0.05 0.21 0.08 0.01 0.04 0.28 0.03 0.01 0.02 0 0 0.01 0.56 0 0 0 0.04 0 0 Bacteria_uc 0.09 0.04 0.05 0 0.02 0.1 0.02 0.05 0.02 0.02 BRC1 0.01 0 0.03 0.27 0.01 0.04 0 0 0 OP11 0 0 0 0.01 0.01 0 0 0 0.01 0.03 0 0.28 0.02 0.01 0.06 0.01 0.04 0.05 0 0.02 0.04 0.05 0 0.01 OD1 0.01 0 0.03 0.01 0 0 0.08 0.02 0.07 0.02 Firmicutes 0.01 0.03 0.02 0.01 0 0 0 0.01 0.02 0.09 Caldithrix_p DQ404828_p NKB19 Proteobacteria_uc 72 SQ2-1 0.07 Verrucomicrobia Bacteroidetes group is the most dominant (26.5%), followed by Alphaproteobacteria, Chloroflexi and Actinobacteria groups SQ1-2 EF092200_p Chlorobi Bacteroidetes group including Sphingobacteria, Flavobacteria and Gammproteobacteria are found in all samples SQ1-1 0.01 0 0.01 0 0 0 0.1 0.15 0 0 0.01 0 0.15 0 0 0 0 0 0.01 0 0.02 0 0 0 0.08 0 0 0 0 0 0.01 0.02 0 0.08 0 0 0 0 0.01 0.02 0.01 0.01 0.01 0 0 0.01 0 0.02 A total of 84,422 sequences were obtained from ten biofloc samples Totally 43 phyla, 105 classes, 263 orders, 606 families and 1,265 genera were found from ten samples Based on OTUs (operational taxonomic units), 351-773 OTUs are identified from each samples Species richness is the higher in samples of matured biofloc water Bacteroidetes group is the most dominant, which is commonly found in sewage treatment plants More studies on microbial characterization and functions in biofloc system are needed 예진수산 73 55 74 jangik@korea.kr 75 THE EFFECTS OF BIOFLOCS GROWN ON DIFFERENT CARBON SOURCES ON SHRIMP IMMUNE RESPONSE AND DISEASE RESISTANCE Julie Ekasari1,2*, Muhammad Hanif Azhar1, Enang Harris Surawidjaja1, Peter De Schryver2 and Peter Bossier2 1 Department of Aquaculture Bogor Agricultural University, Indonesia 2 Laboratory of Aquaculture and Artemia Reference Center Ghent University, Belgium Abstract Biofloc technology (BFT) has been studied in terms of maintaining good water quality in the system and making a nutritional contribution to the diet of cultured animals. This study investigated the effect of different carbon sources used in biofloc-based shrimp culture on selected immune parameters and resistance to IMNV infection. The application of BFT for whiteleg shrimp culture significantly increases the shrimp immune response and survival after challenge with IMNV. Introduction Shrimp farming is one of the most important aquaculture sectors, with a projected annual growth rate of 10.3% (Valderrama and Anderson 2011). Nevertheless the growth of shrimp farming faces challenges, particularly disease outbreaks (Valderrama and Anderson 2011). Biofloc technology (BFT) has been studied in terms of maintaining good water quality in the system and making a nutritional contribution to the diet of cultured animals (Avnimelech 1999). Few studies have been conducted to describe how BFT may stimulate the immune cellular response and antioxidant status of shrimp (de Jesús Becerra-Dorame et al. 2012, Xu and Pan 2013). The objective of this study was to determine the effect of bioflocs grown on different organic carbon sources (molasses, tapioca, tapioca by-products and rice bran) on the immune response and resistance to the infectious myonecrosis virus (IMNV) of whiteleg shrimp Litopenaeus vannamei. Methods 76 The experiment consisted of five treatments (organic input types) with four replicate tanks per treatment. The five treatments were a control without organic carbon addition and with regular water exchange, and four treatments with different organic carbon sources added for biofloc development (molasses, tapioca, tapioca by-products and rice bran) with seawater added only to replace water lost from evaporation. Pacific white shrimp at an initial average body weight of 2.0 ± 0.1 g were randomly distributed to 20 glass tanks (96 L) at 30 per aquarium (125/m2). Each carbon source was added daily at an estimated C/N ratio of 15 to stimulate biofloc formation. After 49 days of rearing, shrimp were challenged by injection with IMNV. Total haemocyte count (THC), phenoloxidase activity and respiratory burst activity were measured on two intermolt shrimp from each tank. The challenge test was performed on the final day (day 49) of the rearing period before injection and again 6 days after IMNV injection, using the procedures of Liu and Chen (2004). Results TABLE 1. Mean values (±SD) of immune indicators of whiteleg shrimp cultured in biofloc systems provided with different carbon sources prior to IMNV challenge and post IMNV challenge (n = 3). Values within the same column indicated with a different superscript are significantly different (P<0.05). Pre-challenge Treatment Negative control Positive control Molasses Tapioca Tapioca byproduct Rice bran Post-challenge THC (× 106 cells mL-1) PO (abs- 100 µL-1) RB (abs- 10 µL-1) 0.470±0.147a 0.214±0.055ab 0.189±0.114b 11.7±0.6a 6.8±2.4b 6.8±2.5b 5.1±0.8b 7.2±1.2b 0.144±0.039a 0.071±0.019b 0.090±0.020ab 0.192±0.090a 0.132±0.008ab 0.257±0.155ab 0.084±0.007c 0.317±0.020a 0.215±0.052ab 0.194±0.055b 0.459±0.183ab 6.5±1.3b 0.127±0.003ab 0.160±0.016b THC (× 106 cells mL-1) PO (abs 100 µL-1) RB (abs 10 µL-1) 11.9±0.7a 0.160±0.026a 0.223±0.115ab 12.0±3.1a 16.6±4.2a 16.2±2.5a 0.491±0.224b 0.603±0.224b 0.277±0.084ab 15.1±4.5a 0.435±0.094ab After 49 days in the experimental period, phenoloxidase activity of shrimp in tanks provided with tapioca by-product and tapioca were significantly greater than that of the control (Table 1). There was no significant difference in total haemocyte count before IMNV challenge among carbon-source treatments. Respiratory burst activity was affected by the different carbon sources before challenge. Following the IMNV challenge, total haemocyte count of shrimp in all carbon- 77 input treatments was less than the negative control although there were no differences among carbon-input treatments. The IMNV challenge induced a significant decrease in phenoloxidase activity for the positive control treatment as compared to the negative control treatment, whereas this was not the case for the carbon treatments. In addition, the phenoloxidase activity of shrimp in tanks provided with tapioca, tapioca by-product and rice bran were significantly greater than the positive control. A similar pattern was observed for the respiratory burst activity, although all treatments with organic carbon addition were significantly greater than the positive control. Following IMNV challenge, survival in the positive control (23%) was significantly less than all other treatments although survival in all carbon treatments was less but not significantly different from the negative control, except for the molasses treatments (Figure 1). FIGURE 1. Mean values of survival (%) of whiteleg shrimp Litopenaeus vannamei in biofloc systems with different carbon sources 6 days after challenge test with infectious myonecrosis virus (IMNV) (n of carbon source treatments = 4; n of negative and positive control = 3). Different letters over the bars indicate significant differences (P<0.05). The application of biofloc technology for whiteleg shrimp culture significantly increased the shrimp immune response and survival following challenge with IMNV. 78 Acknowledgement This research was financially supported by the Flemish Interuniversity Council–University Development Cooperation (VLIR). P. De Schryver is supported as a post-doctoral research fellow by the Fund for Scientific Research (FWO) in Flanders (Belgium). References Avnimelech, Y. 1999. Carbon ⁄ nitrogen ratio as a control element in aquaculture systems. Aquaculture. 176:227-235. de Jesús Becerra-Dorame, MJ, Martinez-Cordova LR, Martínez-Porchas M, Hernández-López J, López-Elías JA, Mendoza-Cano. 2012. Effect of using autotrophic and heterotrophic microbial-based-systems for the pre-growth of Litopenaeus vannamei, on the production performance and selected haemolymph parameters. Aquaculture Research 1-5. Valderrama D, Anderson JL. 2011. Shrimp production review. Global Outlook for Aquaculture Leadership. Shrimp production survey: Issues and Challenges. Santiago, Chile, November 6-9, 2011. Xu WJ, Pan LQ. 2013. Enhancement of immune response and antioxidant status of Litopenaeus vannamei juvenile in biofloc-based culture tanks manipulating high C/N ratio of feed input. Aquaculture. 412-413:117-124. 79 Objective The effects of bioflocs grown on different carbon sources on shrimp immune response and disease resistance • to determine the effect carbon source used in bio‐flocs system on white shrimp immune response (Litopenaeus vannamei) and resistance to the infectious myonecrosis virus (IMNV) Julie Ekasari1,2, Muhammad Hanif Azhar1, Titi Nur Chayati1, Enang H. Surawidjaja1, Peter De Schryver2, Peter Bossier2 1 Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Indonesia 2 Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Belgium Shrimp Disease Materials and Methods • Treatments: 4 local carbon sources and 1 control (clear water) 1. 2. 3. 4. • • • • • Walker and Mohan (2009) What is the contribution of biofloc technology to disease control? Filamen tous Floc • Higher survival and feed efficiency • Better lipid and protein assimilation Microalga e forming Source of Immunostimulants? BIOFLOC bacteria Nitrifiers L. vannamei : 2.02  0.05 g , 100 L tank, 83/m2 Estimated C/N ratio: 15 Culture period 49 days Challenge test with IMNV (Escobedo‐Bonilla et al 2006): 6 days Growth performance What is Biofloc Technology? Total Ammonia N Molasses Tapioca Tapioca by product Rice bran – Contribution of extra protein and lipid – Contribution of endogenous digestive enzymes (Xu and Pan 2012) Grazer 80 Immune Indicators after IMNV challenge Immune Indicators after 49 days • THC, PO and RB reduction in positive control: normal response of disease infection • Higher PO and RB activities in biofloc treatments: faster recovery or constant activity? • Increased THC and PO activity in BFT treatments • Carbon source affected RB activity Evidences of Biofloc immunostimulatory effects Survival after IMNV challenge Higher post challenged survival in BFT treatments than positive control (Jiravanichpaisal et al 2006; Wang et al 2008) Evidences of Biofloc • proPO and PPAEimmunostimulatory expression effects LGBP expression (Kim et al (Kim et al 2013) • PO activity (present study) • phagocytic cativity (Xu & Pan 2013) • RB activity (present study) What is the contribution of biofloc technology in disease control? Disease 2013) • SOD (De Jesus Becerra‐ Dorame et al 2012; Xu & Pan 2013) Pathogen Environment Host • Mas expression level (Kim et al 2013) (Jiravanichpaisal et al 2006; Wang et al 2008) 81 Can the host be protected? Disease Disease • Immunostimulation • Better nutritional condition: efficient nutrient uptake • Bioactive compounds (Ju et al 2008; Crab et al 2010; Xu and Pan 2013) Pathogen – Carotenoids – Chlorophylls – Vitamins , etc Environment Pathogen Environment Host Host BFT as an integral approach in disease control? Can good water quality be maintained? Conclusion • Biofloc system contribute to the enhancement of immune response and survival after IMNV challenge regardless the carbon source • The application of BFT could bring about beneficial effect in disease control and management in shrimp culture BFT treatments: generally lower in dissolved inorganic N Can pathogen presence be reduced? (Crab et al 2010) • Reduced luminescene and viable cell of V harveyi • Increased survival of Artemia after challenged with V. harveyi • disrupting V. harveyi cell‐to‐cell communication • This research was financially supported by the Flemish Interuniversity Council–University Development Cooperation (VLIR). 82 BIOFLOC SYSTEMS MANAGEMENT 83 SHRIMP BIOFLOC TECHNOLOGIES, FEEDS AND GUT HEALTH Craig L. Browdy Novus International 5 Tomotley Ct. Charleston, SC USA 29407 craig.browdy@novusint.com Maintaining shrimp health remains one of the most important challenges for the global shrimp farming industry. Losses from disease continue to mount in many parts of the industry as problems with shrimp viral and bacterial pathogens persist and new diseases continue to emerge. Maintaining health requires careful attention to site selection, seed quality and farm biosecurity as a foundation of an integrated shrimp health management strategy. The present presentation discusses the dynamics of the grow-out system and interactions among shrimp health, biofloc culture system management, feeds and feeding programs. Biofloc systems are based on the concept of cultivating a balanced microbial community within a production unit. In addition to providing key ecosystem services such as waste material cycling and provision of supplemental nutrition to the target crop, properly managed biofloc systems can also contribute to management of disease risk. By eliminating water exchange and inoculating ponds with a healthy and diverse microbial community in a biosecure farming system, excludable and opportunistic pathogen control can be achieved. Through proper management of inputs, target crop density, and organic material during and/or between crops, the grower can achieve a stable balance to maximize benefits. This can provide improved cost efficiencies, more stable production conditions and greater overall environmental sustainability of production. The farm feed program is a crucial foundation for management of culture system dynamics and shrimp health. Feed performance is intimately related with target crop production success in terms of direct nutritional benefit and indirectly by effects on water quality. Feed nutrient inputs drive culture ecosystem efficiencies. One of the keys to any shrimp farming system, and especially to zero-exchange biofloc-based systems, is to maximize efficiencies of incorporation of feed nutrients into shrimp. As the amount of nitrogen in the feed that is not incorporated into shrimp flesh increases, problems with water quality and pond bottom conditions increase. Addition of carbon into biofloc systems can sequester excess nitrogen into bacterial biomass. A portion of this biomass may be assimilated by shrimp with the balance contributing to organic 84 waste in the system. On the other hand, as culture systems intensify and contributions from natural productivity decline, the importance of a well-formulated nutrient-dense diet and appropriate feeding regime increase. Thus, the most efficient feeding programs producing the fastest growing shrimp strains in intensive production use automated, almost continuous feeding to meet the animal’s nutritional requirements while maximizing feed conversion efficiencies and reducing uneaten feed and leaching of nutrients. Today’s shrimp feeds must do more than efficiently meet target nutritional requirements; they must be a vehicle for the delivery of health solutions. A growing body of literature suggests that effective use of feed supplements can fundamentally improve shrimp health by affecting the gut environment and improving overall immunocompetence and fitness. With increasing scrutiny, the high cost of antibiotic use and questionable efficacy of antibiotic treatments against some of the most virulent bacterial diseases, focus must be expanded from use of chemotheraputant treatments to prevention and strengthening the animal before acute disease outbreaks. Although shrimp have no truly adaptive immune response, an increasing body of literature is focusing on the innate immune system of shrimp. Immunostimulants like β-glucans stimulates immune responses in shrimp and, in some cases, improves the response to a disease challenge. The development of tools to better measure and modulate shrimp immune system balance will contribute to maintaining shrimp health under stressful culture conditions. The shrimp gut has limited physical barriers to pathogen infiltration and microbes found in the midgut can typically also be found at lower concentrations in the hepatopancreas. Thus, a key to shrimp health is controlling the concentrations of pathogenic microbes in the shrimp stomach. Gut environment modifiers include several classes of compounds including prebiotics, probiotics and antimicrobials. Prebiotics can selectively provide nutrients to certain classes of microbiota, shifting gut microbial communities while enhancing host immune responses and pathogen binding. Probiotic strains can be fed to enhance favorable bacterial strains and to directly target pathogens. An increasing body of laboratory data and commercial experience suggest benefits of antimicrobial compounds including organic acids and essential oils for controlling primary and secondary bacterial pathogens. Organic acids effectively kill Gram-negative pathogens, 85 including vibrios, and reduce concentrations in the shrimp gut. Essential oil blends can provide a very potent and broader-spectrum antimicrobial activity. The combination of these approaches can be applied to achieve several beneficial effects that collectively allow more effective management and prevention of disease outbreaks: 1) Improve the makeup of the exogenous microbial community by maintaining a healthy and biosecure biofloc system, 2) Maintain the shrimp pond environment and shrimp performance by providing balanced nutrient-dense feeds in highly efficient feeding programs, and 3) Enhance shrimp fitness and immunocompetence while improving gut biota, structure and function through the effective use of selected feed supplements. 86 SHRIMP BIOFLOC TECHNOLOGIES, FEEDS AND GUT HEALTH OK, Is there anything about shrimp health and bioflocs that we haven’t covered? EMS Craig L. Browdy Executive Manager Aquaculture Research Novus International Inc. Biofilms Biofilms Host Pathogen Environment Concentrators Viral versus bacterial shrimp pathogens • Transfer in shrimp – Live, frozen? • Obligate host? • Excludable? • Vertical transmission? • Resistance mechanisms / breeding strategies? • Mutation and gene transfer rates Pathogens Biosecurity 87 • Other spread – Ballast? Currents? • Concentrated in environment • Effect of probiotics • Effects of antimicrobials • Quorum sensing • Biofilms Vibrio fitness and virulence • • genomic plasticity attachment and colonization Host – biofilm formation: flagella, type IV pili, exopolysaccharide synthesis, quorum sensing • • • • • Feeds and Gut Health immune evasion virulence factors nutrient acquisition competition survival in unfavorable biotic and abiotic conditions – viable non culturable state, signaling, colonization, transcriptional regulation, antimicrobials, acid and salt tolerance, polysaccharides Johnson 2013 - Microbial Ecology Feed cost reduction Fast growing shrimp strains Feed cost reduction and health “Feed cost reduction is not only about supplying and Feed cost Economical return for farm: ROI • • • • Formulation Raw material cost Operational efficiencies Finance • Survival: health • FCR & growth • Fish/shrimp quality balancing nutrients for fast growth. Assuring nutrient availability and improving animal health is equally Role of Nutritional Additives ? important” • • • • • More bioavailable micronutrients Attractants and pallatants Gut health modifiers Immunostimulants, nucleotides Enzymes Shrimp digestive system Vibriosis • The anatomy of the shrimp digestive system includes barriers to protect against pathogen infection White Feces Disease Nyan Taw 2010 Gastric Sieve Bell and Lightner 1988 Digestive fluid circulation Ceccaldi 1997 Peritrophic membrane in dissected midgut Wang et al. 2012 Stomach cuticular lining Bell and Lightner 1988 Bacterial colonization in posterior stomach Lightner 1996 88 Lightner AHPNS presentation NACA 2012 Essential oil blend – NEXT Enhance 150 Gut environment modifiers 40 X a a a b • Antibiotics Antimicrobials • Essential Oil blends • Organic acid blends Prebiotics • Oligosacharides Probiotics • Bacillus • Pediococcus Gut Health and Microbial Community Equilibrium 1 mm Challenge mortality HP microbial load active substances found naturally in Oreganum spp Kasetsart University, 2008 Minimum inhibitory concentration against Vibrio parahemolyticus Organic acid blends Treatment Number of total bacteria (CFU/g) Control 1.52 + 1.21 x 107b MeraCid 1% 3.06 + 1.95 x 106a Vibrio parahemolyticus has been identified as the causative agent of Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Syndrome (EMS/AHPNS) affecting shrimp. % 1.0 0.5 0.25 Bacterostatic and bacteriocidal Number of total Vibrio (CFU/g) Control 1.15 + 1.10 x 107b MeraCid 1% 7.74 + 4.16 x 105a Minimum inhibitory concentration (%; pH 7.0) Product 0.125 0.0625 120 100 Survival (%) Treatment 0.063 80 0.0313 60 0.0156 40 20 control 0.250 Meracid 0.5% 0 0 10 20 30 Days 40 50 0.0078 60 V. parahemolyticus used was donated by Dr Dang Thi Hoang Oanh , Can Tho University Nutritional additives and Health: Farm trial In pond cage trial - Growth Trial set up: 1.45 1.80 b ab 1.40 ab 1.35 1.30 1.00 b a 4500.00 ab a Production (kg/ha) Survival (%) 90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40% a ab b 1.40 1.20 1.25 89 a 1.60 a FCT Growth (g/wk) Cage (1x1x1 m) trials in pond. 6 cages per treatment 20 shrimp per cage (20/m²), Initial weight: 10g; 71 days, final weight 23‐24 g Treatments: Control Antibiotic treatment (Enroflox) Next Enhance 150 (30 ppm thymol+carvacrol) Meracid ( 5 kg/ton) b ab 4000.00 3500.00 3000.00 2500.00 2000.00 a a Environment Biofloc systems Phagocytosis Phenoloxidase activity Respiratory burst Aerated Zero Exchange Biofloc Systems • • • • • • • • Resuspension in intensive aquaculture • Prevents precipitation of organic particles to the bottom of the pond • Enhances growth of heterotrophic bacteria using ammonia • Organic matter-bacteria protozoa complex is a source of natural food Solar Aquafarms - ODAS ↓ eutrophication ↓ sedimentation ↓ escapement ↑ nitrogen assimilation ↑ water quality stability ↑ growth factors ↓ production costs ↓ pathogen introduction Microbial Community • Heterotrophs – Nitrogen uptake – lower salinities – Floc substrates – Sludge degradation • Chemoautotrophs – Nitrogen cycling – Nitrification – Denitrification • Photoautotrophs – Growth enhancement – photosynthesis – Nitrogen uptake 90 Solids Management and Microbial Communities • What are the best tools for measuring and describing the complex microbial floc community • How do we establish a diverse and stable community • What is the optimal microbial floc community composition 800 35 700 30 Fishmeal 25 Fishmeal Settled 20 Plant 15 Plant Settled 10 5 Chlorophyll-a (µg L-1) PAR Extinction Coefficient 40 600 500 400 300 1 2 3 4 5 6 7 8 9 Branched and Odd Chain Fatty Acids (µg L-1) Mean Photosynthetic Oxygen Production (mg L-1 h-1) 8 1600 2.0 1.5 1.0 0.5 0.0 Fishmeal Fishmeal Settled Plant Plant Settled Fishmeal Fishmeal Settled Plant Plant Settled 1400 1200 6 4 2 1000 800 0 0 600 1 2 3 4 5 6 7 8 9 10 11 200 0 1 2 3 4 5 6 7 8 9 10 11 12 Week • Shrimp Production – 47% ↓photosynthetically active radiation extinction coefficient – 200% ↑ photosynthetic oxygen production – 65% ↓ final chlorophyll-a – 80% ↓ fatty acid bacterial indicators – – – – No difference in survival 28% ↑ growth rate 41% ↑ biomass 26% ↓ FCR Rat et al 2012 - Aquaculkture Biofloc Influence on Shrimp Growth Recent results from Texas A&M 1.60 1.40 RW1Mean Wt 1.20 RW2 Mean Wt • Low level mortality from enteric and systemic vibriosis. 16S rRNA sequencing: showed presence of Vibrio parahaemolyticus, V. owensii, V. communis, V. alginolyticus • No significant differences in performance except for survival • Shrimp fed the HI-35 feed had higher survival than those fed the EXP potentially due to the presence of Zeigler VPak® and possible contribution to control of Vibrio 1.00 0.80 0.60 0.40 0.20 0.00 4/18 4/25 5/2 5/9 5/16 5/23 5/30 6/6 6/13 Growth rates converged as biofloc communities converged Final Weight (g) HI-35 EXP 27.22 ± 0.85 28.80 ± 1.84 Growth (g/wk) 2.05 ± 0.13 2.16 ± 0.31 Yield (kg/m3) 8.21 ± 0.31 7.79 ± 1.13 FCR Survival (%) 1.59 ± 0.01 1.72 ± 0.08 93.14 ± 3.13a 83.35 ± 2.69b 35 30 Average weight (g) Diatoms in a bacterial matrix 25 20 15 10 5 0 0 Synechococcus dominated (cyanobacteria) 20 40 60 Samocha et al. Unpublished Performance With Sucrose Control Weight Gain 11.33±0.02a 9.98±0.025b FCR 1.67±0.11a 1.8±0.17b Survival 65.7±4.6a 52.3±6.1b • DGGE analysis • Bacillus dominant with sucrose (27.71%±2.83%) • Vibrio sp. Dominant in control group (22.65%±4.49%). • Control vs sucrose (0.5kg/kg feed) as carbohydrate source • No water exchange • Periodic probiotic additions to water 91 12 Week 400 • Effects of settling chambers – Fertilization - Filtration – Sterilization – Inoculation, probiotics – Habitat - Environment Fishmeal Fishmeal Settled Plant Plant Settled 10 Plant 0 2.5 • How do we manipulate the community to maintain optimal composition Fishmeal Settled Plant Settled 10 11 12 Week – For competitive exclusion of pathogens – For target crop growth – For water quality management 12 Fishmeal 200 100 0 Weight (g) Research Questions 80 Feed programs • Feeds are the driver of nutrient inputs into the system – Physical characteristics, leaching – Nutrient quality, digestibility – Nutrient density, formulations – Ingredient costs – Feeding timing, frequency, amounts Approaches Biofloc Feed Formulation Strategies • Design feeds to drive biofloc management • Improve makeup of the exogenous microbial community by actively maintaining a healthy and biosecure biofloc system • Maintain shrimp pond environment quality and shrimp performance by providing balanced nutrient dense feeds in highly efficient feeding programs • Explore opportunities to enhance shrimp fitness and immunocompetence while managing gut biota, structure and function through the effective use of selected feed supplements – Focus on microbial community, C:N ratios, low protein OR • Design feeds to efficiently meet shrimp requirements – High protein nutrient dense formulations – Tight control of feeding rates – Supplemental carbon addition as necessary – Avoid waste nutrient buildup, Phosphorus, Minerals etc. Integrated strategies • Understanding of the pathogens – laboratory models, sensitive diagnostics, sharing of data and isolates • Innovative strategies for assuring shrimp fitness – Feed quality, breeding, resistance, nutrition, gut microbial health , theraputants • Improving culture systems – intensive nursery systems – micro and meiofaunal community management 92 IS IT POSSIBLE TO CONTROL THE BACTERIAL COMPOSITION IN SHRIMP (AND FISH) PONDS? Stephen G. Newman Ph.D. AquaInTech Inc. 6722 162nd Place SW Lynnwood, WA USA 98037 sgnewm@aqua-in-tech.com Ecosystems are complex assemblages of micro and macro organisms. “Stable” ecosystems are really far from stable. They change as inputs and outputs impact available niches and are thus constantly evolving towards new steady states. The rates at which ecosystems change depend on the type of environment and its relative complexity (among other things). While some terrestrial ecosystems are comparatively simple, aquatic ecosystems can be quite complex. Shrimp farm (and fish) ponds are for the most part very complex ecosystems. There are arguments to be made for and against efforts to control the bacterial makeup in production systems. Theoretically, reducing the levels of potential pathogens in these systems could reduce the prevalence of certain types of profit impacting diseases. However, that being said, these are complex systems and it does not seem out of the question that tweaking one aspect of the system could very well result in unforeseen impacts. There is some speculation that this might be a reason that the etiologic agent of AHPNS has become as wide spread as it has. There are many different shrimp production paradigms ranging from extensive, very low-density production systems that approximate the natural ecosystems to extremely high-density systems where many of the variables that are inherent in other more common forms of production such as semi-intensive are controlled. Examples of these controls would be liners that eliminate the soilwater interactions and closed systems where the water quality is self-moderating through biofloc formation. These controls alter the ecology of the ponds although they do not necessarily change the system to make it more stable. Shrimp are typically reared in a state of more or less constant stress. While genetic programs have been successful to some extent in mitigating some of this stress susceptibility, few would 93 argue that crowding animals that evolved in environments where they were never crowded and that many other production constraints are of their very nature stressful. Some are obvious and others less so. Disease is a serious problem affecting all types of agriculture and a great deal of effort is spent on trying to lessen the impact of many of the problems that affect both terrestrial and aquatic systems. Shrimp are not unique in this. Many different types of pathogens impact productivity on a regular basis. Some are serious with large short-term financial impacts that can force shifts in production methods and force changes in the types of animals being farmed, etc. Others can be chronic with long-term impacts that are not as massive. Some even are benign allowing productivity to be maximized with minimal impacts. Historically viral diseases have had the greatest short-term massive impacts with bacterial diseases following closely behind. Most bacteria that affect shrimp are opportunistic pathogens taking advantage of animals that are chronically stressed or infected with other pathogens that are not opportunistic. Viral diseases are managed by exclusion. This begins in the hatchery starting with broodstock that are free of the specific pathogens of concern. Biosecurity measures are taken in ponds as well to try and limit the introduction of vectors and high viral loads in the water. The most common approach to this entails the use of chlorine, a non-selective disinfectant that while highly effective is readily inactivated in the presence of organic matter and converts much of the organic matter in the ponds into assimiliable forms. While the viruses may be killed and to some extent the vectors that can carry high viral loads limited, the natural bacterial populations of the ponds are affected as well. This would be a shift in the ecology of the ponds. In the last decade many companies have been selling products that purport to positively impact the bacterial populations in the ponds with little science to support the claims. While some may actually impact the ponds microbial ecologies it is not likely that any of them affect anything more than a short-term change (thus the need for constant addition). Is it possible to alter the microbial ecology in a meaningful and long-term manner? 94 Much focus has been on the role of vibrios as pathogens of shrimp although the reality is that many different genera of bacteria can affect shrimp. With the recent appearance of a particularly nasty vibrio, which appears to be an obligate ( although not in the classic sense of the term) pathogen, the focus on trying to alter the microbial ecology has increased. With very little real understanding of why this vibrio has been able to move the way that it has and why it is able to occupy the niche that it does, efforts are being made through various mechanisms to try and impact its growth. Although there are likely many potential tools that can be used to mitigate the impact of this particular vibrio, farmers and hatchery personnel should be using techniques to lessen overall vibrio loads. There are many tools that work well in the hatchery ranging from disinfection through mechanical means, filtration through bag filters, drum filtration, UV and ozone filtration, etc. Many different chemical disinfectants, including tamed chlorine compounds, quaternary ammonium compounds and others can significantly reduce overall vibrio loads as well. Some microbial bacterial amendments also work well, although generating home grown preparations is risky. A useful exercise is to evaluate the various inputs into these production systems and determine at which points some control can be implemented. In shrimp farms there are a number of inputs that can impact the overall health of the animals. The use of captive or wild broodstock is one area where a few simple biosecurity measures can be useful in breaking false vertical transmission. Washing and disinfecting adults, eggs and nauplii, using open systems, minimizing the numbers of animals spawned at a single time, etc. are all potential tools. Carry over into the hatchery requires additional steps. Farming inputs can be distinctive and in some cases the only solutions might appear at first onerous, filtration of incoming water and exclusion of potential vectors are among some of the potential solutions. Closing ponds combined with heavy aeration and in some cases fertilizing to allow the formation of particulate biofilm based assemblages of micro and macro-organisms (biofloc) has worked well for some. Many see this as a potential tool to minimize the need to introduce the risks of make up water and lower costs of pumping. Likely though, at this time, the formation of biofloc 95 per se, this does little to exclude vibrios and other potential pathogens. This is not to say that someday we may be able to tailor make biofloc that contains microbes that exclude some of the nastier potential bacterial pathogens. Farms can filter water (to under a hundred microns) using bag filters, and some use drum filters to further exclude particulate material to as low as 25 microns. Various forms of lime, disinfectants, herbal remedies, etc. are all being used in an attempt to periodically alter and reduce bacterial loads. The wide spread use of a myriad of products that would best be characterized as microbial ecology management tools is also an attempt to modify the microbial composition although it has not been shown that this is a stable (and indeed in many cases even worthwhile) change. It is not likely that we can modify our production environments to exclude all vibrios. Nor does this particularly make much sense as they occupy a variety of critical niches and opening one niche creates an opportunity for another microbe to occupy it. We can however better manage stress in these production environments by proper use of the right science based tools. Adequate aeration, filtration, proper fertilization strategies suitable for each environment and genetics to develop domesticated stocks are all viable tools. While it may be a laudable goal and technologically not difficult or expensive to control vibrio loads in the hatchery the extent to which this will be meaningful in the field is dubious. Although if it turns out that the etiologic agent of AHPNS is spread through this vehicle than it will be a critical matter to break the cycle at this point in the process. In conclusion, we must consider that any effort to alter the microbial flora in production systems is not going to be simple and that unforeseen consequences could readily result. Short-term alteration is certainly possible although it remains to be seen if system wide paradigm shifts, such as the through the use of biofloc, will be helpful in mitigating the impact of stress related disease processes, either directly or indirectly. Ensuring that practical controls are in place on all inputs is a meaningful proactive measure to start. 96 Why bother? Is it possible to control the bacterial composition in shrimp (and fish) ponds? • Number one cause of mortality in farmed shrimp is bacteria largely vibrios although many other bacteria can kill shrimp filamentous bacteria in the hatchery, Aeromonas species, Pseudomonas species, etc. • Controlling loads could reduce severity of problems and allow greater productivity Stephen G. Newman Ph.D. President Aquaintech Inc. • On the other hand….. Why bother (2)? Simple answer • Stressed animals have increased susceptibility to disease; culture paradigms that fail to address this will always have problems with disease • Complex ecology‐reactive in nature deliberate change can result in unforeseen impacts such as widespread use of chlorine to disinfect ponds creating new niches • Yes and • No • Very complex subject cannot be dealt with in 20 minutes. • Discussion from the standpoint of impacting the loads of the etiologic agent of AHPNS, characterized as a strain of V. parahaemolyticus Vibrio parahaemolyticus Normal inhabitant of aquatic ecosystems Found in zooplankton and attached to algae Carried by fish, bivalves (filter feeders) Can be carried by birds Found in sediments and water Occupies wide range of environments, wide salinity range, wide pH • Forms non‐culturable but viable forms (NCBV) • • • • • • Vibrio with single flagella Vibrio with many flagella Mixed vibrios on TCBS plates 97 How do bacteria enter the production ecosystem(s)? Tools to control bacterial loads Hatchery Farm EVALUATION OF INPUTS Maturation • Hatchery: through water, air, broodstock (live feeds including bivalves, worms, Artemia biomass, squid, etc.), Artemia cysts, algae, other live feeds, feed, personnel, ??? Reduced levels of potentially problematic bacteria • Farm: through water, air, vectors (algae, zooplankton and phytoplankton), PLs, feed and personnel, ??? Inputs Maturation Inputs • Examination and determination of where one can intervene • What are the best strategies for reduction of microbial loads or altering microbial composition? • Broodstock – From non nuclear breeding facilities such as ponds and open production systems – Live and whole feeds • • • • • Many possible tools though all require monitoring include a variety of approaches ranging from improved production systems design to the use of feed additives such as QSI and other non‐antibiotic compounds Maturation Systems Bivalves‐filter feeders concentrate bacteria Worms‐contaminated with many different bacteria Artemia Biomass‐same thing Squid‐known carriers of vibrios Freezing not always adequate NCBV forms are well documented for many bacteria including vibrio Broodstock held in outdoor ponds lose any SPF status they may have had and are a potential source of pathogens. Broodstock are safer from secure closed indoor systems. Crude and cheap Better design with recirculation 98 Feeds Local caught worms from China pose a strong biosecurity risk Control in Maturation • Use of closed systems modeled after nuclear breeding facilities • Isolation of maturation facilities and hatcheries –not built in proximity to farms • Screening of all inputs with proper strategies for reduction and elimination • Live and frozen feeds need to be tested or produced in environments where there is no possible contamination Cultured worms pose much less of a risk Frozen Artemia Biomass from China produced in ponds poses a risk while biomass from high salinity lakes likely does not – example might be use of worms produced in controlled environments or from sources where there are no warm water vibrios present Control in maturation/hatchery overlap The Hatchery (early stage) • Spawning animals individually reduces cross contamination potential • Proper control of water quality, copious washing with very clean water • Surface disinfection with iodophors and other disinfectants (formaldehyde, quats, etc.) • Facility dry outs • Disinfection of pipes, surfaces, etc. Overlap with maturation eggs and nauplii contaminated by fecal material, fluids expressed during egg laying high nutrient loads in water mass breeding versus individual animal bacteria inside of the eggs is the exception Tank Set ups conducive to pathogen spread Luminescent bacteria –vast majority are not pathogens Common problem in hatchery tanks that proper controls can prevent and eliminate 99 The hatchery after nauplii are stocked into production tanks Hatchery design and layout will affect ability to minimize bacterial loads Construction and design impacts ease of cleaning and ease of ensuring proper biosecurity Water Air Feeds Live Artemia and algae Personnel Design of hatchery impacts ability to run biosecure Air • Aerosols disperse bacteria and nutrients • Design of facility and proximity to nutrient sources impacts potential for airborne contaminants • Filtration of air used in tanks • Sealed buildings or biosecure segregated production units (Artemia, algae, etc.) Feed Major sources of contamination • Live feeds (or frozen) • Artemia high vibrio counts – Artemia – Algae – Worms – Bivalves – Krill – Squid Worms‐although not needed still in common usage many from China • Prepared feeds (pelleted) 100 Tools Farms • Inputs • Hatchery – Animals – Water – Fertilizers – Disinfectants – Pesticides – Bacterial products – Personnel – Impact of neighbors – Disinfection (chlorination, disinfectants) – Proper water treatment (ozone, filtration, etc.) • QC is critical for ensuring success – Proper biosecurity • Limit movement of animals • Wash and surface disinfect eggs and nauplii • Microbial additives, home made and commercial Water source can have a large impact on bacterial loads The type of production system will impact this • Open systems with soil on the bottom of the ponds Extensive Semi‐Intensive Intensive Super Intensive • High water exchange rate systems • High input systems • Closed systems with plastic liners Clean Oceanic water with little nutrient load Tools • • • • • • • • Conclusions Stop vertical transmission stock clean animals Pond water treatments (microbial?) Filtration and removal of vectors Dry out? High water exchange rates Closed ponds Paradigm shift Feed additives • Management of microbial loads and types is achievable in those components of the production system where the inputs can be controlled • Unknown impacts as opening niches creates new opportunities • Controlling AHPNS via this approach ???? Time will tell. 101 Likely that once it is in the environment it is there to stay AHPNS Have cultural practices opened Pandora’s box? 102 HIGH PERFORMING BIOFLOC SYSTEMS USING PROBIOTICS – THE VIEW FROM ASIA AND LATIN AMERICA Olivier Decamp*, Marcos Santos, Hoa Nguyen Duy, Fauzan Bahri and Jaime Munoz Medina INVE Aquaculture 471 Bond Street Tambon Bangpood - Amphur Pakkred Nonthaburi 11120, Thailand o.decamp@inveaquaculture.com High water exchange was former standard practice to maintain suitable water quality in intensive shrimp production systems. Environmental and biosecurity issues led farmers to develop methods relying on reduced or zero water exchange. A number of commercial farms have adopted this approach for all or parts of their shrimp operations: broodstock, hatchery, nursery and/or ongrowing. Through the combination of high stocking density, control of the carbon:nitrogen ratio, adequate aeration (and floc suspension), it is possible to control water quality (ammonia and nitrite) and reduce the risk of disease while lowering the consumption of compound feed. One of the challenges concerns the method to manipulate the complex microbial interactions within the system to optimize production (i.e. shrimp growth and survival rates yield, FCR, feed and aeration cost, etc.). In wastewater treatment, inoculation of the system with suitable microorganisms can lead to better results. In laboratory assays, Bacillus strains can be selected for their ability to inhibit pathogens or produce exoenzymes that help with the degradation of organic waste. The benefits of delivering quality probiotics in shrimp aquaculture is increasingly being reported. However, the mechanisms behind the improvement are not fully explained. We review lab and field data on the effect of the addition of selected strains of Bacillus, including information on floc during system start-up, and the nutritional composition of floc. Another benefit of applying Bacillus probiotics is the control of potentially pathogenic Vibrio, and the associated improved survival. Protocols from commercial operations will be detailed. 103 Functions of probiotics sh a pin ga qu a cu lt ur e t oge t h e r Waste control (production of enzymes) Improvement of ambient microbial community Improvement of gut microflora Improved use of feed Immunostimulation High performing biofloc systems using probiotics – View from Asia and Latin America Olivier Decam p, Marcos Sant os, Fauzan Bahri, and Jaime Munoz Medina Applied in the water Coated on feed at the farm Coated on feed at the feedmill 2 1 Biofloc system – parallel with activated sludge Types of probiotics - Aeration and stirring provide the required oxygen for the process. -This common method of wastewater treatment relies on microbial activity for the reduction of the organic matter. - Microorganisms are seeded (bio-augmentation) and provided with the recycled sludge from previous runs. - This leads to the production of microbial biomass, typically associated with flocs. - These flocs are removed from the system by sedimentation. - A “side-effect” is the drastic reduction in the abundance of intestinal pathogens, i.e. mostly coliforms. Wide range of genera/species used Selection based on availability (for terrestrial animals or humans; for waste water treatment) or performance Widerange of product: - liquid, frozen or dry powder - pure strain, defined mixture, undefined mixture - low or high concentration - requiring brewing, activation or ready-to-use 3 4 Biofloc system – parallel with activated sludge Probiotics in hatchery - Protozoa associated with floc in RAS (Decamp et al., 2006) Nassulida Suctoria Hatchery cycle without water exchange, in 2 production phases. - 1st phase till Mysis 3/early PL. Algae + probiotics - 2nd phase from PL1-Pl3, Sanolife Bacillus at 2.5x105 cfu/ml, followed by daily dosing of 1.5x105 cfu/ml, together with addition of molasses Control of pathogenic bacteria  maintain water quality Pleurostomatida - Protozoa associated with wastewater www.nhm.ac.uk http://www.environmentalleverage.com 5 6 104 Application of probiotic in hatchery Application of probiotic in hatchery Control of pathogenic bacteria Control of water quality Enhancement of immune system of shrimp Materials & Methods 1x105 cfu/ml Sanolife MIC added daily in the water till animals are ready for the challenge with Vibrio harveyi. - N till Z1  Zoea test - Z1 till M1  Mysis test - PL1 till PL10  PL test Animals transferred to new unit for thechallenge test (single applictation of V. harveyi at 107 cfu/ml)  Example from Brazil EFFECT OF PROBIOTIC ADDITION ON THE SURVIVAL OF Litopenaeus vannamei LARVAE SUBMITTED TO INFECTION BY Vibrio spp. Experiments Z , M and PL (4 replicates each) Joana Vogeley*, Bruna do Valle, Roberta Nery, Emanuell Silva, Juliana Interaminense, Kennya Addam, Camila Brito, Marcelo Soares, Nathalia Calazans José Vitor Lima, Roberta Soares, Silvio Peixoto VH PVH Universidade Federal Rural de Pernambuco, Laboratório de Tecnologia em Aquicultura, Recife, PE, 52171-900, Brazil.*joanavogeley@hotmail.com C V. harveyi Probiotic + V. harveyi Control – no Vibrio sp. and probiotic For PVH treatment, Sanolife MIC applied daily at 1x105cfu/ml After 96 hours, survival and TCBS plating 7 8 Application of probiotic in hatchery Application of probiotic in hatchery Materials & Methods Results on activity against Vibrio 1x105 cfu/ml Sanolife MIC added daily in the water till animals are ready for the challenge with Vibrio harveyi. - N till Z1  Zoea test - Z1 till M1  Mysis test - PL1 till PL10  PL test Animals transferred to new unit for thechallenge test (single applictation of V. harveyi at 107 cfu/ml) Direct inhibition Vibrio in challenge test Experiments Z , M and PL (4 replicates each) VH PVH C V. harveyi Probiotic + V. harveyi Control – no Vibrio sp. and probiotic For PVH treatment, Sanolife MIC applied daily at 1x105cfu/ml After 96 hours, survival and TCBS plating d = 12 mm 9 10 Performance of Sanolife PRO-W in nursery Application of probiotic in hatchery Results on survival in the challenge test Nursery rearing of the pink shrimp Farfantepenaeus brasiliensis in a zero exchange aerobic heterotrophic culture system. Three replicate tanks randomly assigned to the 3 probiotic treatments vs control. Other probiotic Other probiotic Benefit of probiotic treatment: - Concentration of presumptive Vibrio spp. significant lower (p< 0.05) - Higher levels of total protein and granular hemocyte Souza et al. 2011. THE USE OF PROBI OTI CS DURI NG THE NURSERY REARI NG OF THE PI NK SHRI MP Farfantepenaeus brasiliensis I N A ZERO EXCHANGE SYSTEM. World Aquaculture 2011 - Meeting Abstract. https:/ / www.was.org/ WasMeetings/ meetings/ ShowAbstract.aspx?Id= 24044 Larvae Zoea and Mysis only exposed to V. harveyi (VH) presented a significantly lower survival than the control (C) 11 105 Biofloc in nursery – control of Vibrio Example of nursery 40 b Vibrio spp. CFU x 102/ml 35 30 25 b b a b 20 a 15 10 a a a a a a a a a a 5 0 10 -5 15 22 30 Time (Days) -10 Control Toyoi Biomin Start-Growth Inve Pro-W *Different superscript letters indicate significant differences Example of nursery protocol Biofloc in ongrowing Mexico: 30 day nursery cycle with min harvest weight 200 mg, with stocking density of PL15 at 15 PL/L Work from FURG (Brazil). Vita et al. Health booster No water exchange 30 days experiment 4 replicates 300/m² tank Control vs Sanolife PRO-W probiotic Use of Bacillus-based water probiotics for L. vannamei under a super-intensive heterotrophic culture system – 300 shrimp/ m 2 16 Probiotics. Use in Biofloc Technology System – required or C:N management enough? Biofloc in ongrowing Results 1. Composition of the biofloc Treatment Control CP (%) 27.06 a Ash (%) 45.63 a FB (%) 13.53 Fat (%) 1.05 a Probiótic 32.42 b 36.70 b 13.94 3.33 b 2. Performance of shrimp Variable Wasielesky et al. 2008. Present status of heterotrophic super-intensive shrimp culture in southern Brazil I n: Aquaculture America 2008,Orlando, Florida Glucose + Bacillus Glycerol Glycerol + Bacillus Crude Protein (% DW) 19 ± 7 20 ± 4 15 ± 3 22 ± 10 Total n-3 PUFA (mg/g DW) 0.3 ± 0.3 0.7 ± 0.3 0.7 ± 0.1 0.9 ± 0.1 Total n-6 PUFA (mg/g DW) 9 ± 11 28 ± 32 12 ± 16 17 ± 16 Treatment Control Probiotic AVG (SD) 97.00 (4.00) 97.00 (3.00) BW (g) Control Probiotic 0.66 (0.21) a 0.78 (0.32) b 0.0010 Nutritional value of bioflocs used as feed for L. vannamei. Bioflocs were grown in different carbonsources with or without the addition of a Bacillus-based probiotic. FCR Control Probiotic 1.02 (0.03) 0.99 (0.09) 0.2843 Crab, R. 2010. PhD thesis. Faculty of Bioscience Engineering. University of Ghent, Belgium. Survival (%) P Glucose 0.9695 17 106 Probiotics. Use in Biofloc Technology System – required or C:N management enough? Bacillus and the immune system The black color is caused by reduced iron Understand synergy between pathogens and increase ability of shrimp to fight pathogens Vibrio count (x 103 cfu/ml) Artificial feed 5.7 ± 0.4 Glucose 1.5 ± 0.2 Glucose + Bacillus 0.6 ± 0.1 Glycerol 1.7 ± 0.1 Glycerol + Bacillus 1.1 ± 0.1 Total Vibrio count in the shrimp culture water at day 20. Shrimp were fed either artificial diet or artificial diet partially replaced with biofloc. Bioflocs were grown in different carbon sources with or without the addition of a Bacillu s-based probiotic. Phuoc et al. 2009. Aquaculture 290:61-68 Clearance of Vibrio from haemolymph of shrimp from control and 2 probiotic treatments ( treat 1 and treat 2 ). Probiotics are commercial Bacillus strains. Test carried out at SBBU, Bangkok, Thailand. Crab, R. 2010. PhD thesis. Faculty of Bioscience Engineering. University of Ghent, Belgium. Protocol from Indonesia Probiotics: Waste control Production of enzymes and degrade waste products Lab test 1. Set-up of biosecurity measures, i.e. crab protection and bird nets. 2. Disinfection of the water (Sanocare PUR 1.2ppm) 3. Water preparation with regular applications over 2 weeks: - dolomite (10ppm, 8X) - sodium nitrate and silicate (Sanolife Nutrilake 3ppm, 6X) - calcium carbonate and magnesium carbonate - Bacillus probiotics (Sanolife PRO-W 100g/ha, daily) The combination of small particles and Sanolife PRO-W are “floc starters”. Beige effluent from PRO-W treated pond Dark effluent from control pond expectations Super-intensive L. vannamei, Indonesia 22 Protocol Protocol - floc 1. Stocking with PL10 animals 2. Control of the environment in order to maintain a balance between algae and bacteria: - Frequent addition of a natural source of nitrate (Sanolife Nutrilake 5ppm) - Frequent addition selected Bacillus (Sanolife PRO-W 1ppm) - Molasses added 2-3 times per week at 10-15Kg/ha - mineral additives coated on feed - Floc created with a combination of Sanolife PRO-W, dolomite and calcium carbonate/magnesium carbonate -Target is 2-3ml/L (Imhoff cones, <2 hours). Much smaller than standard biofloc (15ml/L). - Limited water exchange - Regular siphoning to (1) remove the excess organic matter from the central area; and (2) evaluate shrimp molt and mortalities 23 24 107 Protocol - siphoning Results – autumn 2012 25 Pond Size (m2) Density (PL/m2) Days of Culture Size (pcs/kg) Yield (kg) FCR Productivity (Mt/ha) A B C D E Average A B C D E F Average 2,800 2,900 2,900 2,800 2,300 2,740 3,300 3,000 2,900 3,000 3,200 3,000 3,067 80 65 72 72 78 73 113 133 137 119 120 119 124 88 86 87 86 87 87 104 82 83 97 96 96 93 48 52 50 51 48 50 46 62 62 47 46 48 52 4,110 3,979 3,935 3,741 3,172 3,788 7,527 6,273 5,794 6,423 5,360 5,760 6,188 1.32 1.21 1.28 1.26 1.32 1.28 1.38 1.53 1.69 1.36 1.53 1.44 1.49 14.7 13.77 13.6 13.4 13.8 13.8 22.8 20.9 20.0 21.4 16.7 19.2 20.2 26 Results – January 2013 Pond Size (m2) A B C Average 2,800 2,900 2,800 2,833 Density (PL/m2) 107 105 107 106 Days of Culture 96 94 89 93 Size pcs/kg 45 53 51 50 Results – Phytoplankton Yield (kg) 6,011 5,783 5,504 5,766 FCR 1.30 1.28 1.33 1.30 Productivity (MT/ha) 21.5 20.0 20.0 20.3 Dominated by green algae Water transparency from 40-60 cm in the first weeks to 20-30cm in the last 2 months of the crop Blue-green algae below 10% most of the time. 27 28 Benefit of defined dried probiotics with high concentration sh a pin ga qu a cu lt ur e t oge t h e r Consistent formulation (strains in culture collection) Consistent concentration (no variation between batch) No need to brew  no risk of contamination Easy storage and long shelf-life Reduced labour cost and human error Standard seeding of biofloc system Thank you Olivier Decamp o.decamp@inveaquaculture.com  Right tool for efficient management 29 30 108 THE USE OF A BIOFLOC TECHNOLOGY SYSTEM WITH PROBIOTICS TO LIMIT SHRIMP VIBRIOSES Dariano Krummenauer*, Luis Poersch, Luiz A. Romano, Gabriele R. Lara, Bárbara Hostins and Wilson Wasielesky Jr. Graduate Program in Aquaculture Institute of Oceanography Universidade Federal do Rio Grande (FURG) Rio Grande, RS, Brazil darianok@gmail.com In shrimp farming around the world, diseases caused by different species of the genus Vibrio affect growth and mortality, resulting in a significant drop in productivity. The aim of this study was to analyze the effect of bacterial probiotics on a Litopenaeus vannamei biofloc technology culture system infected with Vibrio parahaemolyticus (VP). Two treatments were compared: a control and commercial probiotics. For the probiotic treatment, a multi-strain probiotic containing Bacillus spp., Enterococcus spp., Thiobacillus spp. and Paracoccus spp. (AquaStar Pond) was applied to the water, and a multi-strain probiotic containing Bacillus spp., Enterococcus spp. and Lactobacillus spp. (AquaStar Growout) was added to feed. Treatments were randomly assigned to six 35,000-L enclosed and lined raceway greenhouses. Each tank was stocked with 10,500 nursed shrimp that were naturally infected by VP. During the 60-day study, water quality and shrimp growth were measured. Growth and survival were significantly greater (P<0.05) in the probiotic treatment group than the control. The feed conversion ratio of shrimp receiving probiotics (1.4) was less (P<0.05) than that of shrimp in the control (2.7). There were no significant differences (P>0.05) in water quality of tanks of either treatment (Table 1). The tested probiotics can control infection of shrimp by VP in a biofloc culture system while improving growth and feed conversion. 109 TABLE 1. Mean survival, growth rate, final weight, final biomass and feed conversion ratio (FCR). Treatment Control Probiotic Survival (%) 52a ± 12 83b ± 7 0.92b ± 0.30 Growth rate (g/week) 0.85a ± 0.20 9.1b ± 2.5 Final weight (g) 8.4a ± 2.3 79b ± 11 Final biomass (kg/tank) 46a ± 6 1.4b ± 0.3 FCR 2.7a ± 0.4 Productivity (kg/m²) 1.3a ± 0.2 2.3a ± 0.5 Different superscripts in the same row indicate significant differences (P<0.05). Acknowledgements The authors are grateful to The Brazilian Council of Research (CNPq), FAPERGS, CAPES, Ministry of Fisheries and Aquaculture (MPA) - Brazil and Centro Oeste Rações S.A. (Guabi) for funding this research. 110 MARINE STATION of AQUACULTURE Biofloc System as an alternative to avoid WSSV – the Laguna Case ‐ Southern Brazil Wilson Wasielesky, Geraldo Fóes, Dariano Krummenauer, Luis Poersch Universidade Federal do Rio Grande – FURG Instituto de Oceanografia, Southern Brazil Shrimp culture research since 1989 Courtesy:Paulo Iribarrem Coastal Lagoon – Laguna Area Report of Laguna WSSV Case South America Brazil Laguna City Santa Catarina State Production area – 1,200 ha Production – 4,000 – 5,000 ton/year 2004 High mortalities due to WSSV 111 Production (x1000 ton) Shrimp production in Santa Catarina State Post larvae from different sources Homemade probiotics 4 Medicines (antibiotics) 3 2 Lemon and Garlic 1 ? 2002 2004 2006 2008 2010 WSSV 2012 Year Note: In Laguna area production was zero last nine years Post larvae from different sources Home made probiotics Lemon and Garlic Why production still remains at zero?? Medicines (antibiotics) WSSV Traditional Policulture (tilapia and shrimps) Different factors. After several shrimp culture attempts, no production was possible until 2010. Crustaceans (maintain the virus in environment) and Birds (spreading) Salt-marshes 112 Traditional Policulture (tilapia and shrimps) Basic structure of the farms Fish Processing Plants • No effluent treatment; • Simultaneous discharge and water pumping. The questions??? Temperature • Laguna area can be a good place for shrimp production? Maybe Latitude – 28oS South America • Cold fronts Laguna • Frequently temperature ranges from 19 – 23 C – Good for WSSV • Laguna area is a good place for WSSV? For sure Laguna area ‐ Santa Catarina State • Biosecurity could be a solution? Maybe Marine Station of Aquaculture Different research lines in BFT systems (2005 – now) Greenhouses • Stocking densities • Aeration systems • Nutrition • Water quality • Reuse of water • Biofilm/substrates Lined ponds • Stocking densities • Aeration systems • Feeding management • Economical analyses • Organic fertilization In 2011 – Meeting between local producer from Laguna and Researchers from University of Rio Grande Marine Station of Aquaculture 113 First step – Biosecurity Facility Project development University and shrimp producer It was selected an earthen pond to build the pilot facility!!!! In that farm shrimp was 100% WSSV infected Lined Ponds Four 500 m2 lined ponds Fence to avoid birds Control of visitors Crab fence 114 Control of the workers Cleaning – Chloride and iodine General view of the pilot system Second step Bioflocs production ‐ Inoculum Water preparation Facility • 1 lined pond for physical treatment (Nets: 200 and 100 micra). • Same lined pond: chemical treatment (Bleach) 30 ppm. • 3 lined ponds for grow-out. • Grow-out ponds were rapidly inoculated with bioflocs according Kipper-Fóes et al (2012). 115 • According to Krummenauer et al (2011) it is necessary at least 1% of aged inoculum for better starting a new microbial community. • According Gaona et al (2012) and Ray et al (2010) better growth results are achieved for shrimp culture when TSS are less than 500mg/L. Bioflocs production ‐ InoculumGreenhouse at Marine Station of Aquaculture, FURG. • Bioflocs from a 120 days shrimp culture were pumped to two tanks, and then the bioflocs were settled and discharged water. • Concentrated bioflocs were placed in two shrimp shipping boxes. • DO was kept between 10 and 20mg/L Settling tank for biofloc sedimentation • To keep DO in suitable concentration hydrogen peroxide were applied according Furtado et al (2013). Biofloc were transferred to 3 ponds to start microbial succession. The amount of supplied material was equivalent to 2%. Pilot system 700 km Marine Station of Aquaculture Methodology  SPF and no SPF Post-larvae (100-118/m2);  Feed – 38 % CP (Guabi™) in trays; Third step  Probiotic (Bacillus sp – Pro-W);  Organic Fertilization (C:N) 15:1 – with molasses (grow‐out) Fertilization when ammonia achieved 1 mg/L;  Monitoring of bioflocs and shrimp. 116 Methodology Methodology Post Larvae and shrimp were monitored by:  Zero exchange water;  Replacement of treated water, due to PCR – Animal healthy Agency (CIDASC) and Ministry of Agriculture (MAPA) evaporation;  Calcium hydroxide to keep suitable pH (7.5-8.0) and alkalinity (>150 mg/L)  Aeration – Paddlewheel- 20-40 HP/ha, Histopathology–Diseases Diagnosis Laboratory, Federal University of Rio Grande– Dr. Luis Romano continuously;  Monitoring of water quality Results – Mean Zootechnical parameters Results Cicle Time (Day) Season Temp (C) Dens (PL/m2) IW (g) FW (g) FCR Survival (%) Productivity (kg/ha) 1 140 fall / winter 17 – 23 100 0.008 10.1 1.32 63 % 6,350 2 100 Spring 21 – 26 118 0.005 9.0 1.25 75 % 7,975 3 60 summer 24 ‐ 29 100 0.900 nursed 8.5 1.15 75 % 6,750 Note: In the same period, 10 local shrimp farms were stocked and WSSV killed all shrimps. Results Results  Histopathology  PCR Gill with its preserved architecture without viral inclusions. H‐E Results were NEGATIVE to WSSV Results were NEGATIVE to WSSV WSSV was not detected Overview of the abdominal region where it is observed: Gastric Chamber (CG), Pyloric foregut (CPIA) and hepatopancreas (HP). Gastric epithelium viral inclusions are observed. H‐E 4 X 117 Conclusions The WSSV remains a problem for producers in different states of Brazil. With the publication of the results several producers are seeking the BFT system as an alternative to viral diseases. Bioflocs Production in WSSV infected areas Biosecurity ACKNOWLEDGEMENTS Thanks for your attention! manow@mikrus.com.br 118 PROBIOTIC EFFECTS OF BIOFLOC TECHNOLOGY: DEPRESSION OF TILAPIA INFECTION BY Streptococcus Yoram Avnimelech Dept. of Civil and Environmental Engineering Technion, 32000 Israel agyoram@tx.technion.ac.il One of the problems of tilapia culture everywhere is the infection of fish by Streptococcus iniae. Infection leads to a reduction in growth rate and fish death, a gradual process over a few weeks, often leading to 30% mortality. Losses due to S. iniae infections in Israel range from 30% (tilapia) to 50% (trout) of the total predicted crop. Biofloc technology (BFT) can be used to support intensive production of tilapia. It is based on maintaining a low water exchange rate, intensive mixing and aeration of the pond, leading to the development of a dense microbial population. Bacteria – in association with algae, detrital material, protozoa and zooplankton – form bioflocs that are harvested by fish, serving as part of the feed supply. Several observations were made on commercial farms, indicating that infection with Streptococcus and fish mortality is low (almost negligible) in biofloc ponds. The goal of the experiment reported here was to objectively and statistically evaluate the effect of biofloc technology on infection of tilapia by Streptococci. This report represents a preliminary study of this effect. The experiment was conducted in the Genosar Intensive Fishculture Experimental Station, located near the Sea of Galilee, Israel. The experiment was conducted in 2-m3 tanks. Each tank was equipped with an airlift pump to create circular water movement, thereby providing good mixing and aeration. A baffle was placed at the bottom of each tank to direct heavy particles toward the central water outlet, enabling efficient drainage of sludge, which was done twice daily. 119 Tanks were stocked with 200 all-male tilapia (Oreochromis spp.) with an average weight of 66 g, resulting in a total biomass of 13.2 kg/tank or 6.1 kg/m3. Fish were fed 25%-protein pellets at 2% body weight daily. Two treatments with four replicates per treatment were tested. As a control, tanks were operated with a conventionally high water exchange rate of 0.5 L/min per kg fish (i.e. about 700% daily water exchange). Biofloc treatment tanks were operated with a limited solids drainage and water exchange of 10%/d. A pretreatment period of 3 weeks enabled the development of a dense microbial community in the biofloc treatment, as indicated by high turbidity and the obvious presence of visible bioflocs. Fifty fish were challenged by injecting 0.2 mL of Streptococcus suspension (5 × 104 bacteria/mL) at the end of the pre-treatment period. After 20 days, all fish were harvested. Healthy, sick and dead fish were counted. The number of sick and dead fish (including those found dead during the experimental period) among the non-challenged population is reported in Table 1. A highly significant difference in respect to sick and total infected fish between treatments (p = 0.015 and 0.017 respectively) was found. The average number of sick and dead fish in the biofloc treatment was 3 ± 1, as compared to 11 ± 5 in the control treatment. TABLE 1. Effect of biofloc-dominated water on infection of tilapia by Streptococcus. Treatment Dead fish Sick fish Total infected Challenged fish Control BFT Non-challenged fish Control BFT 9±9 12 ± 3 7±5 2±2 2±2 4±2 4±2 1±1 11 16 11 ± 5 3±1 The tested system included 25% of challenged fish within a population of non-challenged fish. Challenged, infected fish released streptococci bacteria into the water, thus potentially contributing to a secondary infection of fish. The difference in the secondary infection is a 120 reflection of the extent to which the two different environments protect against this infection. Intuitively, it might be expected that less infection will occur in tanks with high rates of water exchange (7 times per day) and flushing of pathogens, in contrast with biofloc tanks, where only 10% of the water was exchanged each day. The number of dead and sick fish in the control treatment was about four times that of the clearwater, high-exchange tanks. This study demonstrates that naturally occurring bacteria may have a beneficial effect on the health of cultured animals and its resistance to disease. The biofloc system apparently confers some type of protection to fish, perhaps through a probiotic effect. Different modes of action of probiotics are proposed in the literature Among possible mechanisms is the effect of probiotics on water quality, antagonism with pathogens, competition on adhesion sites within the host or a positive effect on host physiological conditions. Dense heterotrophic population (106 - 107 bacteria/mL) may attack the pathogens released to the water by sick and dying fish. Other possibilities may be competition for sites of microbial adherence, sites that may be occupied by the overwhelming heterotrophic population, or a positive effect of bioflocs on the well-being, vigor and immunity of fish. In subsequent work, tilapia growing in biofloc-dominated water had higher levels of immune indicators compared to fish growing in clear water. More research on the microbial interactions and probiotic effects of biofloc technology is warranted. 121 Probiotic effects of bio-floc technology: Depression of tilapia infection by Streptococcus One of the problems of tilapia culturing all over is the infection of the fish by Streptococcus iniae. Several observations were made, indicating that infection with streptococcus and fish mortality is low, almost negligible in bio-flocs ponds Y. Avnimelech & I. Bejerano Yoram Avnimelech Yoram Avnimelech Tilapia (Areochromis sp., all male) were grown in tanks,at a density of ca 7kg m-1 using two treatments: Exchanging water at a rate on 7 time a day (conventional control) and a limited 10% daily exchange, (BFT). The goal of the experiment reported here was to objectively and statistically evaluate the effect of the bio-floc technology on infection of tilapia by streptococci. Yoram Avnimelech Yoram Avnimelech 10% of the fish were challenged by injecting a dense Streptococcus iniae dose. The infected fish were tagged. Fish were sorted to healthy. Sick and dead fish following 20 days. No significant differences were found regarding the infection in the challenged fish . Yoram Avnimelech Yoram Avnimelech 122 Treatment, Tank # Dead fish Sick fish Total infected 1 3 3 6 2 6 6 12 3 14 3 17 4 4 5 9 4.3 (1.5) 11 (4.7) Control, High water exchange However, for the non-challenged fish, the rate of disease in the BFT treatmentwas significantly lower (25%) as that found in the control treatment: Average (+ SD) 6.8 (5.0) Bio-flocs Yoram Avnimelech 1 2 2 4 2 1 2 3 3 4 0 4 4 0 1 1 Average (+ SD) 1.8 (1.7) 1.3 (1.0) 3 (1.4) 0.107 N.S 0.015 0.017 Yoram Avnimelech t-test significanc Other possibilities may be a competition on sites for microbial adherence, or a positive effect of the bio-flocs on the well being and vigor of fish Additionally, there were indications That the immune system of Fish in the bio floc tanks Was more active. The effect demonstrated here could be due to several mechanisms. It is possible that the dense heterotrophic population (1,000,000 - 10,000,000 per ml)), attack the pathogens released to the water by sick and dying fish. Yoram Avnimelech Yoram Avnimelech The work presented here demonstrates that naturally occurring bacteria, may have a beneficiary effect on the health of the cultured animal and its resistance toward disease It was shown that the bio-flocs system provides a protective shell to the fish, through a probiotic effect. Yoram Avnimelech Yoram Avnimelech 123 ROLE OF SELECTIVE BREEDING IN BIOFLOC SHRIMP PRODUCTION AND DISEASE MITIGATION Shaun M. Moss1*, Dustin R. Moss1, Clete A. Otoshi1, Steve M. Arce1, and Donald V. Lightner2 1 Oceanic Institute, Waimanalo, Hawaii USA 2 University of Arizona, Tucson, Arizona USA smoss@oceanicinstitute.org Over the past two decades, selective breeding has been used to improve the performance of farmed-raised shrimp and breeding efforts have focused on traits of commercial importance, including growth and disease resistance. The commercial availability of selectively bred Pacific white shrimp Litopenaeus vannamei has contributed significantly to the dominance of this species in global shrimp production and selective breeding will continue to be an important tool as the industry matures. Furthermore, implementation of effective biosecurity strategies are needed to mitigate the introduction and spread of pathogens within and among shrimp farms, including the adoption and refinement of biofloc systems. In this paper, we discuss the potential role of selective breeding to improve performance of shrimp reared in biofloc systems and how selective breeding can be used to reduce crop loss from disease, especially in the aftermath of the Acute Hepatopancrteatic Necrosis Disease (AHPND) outbreak. In addition, we discuss how inbreeding affects shrimp performance and its potential contributory role in the AHPND outbreak. Selective Breeding for Biofloc Systems A major goal of many selective breeding programs is to improve growth of a particular species. In general, heritability estimates (h2) for shrimp growth are considered moderate to high (h2 ≥ 0.3) and farmed shrimp have responded well to selection for this trait. It is common for shrimp populations to have substantial phenotypic variation (within and among families) for growth. This variation, coupled with large population (or family) sizes, allows high selection intensities, resulting in rapid genetic gain. Published reports of selection responses for growth range from 10-25% per generation. 124 Growth and other commercially important traits may be affected by the interaction between a shrimp genotype and its environment (G×E interaction). If this interaction is significant (i.e. of biological significance), breeders may need to develop unique lines of shrimp for different rearing environments, including biofloc systems. Biofloc systems vary greatly in size, configuration and how they are managed, but possess common characteristics that differ from traditional shrimp ponds, including high shrimp stocking densities, chronic exposure to dissolved nitrogenous wastes, sub-optimal dissolved oxygen concentrations, high bacterial loads, and high total suspended solids concentrations. Importantly, microbial communities inhabiting biofloc systems likely have substantive impacts on shrimp growth and health. Published reports from Oceanic Institute indicate that microbial communities and microbial-detrital aggregates from an intensive shrimp pond can increase shrimp growth rates and affect abundance and species composition of gut microflora, stimulate digestive enzyme activity, and enhance the innate immune response in shrimp. Some of these physiological responses to the environment may be under genetic control and thus may be direct or indirect targets for selection. For example, shrimp breeders selecting for rapid growth in biofloc systems may indirectly be selecting shrimp for their ability to produce a novel suite of digestive enzymes that can more effectively digest biofloc particles, thereby improving growth rate and feed conversion ratio. In addition, shrimp breeders selecting for high survival in biofloc systems may indirectly be selecting for behavioral characteristics (e.g. increased docility) that lead to less physical injury at high stocking densities and/or the ability to tolerate chronic exposure to high levels of nitrogenous wastes. These traits, although important in biofloc systems, may be of less importance in traditional shrimp farming environments. There is little published data on G×E interactions for shrimp growth and no published data specifically comparing family performance of shrimp reared in biofloc systems compared to traditional shrimp ponds. Thus, G×E interactions may be insignificant and families of shrimp that perform well in biofloc systems are the same families that perform well in traditional shrimp ponds. If this is true, biofloc systems still may have an important impact on shrimp breeding programs if they are able to exclude specifically listed pathogens. If biofloc systems are capable of excluding such pathogens through adequate biosecurity measures, shrimp breeders can focus selection efforts on growth and general survival and abandon selection for resistance to 125 excludable pathogens. This is important because the number of traits in a selection program is negatively associated with the selection response for each individual trait. Focusing selection efforts on a small number of traits will allow for more rapid genetic gain for those traits. Selective Breeding in Disease Mitigation Shrimp breeders have been successful in enhancing L. vannamei resistance to Taura Syndrome Virus (TSV) since the mid-1990s, using sib selection and by making breeding decisions based on family survival in orally administered laboratory challenge tests. Currently there are commercially available shrimp families that have >90% survival after TSV exposure in laboratory challenges. Shrimp from high-survival families can be infected with TSV but survive exposure because of their ability to maintain relatively low viral loads. Thus, the term “tolerant” may be more appropriate than “resistant” in this context. Interestingly, in the 1990s, a commercial shrimp company in Mexico developed a line of L. stylirostris that was truly resistant to infectious hypodermal and hematopoietic virus (IHHNV). When shrimp from this resistant line were exposed to IHHNV, the virus was unable to replicate in the host’s cells. Selection for resistance to white spot syndrome virus (WSSV) has been more elusive, although mass selection efforts in Latin America and Asia have made some progress. Similarly, progress has been made to enhance resistance to infectious myonecrosis virus (IMNV) in Brazil. To date, shrimp breeders have focused primarily on resistance to viral pathogens. However, a number of prokaryotic, fungal, and protozoan pathogens continue to negatively impact global shrimp farming. Recently, the pathogen responsible for AHPND was identified as toxic strains of the rod-shaped, Gram-negative bacterium, Vibrio parahaemolyticus. Shrimp exposed to this toxin exhibit a pale-colored and atrophied hepatopancreas and typically die within the first 30-45 days after stocking into ponds. Over the past several years, AHPND has caused catastrophic losses of farmed shrimp in China, Vietnam, Thailand, and Malaysia, and this disease was recently identified in Mexico. There is interest among shrimp breeders to develop lines of shrimp that exhibit high survival after exposure to this bacterial toxin. However, little is known about the quantitative genetics of bacterial toxin resistance in shrimp. Examples from the insect literature indicate that pest insects can be selectively bred for resistance to specific bacterial toxins (e.g. endotoxins produced by Bacillus thuringiensis) by altering the binding site of the 126 toxin, so this approach may work with shrimp. In addition, shrimp breeders may be able to exploit aspects of maturation immunity, because younger shrimp appear to be more susceptible to the toxin than older shrimp. Recently researchers at the University of Arizona (UAZ) developed a molecular-based disease diagnostic tool to identify toxic strains of V. parahaemolyticus and this will serve as an important tool in combating AHPND. Currently UAZ researchers are developing disease-challenge protocols to be used in family-based breeding programs to evaluate toxin resistance in shrimp. The protocols call for immersing shrimp pellets in a culture of toxic V. parahaemolyticus and then feeding the pellets to shrimp. The culture contains a known concentration of bacterial cells, so the bacterial dose per gram of shrimp pellet is known. Preliminary results from dose-response trials suggest that the LD 50 (i.e. the bacterial concentration required to kill 50% of the shrimp) is 105 cells/mL of culture. At 106 cells/mL, all shrimp die. In shrimp ponds, toxic strains of V. parahaemolyticus can reach 108 cells/mL in a 24-hr culture and these strains have very rapid generation times of 8-10 minutes. UAZ researchers are refining disease-challenge protocols so that commercial breeding companies can assess if their shrimp stocks contain innate resistance to this bacterial toxin. If they do, and if toxin resistance is heritable, shrimp breeders may be able to contribute to the battle against AHPND. Inbreeding and the AHPND Outbreak Inbreeding can be defined as the mating of individuals related by ancestry, resulting in a reduction of heterozygosity within a population. Inbreeding depression (IBD) is the effect of inbreeding measured as the reduction in mean phenotypic performance with increasing levels of inbreeding within a population. IBD estimates generally are expressed as the percent change in phenotype per 10% increase in inbreeding. IBD typically is observed in fitness-related traits (e.g. survival and various reproductive traits) and has been well documented in many agricultural plants and animals, including laboratory animals. Published reports from Oceanic Institute indicate that inbreeding has small impacts on shrimp growth (IBD = -2.6 to -3.9%) and grow-out survival (IBD = -3.8%) but larger impacts on hatchery performance traits such as hatch rate (IBD = -12.3%) and hatchery survival (IBD = - 127 11.0%). Oceanic Institute researchers also reported IBD estimates for shrimp survival after exposure to viral pathogens, including the TSV isolate USHI94 (IBD = -8.3%), the TSV isolate USTX95 (IBD = -11.1%), the TSV isolate BZ01 (IBD = -31.4%), and WSSV (IBD = -38.7%). In this latter example, for every 10% increase in inbreeding, shrimp survival will decline by 38.7% when exposed to WSSV. Shrimp, like many aquaculture species, are highly fecund and thus few broodstock are required to produce sufficient numbers of offspring for the next generation. This characteristic, coupled with selective pressures, can lead to genetic bottlenecks and make shrimp highly susceptible to inbreeding, especially if pedigree records are unavailable or incomplete. Additionally many shrimp breeding programs were initiated from a small number of founder stocks, thus making them even more susceptible to inbreeding. Inbred populations tend to be more susceptible to environmental stress due to reduced genetic variability, and this relationship has been observed in plants and animals, including shrimp. Published reports from Oceanic Institute indicate that the effects of IBD are more pronounced with decreasing environmental quality, where environmental quality is defined as the survival of non-inbred shrimp (Figure 1). Inbreeding depression (%) 5 Hatch rate Hatchery survival Growout survival TSV-Americas survival TSV-Belize survival WSSV survival -5 -15 -25 -35 -45 15 35 55 75 95 Survival at F = 0 (%) FIGURE 1. Relationship between IBD for survival traits and mean survival at inbreeding (F) = 0. Survival at F = 0 is a measure of environmental and/or life stage stress (i.e. low survival indicates higher stress). 128 Despite the negative ramifications of IBD, many shrimp breeding companies use inbreeding as a germplasm protection strategy to safeguard their valuable investment. Shrimp hatchery operators often buy broodstock from a few suppliers, who provide limited genetic diversity, and attempt to use these stocks (and their descendants) as breeders for multiple generations. Significant declines in grow-out and hatchery performance are often reported after several generations and this may be attributed, in part, to inbreeding. Despite the common practice of using pond-grown, multi-generation broodstock in commercial hatcheries, the relationship between this practice and the emergence and spread of AHPND is unclear. However, it is incumbent on the shrimp farming industry, and those governments where shrimp farming is important, to safeguard against the potentially huge liability of farming shrimp that do not have the genetic variability to tolerate or exclude emerging pathogens. 129 Presentation Overview ROLE OF SELECTIVE BREEDING IN BIOFLOC SHRIMP PRODUCTION & DISEASE MITIGATION • Selective Breeding in Biofloc Systems • Selective Breeding in Disease Mitigation Shaun M. Moss Dustin R. Moss Clete A. Otoshi Steve M. Arce Donald V. Lightner • Genetic Diversity and Inbreeding Biofloc Systems Heterotrophic System Selective Breeding in Biofloc Systems Photoautotrophic System Biofloc Particle crude protein = 26.0 – 41.9% crude fat = 1.2 – 2.3% ash = 18.3 – 40.7% Selective Breeding for Growth Characteristics of Biofloc Systems  (data from OI research trials)   • High shrimp stocking densities (> 300/m2) Moderate to high heritability (h2 > 0.3) Genetic gains: 3 - 25% per generation High levels of between and within family variation for growth Harvest Weight (g) Top 3 families: 25.13 23.63 24.46 Bottom 3 families: 19.37 18.52 18.57 • High bacterial concentrations (> 1011 cells/L) • High TSS concentrations (> 1,000 mg/L) 130 Growth Rate (g/week) Minimum Size (g) Maximum Size (g) 2.19 2.09 2.07 18.66 15.94 16.56 33.56 30.54 30.45 1.68 1.66 1.59 13.81 11.08 12.95 26.70 24.26 26.34 Selective Breeding in Biofloc Systems Physiological Mechanisms (growth = g/wk) Trial 1 Trial 2 Trial 3 Trial 4 Selected 1.79 1.65 1.69 1.79 Unselected 0.85 1.13 1.09 1.33 % Difference +110 +46 +55 +35 • Hyperphagy = increased food intake • Reduced maintenance demands for energy • Improved nutrient and energy efficiencies 75-m2 biofloc systems stocked at 304 – 410 novel suite of digestive enzymes shrimp/m2 (Glencross et al. 2013) Possible Feeding Mechanisms in Biofloc Systems  Scanning EM photos of maxillipeds  Observed “sweeping” behavior of 3rd maxillipeds when exposed to diatoms  3rd maxillipeds may form a filter‐feeding “net”’ with a mesh size of ~10 μm in 2g shrimp. Selective Breeding in Disease Mitigation  This “net” could trap diatoms, but probably not smaller cells like Nannochloropsis and Synechococcus. (Kent et al. 2011) Taura Syndrome Virus (TSV) • • • • White Spot Syndrome Virus (WSSV) ssRNA virus h2 estimates ~ 0.2 (low - moderate) Good selection response (>10% per generation) Commercially available families exhibit >90% survival in per os laboratory challenges • dsDNA virus • h2 estimates typically < 0.1 (very low) • Mass selection attempts in the Americas 100 Family 80 60 Generation 7 Mean = 84% CV = 13.6% 40 20 0 100 80 Generation 2 Mean = 44% CV = 43.3% Treatment Survival LP-1 Negative control 49 of 50 (98%) LP-2 Negative control 91 of 96 (95%) LP-3 Negative control 68 of 68 (100%) Kona WSSV positive control 0 of 20 (0%) LP-1 (2 tanks) WSSV challenge 24 of 104 (23%) LP-2 (2 tanks) WSSV challenge 74 of 129 (57%) LP-3 (2 tanks) WSSV challenge 34 of 130 (26%) 60 WSSV virion mean size = 275 nm 40 20 0 Modified from Cuéllar-Anjel et al. 2012 Shrimp families 131 Breeding for Bacterial Resistance UAZ’s AHPND Challenge Room • Lessons learned from insect literature • 90-L aquaria and 1000-L tanks • Internal biological filter with crushed oyster shell – pest insects bred for toxin resistance (Bt endotoxin) • Need data on quantitative genetics • Feed pellets immersed in culture of V. parahaemolyticus • Culture contains known concentration of bacterial cells, so bacterial dose/gram feed is known • Preliminary results: – phenotypic variation – heritability estimates – correlation with other traits • Possible pathways for selection – Resistance (host “fights” pathogen and reduces pathogen load) – Tolerance (limit the harm caused by a given pathogen burden – reduced effect of toxin) – – – – Biofloc System Pathogens LD50 = 105 cells/mL All shrimp died at 106 cells/mL 108 cells/mL common in ponds Generation time of 8-10 minutes Biofloc System Pathogens Vibrio spp. - gram-negative bacteria Fusarium spp. - naturally occurring saprophytic - ubiquitous in Biofloc systems - cuticle infection appears as melanized lesions, can also infect a single organ or be systemic - treatment may include medicated feed fungi - infection facilitated by cuticle wounding - infection appears as melanized, nodular lesions on cuticle, gills, or appendages - fungal hyphae visible by wet mount (various UAZ cases – Biofloc systems in USA) (UAZ case from 2008 – Biofloc farm in USA) Number of Traits Selected by Species Challenges with Multi-trait Selection (modified from Gjedrem et al. 2012)  There are always tradeoffs when adding trait(s) to a selection program! Stewart et al. 1999 132 Species # programs Common carp Rohu carp Silver barb Tilapia Nile Tilapia blue Tilapia red Tilapia O. shiranus Channel catfish African catfish Striped catfish Atlantic salmon Chinook salmon Coho salmon Rainbow trout European whitefish Turbot Atlantic cod European seabass Sea bream Freshwater prawn P. monodon P. vannamei Abalone Oysters Mussel 8 1 1 20 2 4 1 1 1 1 13 2 4 13 1 2 3 3 4 2 3 4 3 3 1 # families per program 76 60–70 – 229 90 125 51 200 70 182 280 100 133 206 70 60 110 100 100 82 212 197 210 48 60 Average # traits 2.0 2 1 3.6 2.0 4.0 1.0 4 1 3 5.4 1.5 2.7 5.2 2.0 1.0 4.0 5 6 1 – 2.0 1.7 4.3 3.0 Possible Limited Genetic Diversity • Breeding companies limit diversity of broodstock for germplasm protection (narrow genetic base) Genetic Diversity & Inbreeding • Random loss of diversity (genetic drift) • Inbreeding – High fecundity – Intense selection – Unknown pedigree Inbreeding Inbreeding  Inbreeding can accumulate rapidly in non-pedigreed populations – especially those with a narrow genetic base  Negatively impacts commercially important traits (inbreeding depression) Inbreeding Depression (%) Trait 0 0 Broodstock 1st generation 2nd generation 3rd generation 0 .125 .375 0 0 0 .125 .375 0 0 .188 .250 .125 0 Breeding at GN Hatch Rate 0 0 0 .125 0 0 .250 .312 On-farm breeding -13 Survival – Hatchery -11 Survival – Growout <-4 Survival – TSV-AG -9 Survival – TSV-BG -31 Survival – WSSV -40 Growth – Growout <-4 .5 Inbreeding – Farm Data Inbreeding and Environmental Quality  Inbreeding depression sensitive to environmental quality or life-stage stress IBD (per 10% F ) 10 0 -10 -20 -30 -40 -50 10 20 30 40 50 60 70 80 Survival of non-inbred shrimp Data from R. Doyle via Farallon Aquaculture, Mexico 133 90 Final Thoughts • Selective breeding can be used to improve shrimp performance in biofloc systems, especially to improve growth. • Selective breeding has been effective in producing TSV-resistant shrimp. Breeding for resistance to other viral pathogens (e.g. WSSV, IMNV) has been more challenging. The potential to breed shrimp for resistance to bacterial pathogens is largely unknown. Thank You! • There are trade-offs when adding traits to a selection program. • The genetic diversity of vannamei populations in Asia may be narrow due to the limited diversity of founder populations and inbreeding accumulation. • Use of pond-reared broodstock (e.g. F2, F3, . . . ), coupled with environmental stressors, can lead to BIG problems for shrimp farmers. Shrimp Feces as “Hot Spots“ for Bacteria Bacteria in feces t0 t12h Exp. 1 t0 t12h Exp. 2 Bacteria in water samples • Rapid growth of bacteria on fresh feces thus enriching its protein content • Bacterial turnover time was much faster in feces (1-10 hr) than in BFT water (350 hr) What is the Nutritional Value of Biofloc as a Protein Source? Essential amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine  Assimilation of DON into fecesassociated bacteria is asalvage pathway for feed-N • Fecal bacteria inoculate water column EAAI 0 4 8 Exp. 1 12 0 12 h Exp. 2 AA Score (aa/AA) & EAAI Brown Floc Green Floc Commercial Feed 0.72 1.00 0.92 0.97 0.63 0.88 0.89 1.00 1.00 1.00 0.71 1.00 0.96 1.00 0.73 0.88 0.96 1.00 1.00 1.00 0.81 0.98 1.00 0.98 0.83 1.00 0.90 1.00 1.00 1.00 0.89 0.92 0.95 Essential amino acid scores and EAA indices indicate that: 1) An EAAI close to 1 means that material contains AA profile similar to shrimp tissue. 2) Material with EAAI > 0.9 considered good quality; material with EAAI of 0.8 considered useful; < 0.7 inadequate. 3) Both sources of biofloc represent good quality protein 4) Arginine and lysine were the most limiting AA in the biofloc 5) Scores and indices do not consider digestibility. •AA Score (aa/AA) = (% of EAA in total EAA of floc sample) / (% of EAA in total EAA of shrimp body sample) raceway • EAAI = (aa1/AA1 X aa2/AA2 X ------ X aan/ AAn)1/n i.e. Geometric mean of the AA scores (Penaflorida 1989) 134 (Ju et al. 2008) CO-CULTURE OF FISH AND SHRIMP 135 EFFECTS OF TILAPIA IN CONTROLLING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) Loc H. Tran1,2*, Kevin M. Fitzsimmons2 and Donald V. Lightner1 1 School of Animal and Comparative Biomedical Sciences The University of Arizona, USA 2 Department of Soil, Water and Environmental Sciences The University of Arizona, USA * Corresponding author: thuuloc@email.arizona.edu A laboratory study was conducted in 170-L fiberglass tanks at the University of Arizona to determine the effects of tilapia in controlling the infection and mortality in Pacific white shrimp Litopenaeus vannamei caused by the pathogen causing Acute Hepatopancreatic Necrosis Disease (AHPND). There were five treatments: A. Tanks were prepared without Nile tilapia Oreochromis niloticus for 2 weeks prior to stocking shrimp (negative control), B. Tanks were prepared for 2 weeks with Nile tilapia and then tilapia were removed prior to stocking shrimp, followed by an AHPND challenge test consisting of additions over 10 days of a bacterial suspension of a pathogenic Vibrio parahaemolyticus strain capable of causing AHPND and evaluation of shrimp survival, C. Tanks were prepared for 2 weeks with Nile tilapia and then tilapia were transferred to a cage suspended in each tank prior to stocking of shrimp, followed by the AHPND challenge test, D. Tanks prepared for 2 weeks without Nile tilapia prior to stocking shrimp, followed by the AHPND challenge test, and E. Tanks filled with clear saline water were prepared one day prior to stocking shrimp, followed by the AHPND challenge test (positive control). After 2 weeks of tanks preparation with the main purpose of inducing algal bloom and maintaining a balanced biota community, bacterial density was not significantly different among all treatments except the positive control. The bacterial density in the clear saline water of the positive control was three orders of magnitude less than water in tanks of all other treatments. After 10 days of adding the pathogenic Vibrio suspension into tank water in the challenge tests (except to tanks in the negative control treatment), shrimp survival was significantly different 136 among treatments. Shrimp survival in the negative control (treatment A) was very high (98%), indicating that experimental conditions were conducive to good survival of shrimp. There was no survival of shrimp in the positive control (treatment E) after 3 days of exposure to the bacteria in the challenge test, indicating the very high pathogenicity of the bacterial strain used in this study. Shrimp survival in treatment B (91%) was significantly greater than that of treatments C (7%), D (20%), and E (0%). Although bacteria causing AHPND were isolated from water and shrimp in all treatments where shrimp were challenged with AHPND bacteria, histological analyses showed that infection rates and severities of the pathology in shrimp of the different treatments corresponded to survival rate. The bacterial density in water samples from tanks in treatments B, C and D was less than that following additions during the challenge test. In contrast, bacterial density in the clear saline water of tanks in the positive control indicated a marked bloom of pathogenic bacteria after being added to tanks. This indicates that native microbial communities in water can interact with AHPND-causing bacteria and mitigate the infection caused by this strain of bacteria. The results of this study suggest that good practices to promote a healthy and balance microbial community in shrimp pond water, such as using tilapia in the reservoir of a shrimp farm to induce the development of a health-promoting algal and beneficial bacterial bloom in water prior to filling the pond, would confer beneficial effects in controlling AHPND. 100 90 Survival percentage 80 Negative control (A) 70 Green water induced by tilapia (B) Green water and tilapia in cage (C) Green water induced without tilapia (D) Positive control with clear water (E) 60 50 40 30 20 10 0 Day Day Day Day Day Day Day Day Day Day Day 0 1 2 3 4 5 6 7 8 9 10 Day post-exposure to the AHPND bacteria FIGURE 1. Survival of shrimp in different treatments after exposure to the AHPND bacteria. 137 Preliminary studies suggesting the effects of tilapia and biofloc in controlling the EMS/AHPND infection Some suggestions to prevent EMS/ AHPND Loc H. Tran* , Kevin Fitzsimmons, Donald V. Lightner * thuuloc@email.arizona.edu 1 2 Effects of tilapia in controlling EMS/ AHPND infection Effects of tilapia in controlling EMS/ AHPND infection Survivability of shrimp after exposure to AHPND bacteria Experimental design 100.00 A B Prep. days 14 14 Tilapia during tanks prep. No 3 fish/tank Fertilizer during tank prep. Yes Yes Challenge test Tilapia during challenge test 90.00 Survival percentage Treatment Sterile TSB+ No 3.105 cells/ml No 80.00 Negative control (A) 70.00 Green water induced by tilapia (B) 60.00 Green water and tilapia in cage (C) 50.00 Green water induced without tilapia (D) 40.00 C 14 3 fish/tank Yes 3.105 cells/ml No No D 14 No Yes 3.105 cells/ml E 1 No No 3.105 cells/ml Yes 30.00 Positive control with clear water (E) 20.00 10.00 0.00 Day 0 3 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day post-exposure to the AHPND bacteria Day 7 Day 8 Day 9 Day 10 4 Animal A1, A2, C2, B1, and B2 show normal stomachs, hepatopancreas and midguts (arrows from top to bottom). Meanwhile, animals D1, D2, and C1 show signs of AHPND infection including: empty stomach, pale hepatopancreas, and empty midgut. 5 6 138 Histology of shrimp in treatment B Histology of shrimp in treatments C & D 7 8 9 10 Brief conclusions  The presence of the biota induced during tank aging helped delay mortality caused by EMS/AHPND infection.  Without the presence of the biota induced during tank aging, the density EMS/AHPND bacteria in water increased significantly.  However, to much algae may also be favorable for the EMS/AHPND infection 11 139 Study on effects of biofloc in controlling EMS/ AHPND infection Comments?  Biofloc water as prepared at the Environmental Research Lab (ERL).  Shrimp were grown in biofloc water for 48 hr prior to challenge test.  Both bacterial immersion and bacterial feeding tests were used. A shrimp pond that recently showed EMS/ AHPND near Mazatlan 13 14 Survivability of treatments post-exposure to Vibrio parahaemolyticus Biofloc experimental set up 100 90 80 Percentage 70 60 50 40 30 20 10 0 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day post-exposure 15 Negative control (A) Biofloc+immersion challenge (B) Positive control immersion challenge (D) Positive control feeding challenge (E) Biofloc+feeding challenge (C) 16 This may explain the result! 17 18 P. vannamei (UAZ 13-472A) Negative control, after growing in biofloc water. 10X 140 19 20 P. vannamei (UAZ 13-472F) Positive control, clear water+ immersion challenge. 10X P. vannamei (UAZ 13-472F) Positive control, clear water+ feeding challenge. 10X 21 22 P. vannamei (UAZ 13-472B&C) Biofloc+ immersion challenge with AHPND Vp. 10X P. vannamei (UAZ 13-472D&E) Biofloc+ feeding challenge with AHPND V. 10X General Conclusions  When the microbiota is balanced, there is less likelihood that EMS/ AHPND will occur.  I n contrast, when the microbiota is unbalanced, there is more likelihood that EMS/ AHPND will occur if the pathogen is present.  A healthy microbiota induced by tilapia or biofloc may minimize the EMS/ AHPND infection via water exposure. 23 24 P. vannamei (UAZ 13-472D&E) Biofloc+ feeding challenge with AHPND Vp. 10X 141 Acknowledgement  Sheng Long Biotech, Long An, Vietnam and Minh Phu Seafood Corp. are thanked for their financial sponsorship for these studies. Any question? 142 EXPERIENCE ON Penaeus monodon/RED TILAPIA CO-CULTURE USING A BIOFLOC SYSTEM Boonsirm Withyachumnarnkul1,2*, Compoonut Gerdmusic2, Teerapong Jutipongraksa2, Padmaja J. Pradeep1 and Sage Chaiyapechara3 1 Centex Shrimp, Faculty of Science, Mahidol University, Bangkok, Thailand 2 Shrimp Genetic Improvement Center, Surathani, Thailand 3 National Center for Genetic Engineering and Biotechnology Bangkok, Thailand Background and Rationale The Shrimp Genetic Improvement Center (SGIC), located in Chaiya District, Surat Thani, a province 600 km south of Bangkok, is an organization under the supervision of the National Center for Genetic Engineering and Biotechnology, which is under the umbrella of the National Science and Technology Development Agency (NSTDA), Ministry of Science and Technology, Thailand. The main function of SGIC is to operate and develop a selective breeding program of specific pathogenfree (SPF) stocks of Penaeus monodon with faster growth and disease resistance traits (Tangprasittipap et al. 2010). Currently the center has stocked several full-sib families of SPF P. monodon in its biosecurity facility with variable growth rate. Each family is FIGURE 1. Broodstock grow-out pond in the biosecurity facility of the Shrimp Genetic Improvement Center. Different families of SPF P. monodon are stocked in the same pond until they reach about 30 g, normally 4-5 month in culture. identified using DNA and physical tagging. To compare the performance of SPF P. monodon families, when possible, various families are stocked in the same 800-m2 round pond, which is lined with polyethylene (Figure 1). The initial stocking density is 30 individuals/m2 and growth and survival rates among different families are compared after 3-4 months, when shrimp reach a 143 market size of 30 g. Stocks that have average growth of more than 0.2 g/d at marketable size are selected to produce offspring for commercial distribution. Methodology For confirmation of the growth performance of different families in the biosecurity facility, the same shrimp families were also stocked outside the biosecurity facility in round, plastic ponds of 12-m diameter (113 m2), with 1 m water depth, having bioflocs (15 - 40 mL/L of biofloc volume) (Figure 2). The salinity of water FIGURE 2. Grow-out plastic ponds for testing performance of different families of SPF P. monodon, using biofloc system (inset). was 15 ppt at the beginning and was increased gradually to 30 ppt at 3 months in culture until harvest at 4-5 months. In those plastic ponds, the culture method was divided into two systems: P. monodon in monoculture and P. monodon co-cultured with juvenile red tilapia (Figure 3). The monoculture system was described as above. For the coculture system, red tilapia FIGURE 3. Co-culture pond stocking 2 – 3 g red tilapia in net cages, with SPF P. monodon outside the net cages. The fish are raised for 1 – 2 months until reaching 30 – 50 g of BW (inset) for further stocking in grow-out fingerlings (2-3 g) were stocked in two net-cages (3 m × 2 m × 0.8 m) hanging in the shrimp pond with the bottom of the cage 20-30 cm above the pond bottom. The purpose of setting the net cages this way was to make the entire area of the pond bottom available for shrimp, which 144 have a benthic orientation. Shrimp were fed with standard commercial sinking pellets and tilapia with standard commercial floating pellets. Tilapia could not gain access to the more expensive shrimp pellets. Moreover, fish were not able to feed on stocked shrimp. Fish were stocked at a variable density, ranging from 400-1,500 individuals per cage. They were raised for 1-2 month to 30-50 g (Figure 3), which was the size suitable for stocking commercial grow-out net cages or ponds. The purpose of this co-culture study was to make full use of the pond space. The two species can thrive well at different areas of habitat in the same culture system. Comparison of growth rates of shrimp and fish was made between ponds with shrimp only and those stocked with shrimp and tilapia. Shrimp were stocked at the same density (30/m2) in both treatments. In a separate experiment using biofloc system for tilapia culture at 5 ppt salinity in a 6-m diameter, round, plastic-lined pond, the bacteria profile in the water was examined using culturebased and culture-independent methods. Approximately 50 mL of floc was collected from the pond and transported on ice to the laboratory. Analysis was performed within 24 hrs. Isolation of pure cultures was performed using trypticase soy agar (TSA) or Marine 2216 agar. Identification of ten randomly selected isolates were determined using 16S rDNA sequencing after DNA extraction, PCR amplification of the 16S rDNA, cloning into E. coli, and plasmid extraction. Culture-independent community profiles of bacteria associated with biofloc were determined using PCR-DGGE based on the V3 variable region (Muyzer et al. 1993). The PCR-DGGE products were analyzed on an 8% polyacrylamide gel (25-50% urea-formamide vertical gradient) for 5 hours at 200 V. The resulting gel was strained using SYBR gold and visualized on PharosFXTM Molecular Imager. Results and Discussion Shrimp and fish performance in plastic-lined ponds Shrimp growth in monoculture ponds (0.15 ± 0.04 g/d; n = 6 ponds) was significantly (P<0.05) less than that in co-cultured ponds (0.18 ± 0.04 g/d; n = 7 ponds). In both types of culture, the survival rate was greater than 60%. Water quality (total ammonia nitrogen, total nitrite, alkalinity, pH, nitrate, and dissolved oxygen) was monitored closely and water exchange (3050%) was carried out when necessary. Water quality in both types of culture was maintained at optimum. However, over 20 weeks, the water exchange rate of co-culture ponds (3.3 ± 1.5 times) 145 was significantly (P<0.05) less than that of monoculture ponds (6.3 ± 1.5 times). Shrimp biomass at harvest was 40-70 kg per pond, with an average body weight of 30 g. Shrimp from the cocultured ponds were apparently healthier than those from the monoculture ponds, based on the vigorous activity and the long, unbroken antennae of co-cultured shrimp. Red tilapia in co-culture with P. monodon grew at 0.7-0.9 g/d, with >95% survival and a feed conversion ratio ranging from 0.4-1.0. Tilapia grew equally well at all stocking densities tested. At the highest fish stocking density (3,000 per pond), the ratio of the number of tilapia:shrimp was close to 1:1 and the ratio of biomass of tilapia to shrimp was 2:1 at the first harvest of the fish, when tilapia were 30 g and shrimp were 15 g, and 1:1 at the second harvest, when both tilapia and shrimp were 30 g. Isolation and DGGE community profile of bacteria associated with the biofloc system Most genera of bacteria isolated from biofloc were closely related to common environmental bacteria found associated with soil, plant, or aquatic environments. A few isolates were closely related to known pathogens or bacteria isolated from clinical samples, including Aeromonas veronii, Bacillus circulans or Pseudomonas monteilii (Table 1). Some are potentially beneficial for aquaculture, like Bacillus cereus and Pseudomonas alcaligenes (Chaiyapechara et al. 2012). Conclusions The study suggests that co-culture of P. monodon and red tilapia juveniles in biofloc system does not cause adverse effect on either species. On the contrary, shrimp grew faster in co-culture with tilapia juveniles, probably from feeding on biofloc and with better digestibility of protein (Kuhn et al. 2010). Water exchange rate was also reduced in the co-culture method, possibly leading to better-balanced populations of microorganisms compared to those of the monoculture ponds. Further studies on the population profiles of microorganisms in bioflocs that compare monoculture and co-culture ponds is required. Because of the good performance and health of the shrimp in the co-culture biofloc system, it is possible that this method of culture may be effective against the current problem of acute hepatopancreatic necrosis syndrome (AHPNS) in whiteleg shrimp Litopenaeus vannamei. Our preliminary study on AHPNS challenge revealed promising results supporting this possibility. 146 TABLE 1. Nearest type strain matches of isolates from bioflocs Closest type-strain matches Description Aeromonas veronii (T) X60414 Arthrobacter ureafaciens (T) X80744 Freshwater Gram-negative bacterium Gram-positive soil bacterium, produces sialidase (cleaving sialic acids) Gram-positive soil bacterium, can be used as probiotics Gram-positive bacterium, could be pathogenic Gram-positive bacterium Gram-positive bacterium, psychrophilic from glacier melts Soil bacterium, clinical samples Bacillus cereus (T) AE016877 Bacillus circulans (T) AY043084 Bacillus nealsonii (T) EU656111 Exiguobacterium indicum (T) AJ846291 Microbacterium maritypicum (T) AJ853910 Microbacterium testaceum (T) X77445 Porphyrobacter tepidarius (T) AB033328 Pseudoalteromonas mariniglutinosa (T) AJ507251 Pseudomonas alcaligenes (T) D84006 Pseudomonas monteilii (T) AF064458 Vibrio natriegens (T) X74714 Predominant endophytic bacteria, in plant Photosynthetic, thermophilic bacteria Seawater bacteria Gram-negative bacterium, degrade polycyclic aromatic hydrocarbons Gram-negative bacterium, clinical samples Gram-negative marine bacterium Relevant References Chaiyapechara S, Rungrassamee W, Suriyachay I, Kuncharin Y, Klanchui A, Karoonuthaisiri N, Jiravanuchpaisal P. 2012. Bacterial community associated with the intestinal tract of P. monodon in commercial farms. Microb. Ecol. 63:938-953. Kuhn DD, Lawrence AL, Boardman GD, Patnaik S, Marsh L, Flick GJ. 2010. Evaluation of two types of bioflocs derived from biological treatment of fish effluent as feed ingredients for Pacific white shrimp, Litopenaeus vannamei. Aquaculture 303:28-33. Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700. Tangprasittipap A, Tiensuwan M, Withyachumnarnkul B. 2010. Characterization of candidate genes involved in growth of black tiger shrimp Penaeus monodon. Aquaculture 307:150156. 147 Why co-culture? • The two species stay in different areas of the pond – P. monodon … pond bottom – Red tilapia … all areas, but also OK with the middle and surface areas Experience on Penaeus monodon/Red Tilapia Co-culture using Biofloc System • Brackish water is OK for both species • May have some unknown benefits Boonsirm Withyachumnarnkul Centex Shrimp, Faculty of Science, Mahidol University, Bangkok, Thailand and Shrimp Genetic Improvement Center, Surat Thani, Thailand Aiming for jumbo-sized P. monodon Our aim for red tilapia • 40-100 g BW • For restaurant distributions • For being broodstock (just in case) • Grow from <0.5g BW (after sex reversal) to 50g BW • Acclimatized to brackish and full-strength seawater for further culture in shrimp ponds Pond preparation Why bioflocs? • Round, plastic pond, 12m diameter, 1m deep • Shading, for biosecurity and to prevent blooming of microalgae • Less frequency of water exchange • Both species may grow faster • Reduced FCR for the fish • Air-lift, to make sure of adequate DO and suspension of bioflocs 148 • Set up two net cages, 3mx2mx1m size, into the pond Procedure • The cage bottom is 10-15 cm above the pond bottom, to allow space for P. monodon • Stock 1,000 red tilapias (0.5g BW, after sex reversal) in each cage, feed at 4% BW daily with fish feed (floating pellets) The condition was similar to this aquarium set-up Results and Discussion How to create bioflocs? • Add molasses 0.3 kg weekly (~10% of feed) • Monitor water qualities – – – – – pH, 8.2-8.5 alkalinity, 120-150 ppm total ammonia nitrogen, <1.0 ppm total nitrite , <1.0 ppm DO, 5-6 ppm • Add molasses (0.3 kg) for every 1 ppm increase in TAN • Biofloc volume, 25-30 mL/L pH Shrimp growth rate in SV04 during December 2012-April 2013 Stocking density: 19 pcs/m2 10 9 8 7 6 0 2 4 6 8 10 12 14 16 18 Week of culture Alkalinity (ppm) 200 150 100 50 • TAN and nitrite were fluctuate at the beginning and then stabilized at <1.0 ppm • Nitrate was increased at the end • pH and alkalinity tended to decrease as time passed • Dolomite was added when necessary Water exchange (30-50%) 50 BW (g) CV (%) 40 30 0.5 g Red tilapia 2,000 pcs 37.2 g 20 10 0 0 0 2 4 6 8 10 12 14 16 18 0 Week of culture 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Week of culture 149 Mono-culture Co-culture 0.15 ± 0.04 (6) 0.18 ± 0.04 (7)* P. monodon ADG (g/d) SR (%) FCR Production (kg/pond) Water exchange (times/4 mo) >60 >60 1.5-2.5 1.5-2.5 40-70 40-70 6.3 ± 1.5 (6) 3.3 ± 1.5 (7)* Red tilapia 0.7-0.9 ADG (g/d) >95 SR (%) FCR 0.4-1.0 Production (kg/pond) 100-150 Fish:Shrimp biomass 1:1 Products *P<0.05, compared to mono-culture Strain matches of isolates from bioflocs (by denaturing gradient gel electrophoresis ) Closest type-strain matches Description Aeromonas veronii (T) X60414 Arthrobacter ureafaciens (T) X80744 Freshwater Gram-negative bacterium Gram-positive soil bacterium, produces sialidase (cleaving sialic acids) Bacillus cereus (T) AE016877 Gram-positive soil bacterium, can be used as probiotics Bacillus circulans (T) AY043084 Gram-positive bacterium, could be pathogenic Bacillus nealsonii (T) EU656111 Gram-positive bacterium Gram-positive bacterium, psychrophilic from glacier melts Exiguobacterium indicum (T) AJ846291 Microbacterium maritypicum (T) AJ853910 Soil bacterium, clinical samples Microbacterium testaceum (T) X77445 Predominant endophytic bacteria, in plant Porphyrobacter tepidarius(T) AB033328 Photosynthetic, thermophilic bacteria Pseudoalteromonas mariniglutinosa (T) AJ507251 Seawater bacteria Pseudomonas alcaligenes (T) D84006 Gram-negative bacterium, degrade polycyclic aromatic hydrocarbons Pseudomonas monteilii (T) AF064458 Gram-negative bacterium, clinical samples Vibrio natriegens (T) X74714 Gram-negative marine bacterium Conclusions • P. monodon grew faster in the co-culture with red tilapia, probably from feeding on bioflocs and with better digestibility of protein. • Juvenile red tilapias (50g) are also the main product of this culture system, especially as being SPF. • Water exchange rate was also reduced in the co-culture method, probably by better balanced populations of micro-organisms, compared to those of the mono-culture one. • This model may be suitable for small-scale farmers, as it is profitable and has low risk of failure. Thank you for your attention 150 GREENWATER TECHNOLOGY FOR SHRIMP FARMING: MODES OF ACTION Marc C.J. Verdegem* and Eleonor A. Tendencia Aquaculture and Fisheries Group Department of Animal Sciences, Wageningen University P.O.Box 338, 6700 AH Wageningen, The Netherlands marc.verdegem@wur.nl For more than 15 years, various types of finfish-shrimp integration – referred to as greenwater systems – have been practiced in the Philippines. These methods were developed empirically by farmers with the specific goal of reducing shrimp mortality. In Philippine greenwater systems, shrimp are cultured directly or indirectly in contact with water in which a saline-tolerant fish species is grown. Tilapia is the most commonly used species in greenwater culture. A major feature of the system is that fish metabolic wastes provide nutrients that stimulate phytoplankton production. The phytoplankton is dominated by green algae such as Nannochloropsis sp. and Chlorella sp., hence the name greenwater system. Originally the greenwater system was developed to prevent or control luminous bacterial disease, using two possible culture methods: 1) shrimp and fish cultured in separate ponds and water is circulated between shrimp and fish ponds, and 2) shrimp and fish cultured in the same pond but fish are isolated in a net-pen. After the occurrence of whitespot syndrome virus (WSSV), sometimes the greenwater system was extended by adding a pond stocked with a carnivorous fish species to consume WSSV carriers such as crabs or polychaetes. Numerous experiments demonstrate that the presence of green algae reduces shrimp mortality. For instance, the survival of shrimp exposed to WSSV was better when Chlorella was added to the culture medium, compared to Chlorella-free water. Other explanations are also possible. In shrimp ponds containing cages with tilapia, nitrite oxidizing bacteria and sulfide oxidizing bacteria are more diverse than in monoculture shrimp ponds, potentially allowing for better control of nitrite and hydrogen sulfide concentrations. In another study, tilapia in shrimp enclosures reduced total zooplankton biomass, mainly by reducing rotifer biomass without affecting copepod biomass. The latter effectively prevented excessive phytoplankton blooms and improved water quality. 151 The presence of tilapia, seabass, snapper and rabbitfish reduces luminous Vibrio harveyi counts in shrimp culture water, possibly from bacteria introduced to the system through fish mucus or feces. Fish mucus might also enhance water quality. Mucus from mullet reportedly enhances the development of nitrifying and oxygen-tolerant denitrifying bacteria. In another study, tilapia growth hormone enhances the growth of shrimp postlarvae by interacting with a growth hormone receptor-like protein of postlarvae. Greenwater systems should be further developed. Most shrimp farmers in the Philippines apply extensive polyculture, combining shrimp with milkfish, crab or tilapia in large ponds. Farmers are reluctant to subdivide ponds into smaller units and allocate some of these to fish culture. For them, a solution would be to contain fishes in pens or cages in the shrimp pond. However, it is not yet clear if this is equally or more effective than raising fish and shrimp in separate ponds. More research on integrated greenwater systems is needed to answer this question. 152 GREEN WATER TECHNOLOGY FOR SHRIMP FARMING: MODES OF ACTION Philippines Eleonor A. Tendencia1, Marc Verdegem2 1 Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines and Fisheries Group, Wageningen University, NL 2 Aquaculture • Luminous bacteria • White Spot Syndrome Virus (WSSV)  Restrains production Survey of 174 shrimp farms and other case-studies Survey of 174 shrimp farms and other case-studies  77 farms monoculture – 97 polyculture (8 provinces)  47 variables related to: W SSV r isk fa ct or s • Rainfall - Salinity fluctuation • Low temperature: < 26-27oC - Stocking during the cold months • Temperature fluctuation of 3-4oC in 10 hours (infection) ● Pond site (history and description) ● Period of culture ● Pond preparation methods ● Water management ● Culture methods ● Inputs (feed and others) ● Biosecurity measures • Rapid pH fluctuation • Sharing water with neighbours – same inlet and outlet • • • •  Binary logistic regression; stepwise backward Survey of 174 shrimp farms and other case-studies Removing sludge and depositing on the dike Feeding with mollusc Higher stocking density Larger pond size Interpretation of results  Sludge removal in itself improves culture environment, if W SSV pr ot e ct ive fa ct or • temperature >28oC  Feeding molluscs is a risk (filter feeders)  WSSV carrier disposed with no risk for re-contamination. • Percentage yellow vibrios >50%  Biosecurity measures were not effective (bird nets, crab Also case with polychaetes • Feeding with phytoplankton (Desrina et al. 2013)  Sharing water sources between farms should be avoided. fences, disinfection measures) • High mangrove to pond area ratio • On farms with mangroves in the receiving environment • WSSV infection did not result in disease outbreak; • Water quality in receiving environment with mangroves better; • No difference in culture performance in ponds. 153 Types of greenwater shrimp culture systems Farming strategies practiced by farmers in the Cult ur e syst e m Philippines    1st 5 - 1 5 ppt crop: finfish 2nd crop: shrimp Seabass tilapia   shrimp tilapia shrimp  Low-salinity culture, Crop rotation, Greenwater culture -Modified greenwater culture Extensive shrimp farming milkfish/tilapia-shrimp polyculture Biofloc systems a Addit ive s  Disinfectants  Antibiotics  Probiotics    Ot he r s Fry quality Biosecurity measures Minimum water exchange/closed system b Add predator Greenwater  Numerous pond organisms are carriers/vectors of WSSV  Tilapia metabolic wastes provide nutrients  ● Decapods ● Zooplankton ● Polychaetes stimulating phytoplankton production Tilapia grazing  dominated by green algae (Chlorophyceae), Nannochloropsis sp. and Chlorella sp.  Carnivores reduce potential carriers Species used for co-culture with P. monodon Duo culture tilapia – shrimp?  Size at stocking – predation & shrimp survival (Yuan et al.  Competition for available food: 2010) ● At high tilapia biomass  reduced shrimp growth ● Feeding strategy: ● Only feed shrimp  improved feed efficiency but lower shrimp production (Azaduzzaman et al. 2010) ● Feed fish and shrimp  need for oxygen addition.  Separating fish and shrimp advantageous Evolution to different co-cultures 154 Greenwater improves production levels Comparison of the use and non-use of greenwater : cultured shrimp Pa r a m e t e r GW N on - GW DoC 160 133 Ind. wt (g) Daily wt gain (g) Survival (%) FCR (-) 38 25 0.24 0.19 93 69 1.84+0.21 1.78+0.03  Improved water quality  Improved soil quality  Inhibition of pathogens  Boost shrimp immune response Poor environment GW= cultured using greenwater technique Non_GW= cultured not using the greenwater technique DoC= days of culture ABW= average body weight DWG= daily weight gain SR= survival rate FCR=feed conversion ratio Higher % survival Larger shrimp Susceptible host DISEASE I n cr e a se d pr odu ct ion Virulent pathogen Improved water quality Soil Quality Re fe r e n ce Mugil cephalus secretes skin mucus containing heterothrophically nitrifying and oxygen tolerant denitrifying bacteria Velusamy & Krishnani, 2013 NOB in coastal ponds are sufficiently diverse to continue exploring for bioremediation candidates. Kithiravan et al., 2012 Sulfur oxidizing bacteria isolated from green fish slime maintain sulfide concentration within safe level Kithiravan et al., 2010 Lower nutrient levels in green water ponds Tendencia et al., 2012. Tendencia et al., 2013 Presence of tilapia reduces phytoplankton blooms & enhances copepod presence, but reduced rotifers. Sun et al.,2011 Re fe r e n ce Lower soil available sulfur in greenwater compared to non-greenwater ponds Tendencia et al., 2013 Shrimp immune response boost Greenwater inhibits growth of pathogen Re fe r e n ce Sodium alginate extracts from brown seaweeds retards progression of WSSV Immanuel et al. 2012 Shrimp fed with feed containing macro-algal metabolites control growth of V. alginolyticus & V. fischeri Lipton et al., 2009; Selvin et al., 2012 Extracts of Gracilaria fischeri has anti V. harveyi activity Kanjana et al., 2011 Shrimp fed with herbal and seaweed dietsenriched Artemia lowered V. parahaemolyticus load Immanuel et al. 2004 Lower luminous bacterial count in greenwater compared to non-greenwater; Tendencia Tendencia Tendencia Tendencia Re fe r e n ce et et et et al., al., al., al., 2004; 2005; 2006; 2013 155 Brown seaweeds extracts affect resistance to WSSV in P. monodon postlarvae Immanuel et al., 2010 Extracts of Gracilaria fisheri had immunostimulant activity that could protect P. monodon against V. harveyi Kanjana et al., 2011 Factors affecting efficiency of greenwater Challenges  Greenwater system research • Some farmers are reluctant to divide ponds into smaller units and allocate separate ponds for finfish and crustaceans ● Fish (mollusc, algae) species ● Fish-shrimp biomass ratio ● Culture density ● Feeding strategies • Practice of the “proper” greenwater culture technique • Is greenwater technique effective for extensive shrimp culture?  Foodweb ecology ● Nutrient cycling and turn over efficiencies? ● Community dynamics? ● Shrimp feeding Questions? 156 COMMERCIAL APPLICATIONS 157 BIOFLOC: PAST, PRESENT AND FUTURE Robins McIntosh Charoen Pokphand Foods Public Company Limited C.P. Tower, 27th Floor 313 Silom Road, Bangkok Thailand The world of shrimp culture before 1995 was dominated by intensive culture of P. monodon in Asia and semi-intensive production of L. vannamei in the Americas. World production gradually increased from year to year with interruptions in production caused by new virus or shrimp pathogens making their way into systems every few years. Markets were dominated by traders that used these supply uncertainties to drive prices to extremes, either up or down. Shrimp farming was still in the gambling phase of development. Profits from one successful pond could pay for the failure of several ponds. Meetings of the WAS were dominated by papers concerned with water quality, soil quality and pond preparation, phytoplankton management, and quality criteria for wild post-larvae. The special session on shrimp farming sponsored by WAS in 1995 was titled “Swimming through Troubled Water”. Then, around 1995, there were some culturists that started thinking outside the box and new terms were introduced into our vocabulary and became dominant in discussions at WAS: domestication, shrimp genetics, flocs, microbial ecology, biosecurity, intensification, and protein utilization efficiency, food safety, and traceability. The state-of-the-art began to change rapidly as culturists dared break the established rules of shrimp culture and assembled new technologies into what today we call high-intensity recirculated systems. These systems make possible the reliable production of large quantities of shrimp at lower prices. Today there is little room in shrimp farming for gamblers; the industry has entered the industrial-commercial phase where a pond failure can wipe out the profits from many successful ponds. Past Flocs (although I did not initially identify them as such) first came to my attention around 1990, when I read a paper by the Aquacop that had described a system in the early 1980s in Tahiti and New Caledonia that did not exchange much water and, in this system, microbial flocs developed. Reported production was beyond anything I had experienced. I filed this in the back of my head. 158 Then, later that year, I was visiting a customer farm in Thailand, and the water of a pond he was harvesting was absolutely black with microbial particles and the shrimp being harvested from this black water were as healthy as I had ever seen. Best yet, the yield from this harvest was over 18 t/ha. Most people would have said that this pond had poor water quality but the shrimp were absolutely beautiful. Then, in 1995, Russ Allen and Barry Bowen presented me with a project that seemingly could not be done: develop a shrimp farm at a higher elevation than was typical of shrimp farms and at a distance that was miles from the coast. With such a site, water exchange would not be feasible. To complicate the plan, they wanted to achieve yields that were unheard of at the time. A visit to Waddell Mariculture Center in South Carolina demonstrated that such a system was feasible because they were producing unheard-of yields using very little or no water exchange. How was this possible? My memory brought back that paper by Aquacop and my experience at that harvest in Thailand many years before. The Waddell Mariculture Center had described a system where feeding rates were constant. This made no sense unless you thought in terms of creating a detrital system in the pond. Applying feed at equal rates from start to finish brought to mind bioreactors where equal loading of nitrogen allows bacterial populations to develop to assimilate wastes. So the ideas of the first zero-exchange commercial ponds were born in my head. I would add shrimp feed, but then I would add carbon in a complex form to encourage bacteria that would digest the more complex carbonaceous wastes of shrimp feed. That carbon source became what I termed the grain feed because it was compounded with wheat, corn and soybean at 18% protein. I came up with a feed mix to the pond of 70% shrimp feed and 30% grain feed. Why not provide a lower protein feed in one diet? In hindsight, I could have used just one lower-protein diet but, in those first years, I did not know if the lower-density protein could be digested as efficiently as a high-density protein diet. So I used a complex grain diet to balance the shrimp feed. I never used molasses and to this day only use it sparingly because I think we need complex carbohydrates to encourage the proper bacteria. 159 From the very beginning this new system produced shrimp at yields and efficiencies I had never experienced, but I was learning. This system was for the most part stable with respect to pH, oxygen levels, and transparency. From this I learned a valuable lesson: shrimp respond favorably to stability by growing faster with greater survival. I also learned that there were different color flocs. Some flocs were tea-brown, others black or green. Each color floc represented different challenges and results in operation. The tea-brown color with big fluffy floc particles resulted in the greatest growth and yield. Black flocs always represented problems. So began the process of learning to seed a pond by transferring water with a developed brown floc, making the system more predictable. This system was heterotrophic, and as such produced large amounts of sludge. This sludge had to be removed from the system or excess hydrogen sulfide and other toxins produced at the bottom. To keep big flocs in suspension also required a lot of energy, up to 35 hp/ha to produce 16 t/ha of shrimp. At that time the discussion going on in my head was: what is the function of the floc in this system? Was it nutritional, water purification or pond stability? I believed after awhile that the greatest benefits of floc were from the resultant pond stability and the enhanced ability of the pond to process large amounts of waste to nontoxic forms. So, was there a better way to achieve this while minimizing the negative aspects of floc-based shrimp culture? Present In Thailand the rationale for doing zero or low-rate water exchange was biosecurity: the less water added, the easier it is to exclude viral pathogens. This led to low-exchange systems using a combination of treatment ponds and reservoirs. As a consequence of closing the system and exchanging little water, microbial flocs developed, but the rationale was completely based on providing stability (oxygen and pH) and digestion of wastes inside the pond. Nutrition for shrimp would come from applied feeds that were 32-40% protein with high nutrient levels and digestibility. Flocs that developed were small but with a high proportion of nitrifying bacteria. Smaller flocs produced less sludge. As a consequence, these systems required less energy to keep flocs in suspension. Flocs contained more nitrifiers and less heterotrophic bacteria. However, results were the same: systems were stable and did not require water exchange to maintain healthy pond conditions, even when shrimp post-larvae were stocked at more than 200/m2. In 160 fact, even though massive amounts of visible floc do not develop, the FCR of such systems can be as low at 1.1, confirming that floc is not most useful as a nutrient, but as a mechanism to maintain conditions conductive to excellent shrimp growth. Future We have learned that not all bacterial flocs are equal and, as we progress in our learning, more scientific microbiological concepts are being applied to floc pond development. Flocs are now domesticated. Just as wild broodstock were not the way forward in terms of getting optimum, consistent farm performance, neither are wild flocs. Bacteria can be screened and blended in such a way to form mixes that create best flocs for given pond conditions. Today we have bacteria mixes that can establish a superior nitrifying floc in a pond or flocs that include microbes that produce more omega-3 fatty acids. The development of domesticated flocs selected for shrimp performance will take us to yet another level of shrimp culture advancement. New domesticated flocs, combined with new systems and new genetic stocks of shrimp, will provide a new platform to grow shrimp. I can envisage a day when such systems will make shrimp production possible in any city of the world. Already we have such systems that consistently produce over 10 kg/m3 of shrimp. Biofloc technology is continuing to evolve and that is important. I didn’t know what I was doing 17 years ago, and I don’t know where we will be 17 years from now, but I know it will not look like what we are doing today. The evolution of technology is vital. 161 Belize Aquaculture Floc: Past, Present, Future Goal: 11,500 kgs/cycle‐ 2.5 cycles per year 1997 But land was 6M elevation; 3 km from the water supply Robins McIntosh Charoen Pokphand Foods Public Company C.P. Tower; 27th floor 313 Silom Road, Bangkok Thailand How to do? UMMMMM! Before: My experience Semi Intensive: 1. Water Exchange 2. High density risky Guatemala: Acapolon Farm The Theory I went on! What Happened! Feeding Protocol 17% Protein Grain Diet for Carbon Balancing POND NITROGEN LEVELS DURING CULTURE CYCLE ammonia nitrite nitrate org N 45 40 NITROGEN LEVEL MG/L 35 30 25 20 15 10 5 Hopkins, J.S., C.L. Browdy, P.A. Sandifer and A.D. Stokes. 1995. Effect of two feed protein levels and two feed rate, stocking density combinations on water quality and production in intensive shrimp ponds operated without water exchange. Journal of the World Aquaculture Society 26:93-97, (5). 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CULTURE WEEK 27% high quality shrimp feed 162 It Works! CONSTANT FEED RATE-ALMOST! 1000 HETERO TROPHIC (BELIZE) YIELD (KG/HA) 6,200 13,500 STOCK RATE /M2 60-70 105-150 300 pH 7.9-8.5 6.8-7.2 200 CO2 1-2 10-15 3000 NITRATE 1-2 10-20 0 OXYGEN (AM/PM) 2.0-8.5 3.8-5.5 TRANSPA RENCY (CM) 20-30 30-40 WEEK OF CULTURE New Terms Dec MONTH Was it Heterotrophic Nutrient and microbial dynamics in high‐intensity, zero‐exchange shrimp ponds in Belize Michele A. Burford*, Peter J. Thompsona, Robins P. McIntoshb in my vocabulary Floc Zero exchange Heterotrophic Sludging • The high nutrient concentrations promoted the growth of bacteria, phytoplankton (mostly autotrophic flagellates) and protozoa. Up to 40% of the bacteria were associated with flocculated • There appeared to be scope for increasing bacterial production in these systems by increasing the C:N ratio, and hence C availability for bacterial growth. However, it remains to be established which microbial processes are likely to be promoted, and if the benefits of this outweigh the costs. CSIRO What is the benefit of Floc? Nutrition? Belize System was Carbon Limited PROT=31.5 PROT=22.5 MEAN 78 66 72 60 ORGANIC MATTER % 50 ASH % 21 32 26 PROTEIN % 51 35 43 FAT % 10 15 12.5 Respiration M G OX YGE N C ON S U ME D 70 CONTROL CARBON + NITROGEN+ 40 30 20 10 0 0.5 1 Time 1.5 2 DAYS Was that Bad? 163 PHOSPHOROUS MG/KG 11.5 CALCIUM MG/KG 15870 ARGININE % 2.3 1.61 1.95 METH % 0.61 0.35 0.48 LYSINE % 2.5 1.7 2.1 Mar-01 Jun Sept Dec Mar 6000 Jun 0 9000 Sept FARM MIX 12000 Dec 100 15000 Mar 400 18000 May 500 Sept SHRIMP FEED 600 Kg/Ha 21000 Feb-98 KGS/WEEK OF FEED 700 KGS/HECTARE WEEK I= 180% OF BIOMASS OR 45 KGS/DAY WEEK 20= 2.1% OF BIOMASS OR 135 KGS/DAY 800 • • • • Yield AUTO TROPHIC (GUATEM ALA) ) 900 What is the benefit of Floc? Maintain Good Water Quality Subsequent Studies “The contribution of flocculated material to shrimp (Litopenaeus vannamei) nutrition in a high‐intensity, zero‐exchange system” Michelle Buford, et al. • Provides Surface Area for Nitrification /de‐ nitrification Reactions • Removes Nitrogen and nutrients via Heterotrophic bacterial biomass formation Microbial floc meal as a replacement ingredient for fish meal and soybean protein in shrimp feed David Kuhn et. al What were the Problems! What is the benefit of Floc? • Sludge accumulation from excessive flocs • Energy to maintain floc suspension STABILITY pH Accumulated Sludge Oxygen So many Colors, So many Textures, So much Variation Too Much Floc? Correct C/N: Heterotrophy 164 Floc: Generation II (Asia) Re‐circulated, low exchange ponds in a closed System Pond Biosecurity Farm Biosecurity For Viral Exclusion Driven by Exclusionary Principle of Bio‐security The Domestication of Floc Floc philosophy • Probiotic Bacteria: oxidize bottoms, breakdown wastes • Encourage nitrifiers over heterotrophic bacteria, • Thinner flocs, cleaner bottoms • Only use carbon to start the system; concentrate on quality diets Mix/Culture of Select Microbes Isolation of Microbes Floc in a Bottle Scaling Floc in a Bottle to Farm Small Pond, Super Intensive Consistent Performance High Nitrification Potential Not Dense‐ Cleaner Bottoms Fcr: 1.15 Biomass: 11 kg/m2 And NO EMS 165 Controlling Floc levels for optimum performance Application to Bio‐secure Broodstock, Larval Rearing Nurseries: EMS Aid The Future: Designer Floc Functional Nutrition DOC: 15 8,000‐10,000/m2 The relative enhancement of Penaeus vannamei growth by selected fractions of shrimp pond water Gary Pruder et al What does it mean? Can we design a floc with QSI? Can we design a more nutritious floc? Isolation of Schizochytrium Screening for QSI compounds Schizochytrium cells 40x And so many other ideas! 166 Floc‐ Meister Rule I Floc systems should never be about growing Floc! Floc is a tool for growing a target Species More Efficiently! 167 PRACTICAL MEASURES FOR SHRIMP FARMING DURING AN EMS OUTBREAK Tung Hoang1* and Marc Le Poul2 School of Biotechnology, International University, Vietnam 2 Tomboy Skretting, Tan Tao Industrial Park, HCMC, Vietnam htung@hcmiu.edu.vn 1 EMS Outbreak in Vietnam From 2011 to 2012 the Early Mortality Syndrome (EMS) and white spot disease had cost the shrimp farming industry of Vietnam an estimated loss of US$ 2.65 billion (MARD 2012). EMS started in China in 2009 before reaching four provinces in the Mekong Delta of Vietnam in 2010 (Lightner et al. 2012). EMS then spread to all coastal provinces of Vietnam in 2011 and 2012, and was found in tiger shrimp Penaeus monodon and white-leg shrimp Litopenaeus vannamei. EMS is infectious. The long-distance “jumps” suggest a close association with movements of shrimp broodstock and/or postlarvae across the region and international borders. The occurrence of EMS in grow-out ponds was closely associated with certain suppliers of shrimp broodstock or postlarvae. In 2012, EMS was also found in shrimp hatcheries. Furthermore, during the outbreak of EMS, vibriosis in shrimp hatcheries became much more difficult to control, suggesting that Vibrio could be the causative agent of EMS. It is hypothesized that better control of Vibrio in hatcheries by probiotics or antibiotics did not allow EMS outbreaks. However, once stocked into grow-out ponds, postlarvae carrying pathogens are not well protected and EMS could break out easily. Mortality of older shrimps by EMS is possible because causative agents were previously anchored in the pond environment. The outbreak of EMS has prompted many farmers to look for “magic” solutions or to drastically change established farming protocols without much consideration of the consequences. Production cost increased accordingly from utilization of new chemotherapeutants that, as used, are not often proportional to success. Interestingly, there were shrimp farms that applied no change, but managed to produce shrimp. Since early 2011, shrimp ponds with low salinities, a high level of DO maintained throughout the production cycle, a clean pond bottom, using groundwater and periodic application of disinfectants or probiotics were productive and 168 successful, even in EMS-infected areas. These findings suggest that EMS can be controlled with good farming strategies and pond management. Preventive Strategies Effective control of EMS requires in-depth understanding about the pathogen, hosts, its epidemiology and specific conditions for outbreak. Tran et al. (2013a) have identified a specific strain of Vibrio parahaemolyticus colonizing the digestive tract of infected shrimps as the causative agent of EMS. However, the hepatopancreas of infected shrimps is destroyed only when the colonizing V. parahaemolyticus is attacked by a bacteriophage and releases toxin. These findings point out the need to characterize the identified pathogens genetically for diagnostic purposes. To control EMS, we could keep either Vibrio or the bacteriophage from cultured shrimp, or more effectively not allow transmission through contact. Theoretically, studies on genome sequencing, genome annotation and communication mechanisms of these two pathogens could help develop a ligand that blocks them from contacting with each other (Dr. Ly Le, pers. comm.). To avoid the bacteriophage, postlarvae must be carefully checked before stocking. In addition, once the intermediate hosts of the bacteriophage are identified, they must be removed from the farming system. For the time being some farmers use antibiotics to control Vibrio and thus EMS. However, previous research has reported a high multiple-resistance index of V. parahaemolyticus to different antibiotics. The use or misuse of antibiotics is thus dangerous and could create more problems in the near future. Practical Measures Diseases can only break out in specific conditions. For example Akazawa (2013) reported that high pH (8.5-8.8) of pond water was associated with EMS outbreak in Malaysia. Thus, good pond management could prevent shrimp diseases. Farming strategies. Suitable farming strategies should be seriously considered to better cope with increasing incidence of virulent diseases. Shrimp farms should either scale back to improved extensive farming or, where capital and technical assistance allows, intensify the 169 production system for better management. Lower stocking densities certainly reduce the risks of disease outbreak, thus enhancing farming success. System design and operation. Ponds should be smaller (2,000-4,000 m2), with a square shape and round corners, a minimum depth of 1.7 m, and sufficient aeration. Paddlewheel aerators should be more powerful and extend much farther toward the pond center than normal. This will help provide enough oxygen and keep the pond bottom cleaned of sludge and minimize the occurrence of anaerobic spots. The use of automated feeders for feeding is highly recommended from the second month onwards. This reduces feed costs by 10-15% and the rate of waste accumulation in the pond. Quality of postlarvae. Until a reliable diagnostic kit is commercialized to screen for the causative agents of EMS, shrimp farmers should buy postlarvae from hatcheries that are wellestablished with a good reputation. Furthermore, postlarvae that have been infected by vibriosis during larval rearing should not be used for stocking because of the biosecurity risk of bringing Vibrio into the farming system. Splitting the production cycle into two stages – advanced nursing of postlarvae and grow-out production – is also recommended. For this option, stress-free transfer methods from nursing ponds to production ponds are considered a key for success. Daily pond management. Dissolved oxygen should be maintained at high levels throughout the production cycle. Biofloc technology should be applied for the advanced nursing period, using pond water from a previous crop. Probiotics should be supplied by reliable sources and mass cultured on the farm for daily application to production ponds. Finally, sludge should be removed more frequently to keep the pond bottom clean and remove the associated oxygen demand. 170 Sh a r in g e x pe r ie n ce fr om pr a ct ica l poin t of vie w Pr a ct ica l m e a su r e s for sh r im p fa r m in g in a n EM S ou t br e a k  EMS out break in Viet nam  St rat egies for prevent ion  Pract ical m easures Tung Hoang 1 * & Marc Le Poul 2 1 School of Biot echnology, I nt ernat ional Universit y VNUHCM 2 Tom boy Skret t ing, HoChiMinh Cit y, Viet nam htung@hcmiu.edu.vn 1 htung@hcmiu.edu.vn 2 EMS: SOS! 2009 EMS EMS 2012 (19) EMS 2011 (10) EMS 2010 (4) Economical loss by EMS and WSD: 2010 (87k mt or US$ 484 mil), 2011 (280k mt or US$ 1.6 bil) and 2012 (200k mt or US$1.05 bil) 3 4 Bot h P. m onodon and L. vannam ei are infect ed. Farm ers claim P. m onodon is m ore suscept ible t o EMS Difficult t o m aint ain a healt hy bloom of algae. I nfect ion and m ort alit y were m ost ly observed in t he first m ont h at t he beginning, but could happen up t o 90 days of cult ure. Filam ent ous algae develop on t he pond bot t om . 30- 40% infect ed shrim ps m ay recover, but feeding t hese could t rigger m ort alit y. Also found in hat cheries since 2012 5 171 Ant obiot ics could help prevent but im proper use is of concern. Daily application (for 5 – 10 ha) Som e herbal ext ract s were effect ive for 2030% of t he cases Bacillus subtilis, B. amyloliquefaciens, B. pumillus, B. cereus (Hoang & Hoang 2012) htung@hcmiu.edu.vn 7 Indonesia (photo by Harris) htung@hcmiu.edu.vn 8 Le ss pr oble m w it h EM S No Changed supplier of broodst ock ( and PLs) + long- arm paddlewheel Successful harvest s aft er 82 days of cult ure htung@hcmiu.edu.vn OBSERVATI ON S I M PLI CATI ON 1 Using underground wat er wit h low salinit ies ( 3 – 5 ppt ) 2 Applicat ion of ant ibiot ics or ant ibact erial ext ract s or probiot ics ( biofloc) Causat ive agent s: bact eria likely and could be Vibrio. 3 Clean bot t om ( e.g. lim it ed sludge, no filam ent ous algae) 4 High level of DO ent irely during t he crop 5 Post larvae qualit y ( i.e. lum inescent diseases in hat chery) and source of supply 6 St rengt hened biosecurit y ( i.e. st rict ly followed t he est ablished farm ing prot ocol) Probably cont rolled in hat cheries by different chem ot herapeut ant s. Out break once in grow- out ponds wit h less cont rol. htung@hcmiu.edu.vn 9 10 WB/GAA EMS mission – Vietnam July 2012 11 12 172 St r a t e gie s for pr e ve n t ion Th e ca u sa t ive a ge n t s?  A strain of Vibrio parahaemolyticus (Tran et al. 2013) + a bacteriophage Hatchery Grow-out systems  More than one strain involved in EMS/AHPNS? PCR-based diagnostic tools 13 14 I nt ensificat ion Sm aller pond, adequat ely equipped, great er cont rol, high densit ies, high yield and good profit RI SKS ZONE RI SKS ZONE RI SKS ZONE RI SKS ZONE I m proved ext ensive ( 5 – 10/ m 2 ) , m ore nat ural, low invest m ent OR Pr e ve n t in g st r e sse s … t h r ou gh good pon d m a n a ge m e n t Sem i- int ensive ( 30- 40/ m 2 ) wit h am ple equipm ent 15 15 16 Pr a ct ica l m e a su r e s Pond management  Keep bottom clean  High DO  Beneficial bacteria 3,000 – 3,500 m 2  pH: 7,0 – 7,5 SPF postlarvae 28hp of paddle wheels plus bot t om diffusers  Low salinity 400/ m 2 , 3.5- m ont h crop wit h part ial harvest Rem oval of wast es aft er each feeding Avoid stress, control the causative agents by ecological approach 50 – 60 t ons/ ha/ crop htung@hcmiu.edu.vn 17 htung@hcmiu.edu.vn 173 18 19 htung@hcmiu.edu.vn 20 Tw o- ph a se s gr ow ou t Thank you for your attention!!! Bet t er cont rol in t he first m ont h Biofloc Cost- effect ive in operat ion Fat er t urnover rat e ( 30 days + 45 days) Challenges: m ovem ent of shrim ps bet ween ponds wit hout st ress htung@hcmiu.edu.vn 21 htung@hcmiu.edu.vn 174 22 SHRIMP FARMING: BIOFLOC AS BIOSECURITY? Nyan Taw Consultant Blue Archipelago BHD T3-9, KPMG Tower, 8 First Avenue, Bandar Utama 47800 PJ Selangor, Malaysia nyan.taw@bluearchipelago.com; nyan.taw@gmail.com With emerging new viral diseases, such as EMS in Asia, biosecurity has become essential for sustainable production in shrimp farming. Farm biosecurity begins with farm design and construction. Shrimp farm layouts have evolved from simple pond-based flow-through systems during the 1980s to modular systems now. Modular systems use reservoirs to treat incoming water to provide the biosecurity required to control emerging viral issues (Taw 2005, 2008, 2011). With biosecure farm design and construction, a biosecure operational system needs to be implemented (Taw 2010, 2012 & 2013). Biofloc, a very recent technology, offers promise for stable and sustainable production because the system has internal nitrification within culture ponds operated with zero water exchange (Avnimelech 2000, 2005a, 2005b, Avnimelech, et al. 2012). The technology has been successfully applied commercially in Belize by Belize Aquaculture Limited (McIntosh, 2000a, 2000b,2000c, 2001). It also has been applied with success in shrimp farming in Indonesia, Malaysia (Taw 2004, 2005, 2008, 2010, 2011, 2012) and recently successfully commercialized in Malaysia (Taw et al. 2013). The combination of two technologies – partial harvesting and biofloc – has been studied in northern Sumatra, Indonesia (Taw et al. 2008). Applying biosecure and biofloc technologies appear to allow sustainable farmed shrimp production. The technology was applied in Indonesia without incidence of WSSV, where it was a threat to shrimp farmers during the early 2000s. During the late 2000s, IMNV outbreaks in Indonesia caused huge losses to shrimp farmers. During this period, a small shrimp farm in northern Bali survived by using biofloc technology (Taw and Setio 2013). In Malaysia, biosecure and biofloc technology has been applied at the iSHARP shrimp farm since October 2011 and has been operating successfully to date (October 2013) without any incidence of EMS (Taw et al. 175 2013). Very recently a trial using post-larvae imported from Thailand and adjacent to a soft crab farm was successfully conducted in Myanmar in earthen ponds using biofloc technology (Taw and Tun 2013). According to In-Kwon (2012) there are more than 2,000 bacterial species in well-developed biofloc water. Biofloc may enhance immune activity based on mRNA expression of six immunerelated genes – ProPO1, ProPO2, PPAE, ran, mas and SP1. This could be one of the reasons for an enhanced immune system in shrimp that consume biofloc. The EMS outbreaks were very serious in China, Vietnam, Malaysia and Thailand. Between 50 to 80% of shrimp farms suffered from EMS in these countries. Biofloc technology as biosecurity has somehow prevented viral outbreaks on shrimp farms. Possible factors contributing to biosecurity using biofloc technology are provided in Table 1. TABLE 1. Biofloc as biosecurity. Biofloc system Biosecurity Zero water exchange (topping up only for water lost due to siphoning and evaporation). Low risk of virus entering culture ponds through water source. Aeration for 24 hours in accordance with pond carrying capacity (full or semibiofloc) to have biofloc suspended in pond water. Stable dissolved oxygen (DO) Phytoplankton (algae) bloom. Crash nonexistent as biofloc does not depend on sun light for photosynthesis. Stable environment. Low stress for shrimp Stable culture water environment – DO, pH, TAN, nitrogen, NH 3 , etc. Stable environment. Low stress for shrimp Extra natural live feed – biofloc with unicellular protein. Extra nutritious feed. Biofloc induces six immune related genes. May enhance immune activity in shrimp. – healthier shrimp. – healthier shrimp. – healthier shrimp. 176 INTRODUCTION SHRIMP FARMING: BIOFLOC AS BIOSECURTY ? Biofloc, a very recent technology seem a very promising for stable and sustainable production as the system has self nitrification process within culture ponds with zero water exchange (Yoram, 2000, 2005a&b & Yoram, et at 2012). The technology has been successfully applied commercially in Belize by Belize aquaculture (McIntosh, 2000a, b & c, 2001). It also has been applied with success in shrimp farming in Indonesia, Malaysia (Nyan Taw 2004, 2005, 2008, 2010, 2011 &, 2012) and recently successfully commercialized in Malaysia (Nyan Taw, et.at 2013). The combination of two technologies, partial harvesting and biofloc, has been studied in northern Sumatra, Indonesia (Nyan Taw 2008 et. al). Recently, a semi-biolfoc applied in earthen ponds in Myanmar was published (Taw & Tun, 2013) and biofloc technology used successfully in small family own farm in Bali, Indonesia will be published in early 2014 (Taw & Setio, 2014) Nyan Taw, Ph.D. Technical Consultant Blue Archipelago BHD MALAYSIA nyan.taw@bluearchipelago.com; nyan.taw@gmail.com With emerging viral problems and rising costs for energy, biosecure farm design with biofloc technology appears to be an answer for sustainable production. BIOFLOC BIOFLOC FLOC COMMUNITIES AND SIZE Biofloc color in pond & under microscope 100 µ The biofloc Green Color Defined as macroaggregates – diatoms, macroalgae, fecal pellets, exoskeleton, remains of dead organisms, bacteria, protest and invertebrates. (Decamp, O., et al 2002) As Natural Feed (filter feeders – L. vannamie Brown Green & Tilapia) : It is possible that microbial protein has a higher availability than feed protein (Yoram, 2005) Brown Color BIOFLOC TECHNOLOGY CONCEPT SHRIMP FARMING IN BIOFLOC SUMMARY Avg . F/ D, GP Co nsum p tio n & Growth Pe rfo rm a nc e GP (kg), F/D (kg) High stocking density ‐ over 130 – 150 PL10/m2 2. High aeration – 28 to 32 HP/ha PWAs 3. Paddle wheel position in ponds (control biofloc & sludge by siphoning) 4. Biofloc control at <15 ml/L 5. HDPE / Concrete lined ponds 6. Grain (pellet) 7. Molasses 8 C&N ratio >15 9. Expected production 20–25 MT/ha/crop with 18‐20 gms shrimp 1. MBW (g) 20 160 F/D GP MBW 18 140 16 120 14 100 12 80 10 8 60 6 40 4 20 2 0 0 1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134 DOC (da ys) Feed & grain application & Growth High aeration & PWAs position High densit y Biofloc technology is a system that has a self-nutrification process within culture pond water with zero water exchange (Yoram, 2012) Biofloc Grain pellet 177 Bioflocs Dark Vannamei Red Vannamei MODULE OPERATION Water treatment system (Control WSSV) Physical barrier for viral carriers – use 250 micron screen net Chemical application – kill viral carriers. Apply crusticides Treatment Reservoirs ponds Kill free water bore virus (aging) ‐ dies in 72 hrs without host Treated water ready for use for culture (apply same procedure) Culture ponds Clean animals – SPF or SPR or PCR check Clean Ponds–clean or oxidized pond bottom Clean Water– treated water & nets (250 micron) Prevent carriers (fence) Strict security to avoid biosecurity breaches POND WATER PREPARATION BASIC OPERATION PROCEDURE (SUMMARY) For already treated water initially in culture ponds and series of treatment reservoirs for later use> Basic Procedure (Summary) Parameters Fill in Water Closed Water Quarantine, Treatment and Culture ponds (Max. level > 120 cm) DOC ( Apply Crustacide To Kill WSSV Carrier Bio assay Check toxicity Aging water To Kill WSSV in water Screening: 250 – 300 micron. Prevent carrier entering module 1 - 45 All ponds/0.5 – 1.0 ppm Water exchange Water source Yes - approximately 5–10 days All ponds minimum 3 days Yes – at all critical points: Main inlet, between QP & TP; TP & Main TP or Supply canal. TP & CP No Within module only Semi Closed Water Started fill in to Quarantine, Treatment and Culture ponds (optimum level >100 cm) 46 – 90 Use whenever possible Open Water Started fill in to Quarantine, Treatment and Culture ponds (Max level > 120 cm). Shrimp will be cultured in less or no water exchange system. Water will be added only to replace the loose water by siphon or evaporation. Water level starts with maximum and maintain at optimum level. The system is to promote and switch from autotrophic to heterotrophic condition. Water quality management during culture operation 91 - harvest No use Yes – approximately 5-10 days Minimum 3 days No need Minimum 3 days Yes – at all critical points: Main inlet, between QP & TP; TP & Main TP or Supply canal. TP & CP Minimum (topping up only) Start open to outside Yes – at all critical points: Main inlet, between QP & TP; TP & Main TP or Supply canal. TP & CP Maximum Open to outside Day Activity 1 Urea 8 kg & TSP 1 kg Grain pellet 30 kg & Dolomite 50 kg 2 Tea seed cake 15 ppm 4 Grain pellet 30 kg & Dolomite 50 kg 6 Grain pellet 30 kg & Dolomite 50 kg 8 Grain pellet 50 kg, Molasses 8 kg & Kaolin 50 kg 10 Grain pellet 50 kg 12 Kaolin 50 kg Culture System (for 0.5 ha HDPE lined pond) Nyan Taw AA 2006, LV POND WATER MANAGEMENT DURING OPERATION SIPHONING PONDS BY GRAVITY OR PUMPS 1. Dissolved Oxygen: Maintained at level >4 ppm. 2. Alkalinity: Maintained at level > 70 ppm by applied 50 kg/pond Hydrated lime (Ca(OH)2) every week. 3. Kaolin: Apply 50 kg/pond once every three week. 4. Molasses: Apply 8.0 kg/pond three times a week Siphon tip Water pump on float 5. Grain Pellet: Apply together with feed from 10-20% during earlier stage and increase to 20-50 % during late stage (see standard). 6. Sludge: Sludge accumulation in pond bottom need to be removed periodically by siphoning. 7. Management: All applications need to be adjusted by Shrimp growth, Biomass, Feed per day and Biofloc volume. Gravity ‐ central drain Culture System (for 0.5 ha HDPE lined pond) 178 SHRIMP FARM, INDONESIA FEED AND GRAIN PELLET USED BIOFLOC TECHNOLOGY APPLIED IN COMPANY ASSET PONDS (KG /DAY) NO VIRAL (WSSV) OUTBREAKS: 2003-2005 Floc System Production R&D, Trial and Company Commercial Ponds Period 2003 - 2005 40.0 35.0 Number of pond 30.0 25.0 20.0 15.0 10.0 5.0 0.0 Full Biofloc < 2,0 Stock ‐ 130 PL10/m2; 15 HP/0.5 ha; DoC 90‐110 Production ‐ 10‐12 MT/0.5 ha; MBW 18‐20 gram SR 80%; FCR 1.2 (feed ); Carrying Capacity ‐ 650‐750 kg/HP 2,02,4 2,53,0 3,03,5 3,54,0 4,04,9 5,05,9 6,06,9 7,07,9 8,08,9 9,09,9 10,0- 11,0- >12.0 10,9 11,9 Production range (kg/5000m 2) R&D. Densit y 100-200 pcs/ m2, M BW 16.41 g, B iomass 9.905 kg, SR 81.7 %, FCR 1.29 (number of ponds = 46) TRIAL. Densit y 140 pcs/m2 , M B W 16.56 g, B iomass 10.082 kg, SR 87.0 %, FCR 1.42 (number of ponds = 13) CCP. Densit y 130 pcs/ m2 (st andard), M B W 16.99 g, Biomass 9.557 kg, SR 85.5 %, FCR 1.21 (number of ponds = 131) PERFORMANCE –BIOFLOC & SEMI BIOFLOC P. monodon CULTURED IN BIOFLOC Acar Beru, Blue Archipelago, Malaysia Growth No Viral (WSSV) outbreaks 20 15 10 Grams From: 5 David M. Smith, et al, 2008 DoC 40 50 60 70 80 90 100 110 PRODUCTION PERFORMANCE OF ARCA BIRU FARM Production Parameter No of Ponds PWA Energy (Hp) Stocking Density DOC (days) SR (%) MBW (gr) FCR (x) ADG (gr/day) Avg Harvest tonnage (kg) Production (Kg/Ha) Prod per power input (Kg/Hp) i-SHARP SHRIMP FARM PROJECT Malaysia - Semi and Full biofloc Phase one 0 Density 80 (Dike) Density 110 (Full) Density 130 Biofloc Development of protocols for the culture of black tiger shrimp, Penaeus monodon,in “zero”water exchange production ponds Modules in operation System/size/type Biofloc 0.4 ha HDPE Semi‐Biofloc 0.8 ha HDPE Conven 0.8 ha HDPE Dyke 2 19 119 14 24 20 130 110 83 90 101 111 89.16 81.35 83.19 18.78 18.31 17.80 1.39 1.58 1.77 0.21 0.18 0.16 9,006 12,950 9,616 22,514 16,188 12,019 643 540 481 Nyan Taw, et.al. GAA March/April 2011 ISHARP BLUE ARCHIPELAGO, MALAYSIA Paddle wheel aerators position SEMI-BIOFLOC PERFORMANCE No EMS or WSSV outbreaks – October 2011-July 2013 HDPE Lined modules Nyan Taw et. al. GAA Jan/Feb 2013 Production Performance CYCLE Trial & 1 for Modules 1 & 2 Production Parameter No of ponds Paddle Wheels Aerators (HP) Days of Culture (DoC) Survival Rate (%) MBW (grams) FCR Average Production (kg/pond) Average Production (kg/ha) Prod per power Input (Kg/Hp) 179 CYCLE Trial ‐ Modules 1 & 2 Density 40/m2 Density 60/m2 Density 80/m2 Density 130/m2 20 16 8 BFT 4 BFT* 12 12 12 16 113 108 94 88 112.23 101.22 106.05 69.56 21.65 17.41 13.86 12.56 1.34 1.47 1.32 1.74 4,875 5,294 5,828 5,677 9,749 10,587 11,655 11,354 406 441 486 355 CYCLE 1 ‐Modules 1 & 2 Density 100/m2 Density 100/m2 24 BFT 24 BFT 12 12 100 99 97.30 104.92 16.05 16.31 1.39 1.26 7,714 8,547 15,428 17,093 643 712 BIOSECURITY & BIOFLOC MANAGED TO PASS THROUGH MONSOON ? BIOFLOC IN BALI, INDONESIA Family Owned Farm 60.00 Rain (mm),Salinity (ppt) & Temperature (C) Northern Coast of Bali, Indonesia 20 iSHARP Project, Malaysia 15 50.00 Normal… Intense… 10 Rain water Salinity g Temperature 5 40.00 0 42 50 58 64 72 79 Days of Culture 86 93 97 30.00 SHRIMP FARM BALI ‐ Biofloc technology 20.00 10.00 ‐ 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Oct‐11 Nov‐11 Dec‐11 Jan‐12 Feb‐12 Mar‐12 Apr‐12 May‐12 Jun‐12 Jul‐12 NO VIRAL (IMNV or WSSV) OUTBREAKS 2009-2013 Aug‐12 Month/ Week CYCLE Trial - Modules 1 & 2 CYCLE 1 - Modules 1 & 2 Nyan Taw et. al. GAA Jan/Feb 2013 A3 2,600 148 18 97 18.12 1.35 104 7,281 28,004 405 F1 2,800 150 16 97 15.32 1.49 101 6,388 22,814 399 F2 2,800 145 18 95 17.3 1.29 106 7,682 27,436 427 E1 1,000 150 6 95 16.48 1.46 94.7 2,345 23,450 391 E2* 750 180 4 45 4 B1 2,000 155 12 82 19.5 1.2 103.9 6,307 31,535 526 * Aeration problem ‐ DO dropped <1.0ppm Farm total Production: 53,472 kg (26,736 kg/ha) In this cycle ponds B1,B2, B3,C1,C2 & C3 intense control less DOC to just over 80 days‐ more cycles/year B2 2,000 155 12 82 18.5 1.4 94 5,399 26,995 450 B3 2,000 155 12 81 16 1.25 92.9 4,622 23,110 385 C1 600 175 12 82 14.68 1.35 97.4 1,503 25,050 376 Production Performance Shrimp Ponds 50,000 45,000 40,000 35,000 30,000 Shrimp sampling STOCK VANNAMEI PL IN MARCH 2013 25,000 20,000 Two modules-earthen ponds: one module consisted of 1 reservoir and 3 production ponds 15,000 10,000 5,000 Kg 0 No viral outbreaks Belize, C America 2000 L. vanamei Post Larvae imported from Thailand by Air Lampung Indo 2003‐05 Medan, Indo 2008 Commercial Kg/ha Java, Indo 2008 N Bali, Indo 2009 BAB Malaysia 2010 Max Record Kg/ha Taw & Tun, 2013 NITRIFICATION SEQUENCE ALGAE & BIOFLOC Units-ml/L Algae and Biofloc in Pond Water Avnimelech et al 2012 Avnimelech et. at, 2012 (data from experimental pond Dor, Israel) 180 C2 600 175 6 82 19.72 1.1 98.5 2,050 34,167 342 C3 600 175 4 81 18.48 1.14 101.9 1,981 33,017 495 Taw & Setio, 2014 (to be published in Jan‐Feb GAA 2014) BIOFLOC IN SHRIMP FARMING SHRIMP CULTURE - SEMI-BIOFLOC IN MYANMAR Earthen pond bedside Soft Shell Crab Farm Soft Shell Crab Farm Culture period August‐ November 2012 Pond A2 Pond Size (M2) 2,400 PL Stocking Density (No/m2) 170 Aeration (hp) 18 Days of Culture 97 Body weight (gm) 18.4 Feed Conversion ratio 1.26 Survival (%) 105.8 Production (kg)/pond 7,914 Production (kg/ha) 32,976 Production/power input (kg/hp) 440 BAB Malaysia 2010 Trial 45 8 40 10 7 35 8 6 30 6 5 25 4 20 ( DO , pH , Temp , Trans , PO4-P , ( DO , pH , Temp , Trans , PO4-P , CO2 , TAN , NH3-N , NO2-N , NO3-N , CO2 , TAN , NH3-N , NO2-N , NO3-N , Sal , Alk , Chlor-a , Floc , Bact , etc. ) Sal , Alk , Chlor-a , Floc , Bact , etc. ) 3 15 11 10 9 8 7 6 5 4 3 2 1 0 11 21 PO4-P (ppm) NO2-N (ppm) Chlorophyl a (µg/l) 31 41 Sal (ppt) CO2 (ppm) 51 61 71 DOC (DAYS) TAN (ppm) Total Alk (ppm) 81 91 101 111 Vannamei STD 131pcs./ m2 81 1 DOC (DAYS) Temp (pm) 49 57 65 73 81 89 97 105 113 121 DO Autotrophic PM DO Heterotrophic PM DOC ( day ) PH comparison between Autotrophic and Heterotrophic System 10 9 pH – Algae & Biofloc 8 F/ D (kg), Grain Pellet (kg), 160 14 140 12 120 10 100 8 80 6 pH Autotrop am PH Heterotrop am pH Autotrop pm PH Heterotrop pm 97 16 7 115 180 91 200 18 109 20 85 SHRIMP PERFORMANCE 72.01.10 79 Temp(am) 41 103 Trans (cm) MBW (g), Floc (ml/ 1L) DO (pm) 33 73 pH (pm) 25 67 pH (am) 17 61 DO( am) 9 DO Autotrophic AM DO Heterotrophic AM 91 101 111 Vannamei STD 131 pcs./m2 Production : 8,971.8 kg; MBW : 16.36 g SR: 83.9 % FCR: 1.0 ADG: 0.14 DOC: 113 55 71 49 61 43 51 37 41 (ppm) 31 31 12 21 25 13 11 19 14 0 7 500 475 450 425 400 375 350 325 300 275 250 225 200 175 150 125 100 75 50 25 0 15 2 5 1 Alkalinity – Algae & Biofloc 4 10 1 Dissolved Oxygen Comparison between Autotrophic and Heterotrophic System 12 1 ALKALINITY, Chlorophyl a (ug/l) WATER PARAMETER 72.01.10 2 - POND WATER ENVIRONMENT ( ppm ) 14 TEMP 50 9 PO4-P, TAN, CO2 NO2-N, SALINITY(X10) 1 WATER PARAMETER 72.01.10 10 13 DO, pH, TRANSPARENCY ( X 10 ) Pond Pond Environment Environment DOC (day) ppm 160 6 60 4 40 2 20 0 0 140 120 DO – Algae & Biofloc 100 80 60 40 1 11 21 31 41 Product ion : 8,971.8 kg; MBW : 16.36 g SR: 83.9 % 51 61 DOC (DAYS) 71 81 91 101 FCR: 1.0ADG: 0.14 DOC: 113 Pr oduct ion : 8 , 9 7 1. 8 kg; M BW : 16 . 3 6 g SR: 8 3 . 9 % Floc (ml/ 1L) MBW (g) Feed/ day (kg) Grain Pellet (kg) FCR: 1. 0 ADG: 0 . 14 DOC: 113 Heterotrophic 20 Chandaeng, S. et al.(2005): Taw, N (2006) 111 Van n ameiST D 13 1 pcs. / m2 25 Autotrophic 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 DOC (day) BIOFLOC MAY ENHANCE IMMUNE ACTIVITY More than 2,000 bacterial species were found in well‐developed biofloc water Biolfocs may enhance immune activity, based on mRNA expression of six immune‐related genes. ProPO1, proPO2, PPAE, ran, mas and SP1 From – In‐Kwon Jang, IWA International Water Congress, 2012, Busan, Korea EMS (Early Mortality Syndrome)/ AHPNS (Acute Hepatopancreatic Necrosis Syndrome) SHRIMP DISEASES & BIOFLOC EMS/AHPNS Dr. Lightner, GOAL 2012 181 EMS/AHPNS Spreading in Asia & SE Asia SOME ARTICLES ON EMS/AHPNS Dr. Lim, GOAL 2012 RECENT DEVELOPMENTS PROPOSED ACTIONS FOR EMS PCR for EMS/AHPND POSSIBLE SOLUTION FOR EMS/AHPNS AT FARM LEVEL (EMS Panel GOAL 2012 Bangkok) DOF Malaysia EMS Seminar KL, Malaysia March2013 BIOFLOC BASIC MANAGEMENT CONCEPTS FOR SHRIMP CULTURE 1 WHY BIOFLOC AS BIOSECURITY Semi‐biofloc to Full biofloc system feasible 1. 2 Use treated water only 3 Zero water exchange (only topping up) 4 Earthen to HDPE full or semi-lined ponds 5 Aerators to have pond water (biofloc) in suspension (22-24 hrs) 6 Correct aerators’ position and number very important 7 Excess sludge need to be removed –specially for full biofloc 8 9 BIOFLOC as possible solution for EMS/AHPNS control at farm level 2. BIOFLOC SYSTEM BIOSECURITY Zero water exchange (topping up only for water lost due to siphoning & evaporation) Use treated water only – through reservoirs Low risk of virus entering culture ponds through water source Modular system 3. Stable dissolved Oxygen (DO). Healthier Aeration full 22-24 hours in accordance with pond carry capacity (full or semi-biofloc) to have shrimps biofloc suspended in pond water. 4. Phytoplankton (algae) bloom & crash nonexistent as biofloc does not depend on sun light for photosynthesis. Stable environment. Low stress for shrimps – healthier shrimps Biofloc volume control (<15 ml/L) 5. Control C/N ratio to above >15 6. Stable culture water environment – DO and pH. Extra natural live feed – biofloc with unicellular protein (protein 30 -50%) More than 2,000 bacterial species were found in well-developed biofloc water Stable environment. Low stress for shrimps – healthier shrimps Extra nutritious natural feed 10 Molasses & Grain pellet required (Carbon source) 7. 11 Operate in accordance with Carrying capacity of pond essential (species/stocking density/pond type/operating system) 8. 182 Biofloc contains six immune related genes Possibly a probiotic affect May enhance immune activity in shrimps BIOFLOC TECHNOLOGY WORLD WIDE ACKNOWLEDGEMENTS The author would like to sincerely thank the following: Israel Myanmar Vietnam Malaysia Guatemala Beliz Nicaragua THANK YOU Mr. Abu Bakar Ibrahim (CEO) and Mr. Christopher Lim (COO), Blue Archipelago for their interest and support. The staff and members of Blue Archipelago, Malaysia for their support. New Caleonia 183 INTRODUCTION TO THE MIXOTROPHIC SYSTEM: AN INTENSIVE SHRIMP FARMING MANAGEMENT TECHNOLOGY Farshad Shishehchian Blue Aqua International Pte Ltd. Penthouse level, Suntec Tower 3, 8 Temasek Boulevard, Singapore, 039878 Blue Aqua International (Thailand) Co., Ltd. 599/114 Rachadapisek Road, Jatujak, Bangkok, 10900, Thailand info@blueaquaint.com; Tel. +66 2 1921787-8 www.blueaquaint.com This presentation briefly discusses the key issues faced by shrimp producers due to intensification. It describes the main changes in water and soil quality, besides the impact of feeding in the pond environment. The first part describes the most important factors of a biosecurity plan implementation. Feeding management is discussed more in detail in the second part, while the benefits of automatic feeding briefly described. The third part presents the use of minerals and probiotics, as a bioremediation tool and their role during pond preparation. These three parts serve to introduce the zero water exchange, mixotrophic system, developed by Blue Aqua International, a pond management system widely used in Southeast Asian countries. 184 Outlook • Introduction • Disease management: environmental causes • MixotrophicTM System, a natural pond management technology: Shrimp Fa rm Ma na g e me nt Using the Mixo tro p hic Syste m ‐ Main features ‐ Stages or phases ‐ Control parameters ‐ Benefits Dr. Fa rsha d Shishe hc hia n Ph.D., Te rre stria l a nd Aq ua tic Ec o lo g y Blue Aq ua Inte rna tio na l Pre sid e nt & C EO © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d • BAI’s experience worldwide • Conclusion © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 185 © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 186 p H De finitio n p H o r p o te ntia l Hydro g e n is a me a sure o f a c id ity (hyd ro g e n io ns) o r b a sic ity o f a n a q ue o us so lutio n p H sc a le ra ng e s fro m 0 to 14 a nd a so lutio n a c c o rd ing to its p H is d e fine d in ne utra l, a c id ic o r b a sic ©©2012 2013 Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd pH Rang e pH Rang e p H ra ng e fo r a q ua c ulture p urp o se s: 6.5 - 9.0 p H stro ng ly influe nc e d b y p ho to synthe sis a nd re sp ira tio n DENSE BLO O M De a th De sire d ra ng e fo r fish pro duc tio n 10 De a th 9 4.0 6.5 7 9.0 11 14 8 pH > 9.0 Ammonium converted into ammonia and BGA toxins negative effects SPARSE BLO O M 7 pH < 6.5 Heavy metal release from sediments Sunrise Sunse t Sunrise Optimal pH in the pond: 7.5 - 8.5 ©©2012 2012Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd ©©2012 2012 Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd pH b uffe ring with a lka linity pH 11 Lo w a lka linity wa te r induc e s b ro a d pH fluc tua tio ns induc ing shrim p stre ss, re duc e d g ro wth a nd e ve n m o rta lity 10 To ta l Alka linity is the c a p a c ity o f wa te r to ne utra lize a c id s (HC O 3-, C O 3- a nd O H-), thus its b uffe ring c a p a c ity Hig h a lka linity le ve ls with hig h pH a ffe c t shrim p m o lting a s we ll (e xc e ss sa lt lo se s) 9 8 7 Lo w Alka linity Wa te r 6 Exp re sse d a s millig ra ms p e r lite r (p p m) o f e q uiva le nt c a lc ium c a rb o na te (C a C O 3) Hig h Alka linity Wa te r 5 Ea rly Mo rning ©©2012 2013Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd ©©2012 2012 Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd 187 Mid Afte rno o n Ea rly Mo rning Alkalinity and Hardne ss a lka linity ha rdne ss To ta l titra ta b le b a se s To ta l diva le nt sa lts b ic a rb o na te HC O 3 - c a rb o na te c a lc ium m a g ne sium C O 23- C a 2+ Mg 2+ C a lc ium b ic a rb o na te Alka linity me a sure me nt a nd ha rd ne ss c o nc e p t Ma g ne sium b ic a rb o na te Ma g ne sium c a rb o na te CaCO3 Mg ( HC O 3 ) 2 Mg C O 3 C a ( HC O 3 ) 2 ©©2012 2012Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd ©©2012 2013 Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd O RP O RP O xid a tio n-Re d uc tio n Po te ntia l Me a sure o f the c le a nline ss o f wa te r a nd its a b ility to d o wn c o nta mina nts C a lc ium c a rb o na te In o xid a tive c o nd itio ns, p o sitive O RP le ve ls (a b o ve 0 mV), the hig he r the O RP le ve l, the hig he r a b ility the wa te r ha s to d e stro y fo re ig n c o nta mina nts suc h a s mic ro b e s, o r c a rb o n b a se d c o nta mina nts the b re a k Ra ng e o f –2,000 to + 2,000 a nd millivo lts (mV) units O RP Le ve l (m V) Applic a tio n 0- 150 No pra c tic a l use 150- 250 Aq ua c ulture 250- 350 C o o ling To we rs 400- 475 Swim m ing po o ls 450- 600 Ho t Tub s 600 Wa te r Disinfe c tio n 800 Wa te r Ste riliza tio n Me te rs me a sure e le c tric a l p o te ntia l, ind ire c t me a sure me nt o f d isso lve d o xyg e n ©©2012 2012Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd ©©2012 2012 Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd O RP MIXO TRO PHIC TM SYSTEM O RP me a sure me nt PATENT PENDING IN 144 COUNTRIES (PCT) Me a sure me nt o f o xid a nt / a ntio xid a nt p o te ntia l o r a c tivity Pro b e in o zo nize d wa te r g e ne ra te s sma ll vo lta g e G o ld o r p la tinum e le c tro d e re ve rsib ly lo o se s its e le c tro ns to the o xid ize r G e ne ra te d vo lta g e is c o mp a re d to re fe re nc e silve r e le c tro d e in a silve r sa lt so lutio n ©©2012 2012Blue Blue Aq Aqua ua Inte Interna rnatio tiona nallAll AllRig Rights htsRe Rese serve rvedd © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 188 Phy to plankto n m anag e m e nt Type N:P Nitrogen-fixing BGA 42-125 Green algae ~30 Diatom ~10 Red Algae ~10 Dinophyceae ~12 Blue Green Algae <10 N:P ra tio Pro vid ing e sse ntia l nutrie nts a nd b a la nc ing the m fo r: • Nutritio us a nd he a lthy p hyto p la nkto n g ro wth. • O ve rb lo o m a nd d ie -o ff p re ve ntio n. • Blue G re e n Alg a e g ro wth p re ve ntio n. © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d Enviro nme nta l mo d ula tio n O RP pH Ene rg y Po nd p re p a ra tio n Pro b io tic applic atio n Mine ra l a p p lic a tio n (N:P ra tio ) Initia l c ulture with lo w fe e d a p p lic a tio n Ino rg a nic ma tte r Natural fo o d (Phy to plankto n , Zo o plankto n and Be ntho s) Nutrie nts C :N ra tio N:P ra tio Sto c king Ha rve st © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d O rg a nic ma tte r b uild up Pro b io tic a p p lic a tio n (C :N ra tio ) Mid - a nd la te -c ulture Ino rg a nic ma tte r O rg a nic ma tte r Phyto p la nkto n p ha se Phyto p la nkto np ro b io tic p ha se Sto c king Ha rve st © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 189 O rg a nic ma tte r Pro b io tic p ha se Po nd p re p a ra tio n b a se d on pH sta b iliza tio n, d isinfe c tio n a nd b io a va ila b le mine ra l sup p ly. Ab und a nt na tura l fo o d b a sis, g re e n a lg a e a nd d ia to ms, o b ta ine d N:P ra tio thro ug h mo d ula tio n. Wa te r a nd p o nd b o tto m sta b iliza tio n fo r PL sto c king with no stre ss ind uc tio n. G ra d ua l o rg a nic ma tte r a c c umula tio n d ue to fe e d ing is d e c o mp o se d a e ro b ic a nd by fa c ulta tive a na e ro b ic b a c te ria . Pro b io tic a p p lic a tio n ra te inc re a se to b re a k d o wn e xc e ss o rg a nic a vo id ma tte r a nd a na e ro b ic / H2S c o nd itio ns. Pre ve ntio n o f e xc e ssive p hyto p la nkto n b lo o m a nd d ie -o ff. Phyto p la nkto n p ha se © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d Phyto p la nkto n-p ro b io tic p ha se © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d Le ss Nitrific a tio n e nha nc e me nt to re d uc e to xic e ffe c ts of nitro g e no us c o mp o und s, fa c ilita te d b y a hig h O RP o f +100 a nd +350 mV. p hyto p la nkto n wa te r, b a c te ria a nd b a c te ria p ro mo te d e nsure in he te ro tro p hic nitrifying sta b le to wa te r q ua lity. Sup p re ssio n of p a tho g e nic b a c te ria . Hig h o rg a nic lo a d a nd b a c te ria l a c tivity. Ele va te d to xic nitro g e no us c o mp o und s re q uire a hig h ra te o f a mmo nific a tio n Phyto p la nkto n-p ro b io tic p ha se a nd Pro b io tic p ha se nitrific a tio n. © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d Ste p -b y-ste p Pond preparation Pathogen suppression Phytoplankton bloom Nutrient balanced system Water quality and pond bottom stabilization Probiotic application © 2012 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 190 Mixo tro phic Syste m Be ne fits Mixo tro phic Syste m Ma na g e s the Po nd to : Have an e c o lo g ic ally b alanc e d sy ste m Minim ize fluc tuatio ns o f wate r and so il q uality Inc re ase o p tim um c arry ing c apac ity Re duc e shrim p/ fish stre ss All at e c o no m ic al c o st and e asy m anag e m e nt © 2012 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d Tha nk Yo u Blue Aq ua Inte rna tio na l Blue Aqua Mobile Application AVAILABLE NOW! http://www.blueaquaint.com/appnews/bb/ http://www.blueaquaint.com/appnews/android/ © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d © 2012 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d O ffic e s in Tha ila nd, India , Sing a p o re , a nd USA C o nta c t us: Dr. Farshad Shishe hc hian www.b lue a q ua int.c o m fa rsha d .shishe hc hia n@ b lue a q ua int.c o m fa c e b o o k: Blue a q ua Int https:/ / www.yo utub e .c o m / use r/ Blue Aq ua Int © 2013 Blue Aq ua Inte rna tio na l All Rig hts Re se rve d 191 List of Participants Ibrahim Lim Victoria Saleh Steve Christian Yoram Nasser Sun-hye David Roger Alberto Rudi Mohd Fariduddin Suryakumar Peter Craig Luận Leo Emilie Nihar Ethan Ong Dave Carlos Valeriano Oanh Quang Ahmad Ahouf Jimmy Alday-Sanz Alodirji Arce Armijos Avnimelech Ayaril Bae Bal Barnard Bayas Bijnens Bin Othman Boriah Bossier Browdy Bùi Công Cababasay Cardona Chattopadhyay Ranjan Cheung Chin Peng Chua Co Corre Jr. Dang Thi Hoang Dang Xuan Blue Archipelago Venture farms Pescanova Ministry of Agriculture Oceanic Institute EKO FARMING, S.A Technion National Prawn Company Japan Int. Res. Center for Agriculture Science DuPont-Danisco Singapore RMB AQUA SYAQUA SIAM INVE Blue Archipelago Hitide Seafarms Ghent University Novus International Quoc Viet Co. Ltd Blue Archipelago IFREMER West Bengal University of Animal & Fishery Science Zhen Penn Sdn Bhd kwestec Oversea Feeds Corporation U.P. Visayas - Fisheries/Ocean Science TOMBOY 192 venturefarms@hotmail.sg victoria_alday@yahoo.com s.soma@hotmail.com sarce@oceanicinstitute.org info@ekofarming.com agyoram@tx.technion.ac.il nasserakv@robian.com.sa baesh@jireas.go.jp david.bal@dupont.com rogbarmard@gmail.com alberto.baya@goldcoin_th.com rudi@inveasia.co.th fariduddin@dof.gov.my hitide.seafarms@gmail.com peter.bossier@ugent.be browdyc@gmail.com luan.bui@qvseafood.com leo@bluearchipelago.com emilie.cardona@ifremer.fr nrchatterjee40@gmail.com ethancheung2013@gmail.com fa_ocp@yahoo.com kwestec@gmail.com cebuoversea@yahoo.com profcorre@yahoo.com dthoanh@ctu.edu.vn Giap Sukenda Timothy Felipe David Thai Vinh Julie Pedro Kevin Anil Ana Carolina Khang Phoung Siti Lim John Mohammad Mario Tan Tung Long Dac In-Kwon Michael Werner Daniel Ana Su Kyoung Apirux Geraldo Dao Huy Darja Dejager do Nascimento Vieira Drahos Du Quoc Du Quoc Ekasari Encarnacao Fitz Simmons Ghanekar Guerrelhas Ha Thanh Hai Dang Halijah Musa Ham Min Hargreaves Hazzaa Hoffmann Hong Xin Hoang Huynh Huynh Tam Jang Janssens Jost Jun Kamil Kim Kimawanit Kipper Fóes National University of Singapore Bogor Agricultural University co3 Consulting UFSC / LCM Novozymes Biologicals, Inc. Intron Intron Bogor Agricultural University BIOMIN University of Arizona Ecosecure Systems Aquatec Aquacultura Ltda BIOMIN BIOMIN Kembang Subur Sdn Bhd Blue Archipelago Ministry of Agriculture INVE Shanghai Ocean University, China International University, Vietnam Viet Uc TOMBOY Korean National Fisheries R&D Institute INVE Camanor Produtos Marinhos Ltda. Myung Sun Co.Ltd Kembang Subur Sdn Bhd NFRDI - Korea TRF Feed Mill Co. Ltd FURG - BRAZIL 193 daogiap@gmail.com snkenda67@gmail.com dejagert@co3.ca felipe.vieira@UFSC.br Ddra@novozymes.com j_ekasari@ipb.ac.id Kevfitz@ag.arizona.edu anilghanekar@yahoo.com anaguerrelhas@aquatec.com.br halijah@bluearchipelago.com jhargreaves01@yahoo.com hazzaamohamad@yahoo.com hxtan@shou.edu.cn htung@hcmiu.edu.vn Dienluong@vietuc.net jangik@korea.kr werner.jost@camanor.com.br wangkijun@daum.net ana_klantan@yahoo.com jong-hwa@nanmail.net Apiruxkim@gmail.com geraldofoes@gmail.com Are Pakorn Marcos Dariano Poh Ren Leong Mew Luc Tran Marc Che King Jeffrey Ramir Michael Donald Eng Soon Jairo Jeff Dien Guozhi Thanh Sonny Phuoc KOK FOONG Pong Anirak Robins Mohd Afid Zuridah Aufar Natrah Fatin Dustin Klevan Konwuttikai Kroupa Krummenauer Ku Lang Le Le My Huyen Le Poul Lee Lee Lee Leger Lighter Lim Llanos Lotz Luong Tuan Luong Tuan Luong Tuan Ly Ly Vinh MAH Marfoo Masa McIntosh Md. Razi Merican Misbahudin Mohd Ikhsan Moss PHARMAP AS - Norway BIOMIN Areation Industries International FURG - BRAZIL BioTake Co., Ltd. 3LP Global Sdn Bhd Tam Quan Intron TOMBOY Blue Archipelago Berhad Kembang Subur Sdn Bhd Zeigler Bros., Inc. TOMBOY University of Arizona BIOMIN Consultant Gulfcast Research Viet Uc Shanghai Ocean University, China Intron Wanshida Ocean Bio-Tech Intron ECONION MARKETING SDN BHD Asia Aquatic Global Gen Co. Ltd - Thailand Charoen Pokphand Foods Public Company Ltd. Blue Archipelago Aquaculture Asia Pacific Magazine Kembang Subur Sdn Bhd University Putra Malaysia Oceanic Institute 194 are.klevan@pharmaq.no Marcos.Kroupa@aireo2.com darianok@gmail.com Luc.le@taco.com.vn cklee@bluearchipelago.com jeffery.kssb@gmail.com Ramir.lee@zeiglerfeed.com dvl@u.arizona.edu jairo_llanos@hotmail.com Jeff.lotz@usm.fbu Dienluong@vietuc.net gzhluo@shou.edu.vn bobogoseafood@yahoo.com kokfoong_86@hotmail.com asiaayunhvs@gmail.com apirak_masa@hotmail.com Zuridah@aquaasiapac.com f.13amps@gmail.com natrah@upm.edu.my dmoss@oceanicinstitute.org Shaun Karu Karunanithi Thaung Nancy Shanmugam Stephen Huat Tuấn Truong Hoa Nghị Dinh Tung Tam Vinh Toan Binh Tu Công Hoài Huy Hoang Hoa Nhiem Tấn Tuan Nguyen Alberto Tippawam Anthony Moss Muthusamy Muthusamy Myo Min Nevejan Neviliappan Newman Ng Teng Ngô Quốc Nguyen Nguyen Duy Nguyễn Hữu Nguyen Khai Nguyen Minh Nguyen Minh Tam Nguyen Ngoc Phu Nguyen Quang Nguyen Thanh Nguyen Thanh Nguyễn Thành Nguyễn Thị Nguyen Trong Nguyen Van Nguyen Van Nguyen Van Nguyễn Văn Nguyen Viet Nguyen Xuan Nomura Noppakit Ostrowski Oceanic Institute Syndel Asia Sdn-Bhd Aquaculture Asia Pacific Magazine Myanmar Frontline Lab for Aquaculture & ARC (UGent) Ngee Ann Polytechnic AquaInTech Inc. Kembang Subur Sdn Bhd Quoc Viet Viet Uc Branch of Cargill Co., Ltd at Tien Giang Quoc Viet Co. Ltd Vemedim Co. Intron BIOMIN Vemedim Co. International University Quoc Viet Co. Ltd Aeration Industries International Huy Thuan Co. Can Tho University Intron ANABIO Research & Development JSC Tuan Phu Shrimp farm TOMBOY Piscicultura da Fonte BIOMIN Wanshida Ocean Bio-Tech 195 smoss@oceanicinstitute.org Kkarusara@hotmail.com nancy.nevejan@ugent.be sne2@np.edu.sg sgnewm@aqua-in-tech.com kembang_subur@yahoo.com.my Dienluong@vietuc.net hoaria2@yahoo.com nghiqv099@yahoo.com nguyentoan146148@gmail.com binhmatkieng13@gmail.com binhmatkieng13@gmail.com cong.nguyen@qvseafood.com Marcos.Kroupa@aireo2.com huyleader@huythuan.com.vn binhmatkieng13@gmail.com cman@ctu.edu.vn duyennt10686@gmail.com albertonomura@globo.com aostrowski808@yahoo.com Mohmmed Edoardo Brian Kumuda Aiyappa Vorapong Luiz Hung Anh Trung Đạt Minh Sita Ipung Thao KP Afiq Aizee Gahang Yoon Patricia Theerapong Ivanpedro Arjen Ron Goncalo Bruno Teguh Farshad Mitch Kenneth Othaibi Pantanella Park Patra Pattada Pattrakulchai Peregrino Pham Manh Pham Minh Pham Thanh Phạm Tiến Phan Cong Poonsawat Purwanto Hari Quach Thanh Raveesha Razi Reduan Rhee Rico Ritmak Rodriguez Roem Salzberg Santos Sarwana Scopel Setyono Shichehchian Smith Soderhall Ministry of Agriculture AIT Myung Sun Co.Ltd BIOMIN ASAP, India Branch of Cargill Co. Ltd at Tien Giang Camanor Produtos Marinhos Ltda. Vemedim Co. NOVUS Branch of Cargill Co. Ltd at Tien Giang ANABIO Research & Development JSC Apc.Co.Ltd Cargill Feed & Nutrition Blue Archipelago Intron ASAP Blue Archipelago Kembang Subur Sdn Bhd NFRDI - Korea Santeh Feeds Corporation SITTO VN Co. Selecta TOMBOY AZARA CAPITAL BIOMIN PT.STP.JAPF - COMFEED MCR Aquaculture Ltd. PT.STP.JAPF - COMFEED Blue Aqua International Pte Ltd. Shrimp Improvement Systems Uppsala University 196 odaiby@yahoo.com edpantanella@gmail.com bhpark_ms@hanmail.net piaiyappa@gmail.com Vorapong_Pattrakulchai@cargill.com luiz.peregrino@camanor.com.br p.mhung@vemedim.vn minhanh.pham@novusint.com Ngochuong_Dao@cargill.com duyennt10686@gmail.com infoapc.vn@gmail.com Sita-Poonsawat@cargill.com ravsh727@gmail.com afiq@bluearchipelago.com aizeereduan@yahoo.com jong-hwa@nanmail.net thuyloansitto@gmail.com ivanpr@selecta.com.mx arjen.roem@nutreco.com Ron@azaracapital.com sarwana@sapbwi.japtacomfeed.co.id brscopel@hotmail.com teguh@stplpg.japtacomfeed.co.id info@blueaquaint.com AquaSmithUSA@yahoo.com Kenneth.Soderhall@ebc.uu.se Patrick Gede Undang Agus Joko Yuri Philippe Justin Seong Lim Steven Alanox Nyan Ana Paula Eleonor Lovela Huy Loc Su Luan Le Loan Alexander Eng Huan Marc C.J. Andre Boonkob Borphit Figo Trung Tung Wilson Sorgeloos Suantika Supriatna Suryawinadi Susilo Sutanto Tacon Tan Tan Tan Tan Chien Loon Taw Teixeira Tendencia Tinambunan Tran Tran Tran Anh Hoang Tran Dinh Tran Huu Tran Thi Thuy Tseng Ung Verdegem Viana Viriyapongsutee Viriyapongsutee Vo Vo Minh Vo Thanh Wasielesky Ghent University - Belgium PT.STP.JAPF - COMFEED PT.STP.JAPF - COMFEED PT.STP.JAPF - COMFEED BIOMIN CP Prima Indonesia Lesaffre Feed Additives BIOMIN BIOMIN BIOMIN ECONION MARKETING SDN BHD Blue Archipelago Aquatec Aquacultura Ltda Southeast Asian Fisheries Development Center Santeh Feeds Corporation Pentair Aquatic Eco-system University of Arizona TOMBOY AIT Can Tho University SITTO VN Co. Wanshida Ocean Bio-Tech BioValence Wageningen University Poli-Nutri BIOMIN BIOMIN Patrick.sorgeloos@ugent.be gsuantika@sith.itb.ac.id oendangsp@gmail.com agussunjawin.dil@gmail.com yuri.sutanto@cpp.co.id PTN@lesaffre.fr kokfoong_86@hotmail.com nyan.taw@bluearchipelago.com anapaula@aquatec.com.br gigi@seafdec.org.ph huytran@pentair.com thuuloc@email.arizona.edu tdluanait@yahoo.com thuyloansitto@gmail.com alexanderlct@163.com huanung@yahoo.com marc.verdegem@wur.nl andre.viana@polinutri.com.br binhmatkieng13@gmail.com Vemedim Co. PHARMAQ - VN Federal University of Rio Grande 197 are.klevan@pharmaq.no manow@mikrus.com.br Barbara Boonsirm Keen Sh Min Hang-seak Jong-Hwa Sandra Teresa Jrasong Weber Withyachumnarnkul Wong Yew Yoon Yoon Zainathan Catherine Zhang Zhang BIOMIN Faculty of Science, Mahidol University Kwestec BioValence Chunssu Fisheries NFRDI - Korea University Malaysia Terengganu BIOMIN Institute of Southsea Fisheries, China 198 wboonsirm@yahoo.com kwestec@gmail.com shermynn.yew@biovalence.com.my hangxi@hanmai.net jong-hwa@nanmail.net sandra@umt.edu.my Jrasongzhang@hotmail.com