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
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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
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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
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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
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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.
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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
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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 -80C
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 (4C at CTU)
5. 60 frozen samples are kept at -80C 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
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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
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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
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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
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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
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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/
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© 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