Chapter 21 REPTILES AS ANIMAL MODELS: EXAMPLES OF THEIR UTILITY IN GENETICS, IMMUNOLOGY AND TOXICOLOGY Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet, Hugo H. Ortega and Marta D. Mudry Universidad Nacional del Litoral ABSTRACT Historically, animals used as experimental models have contributed to the knowledge of multiple aspects of organisms’ biology and wildlife, providing valuable information about physiological processes, events, environmental situations, and even human interactions. Alternatives to animal testing are primarily based on biochemical assays or experiments with cells/organs cultures, typically far more sophisticated and specific than in vivo approaches. However, the whole organism allows for inferences about particular species and its situation in natural habitats. Sometimes, it is not possible to study directly the species of interest, making it necessary to identify the closest related species that can be used as a model organism. Reptiles may be good and interesting models as they respond both behaviorally and physiologically to environmental or experimental conditions. This chapter specifically describes the utility of crocodiles, lizards, and turtles as animal models in studies of genetics, immunology, and toxicology. The increased interest in reptile genomics is evident by newly sequenced genomes, by the establishment of significant genomic resources for some reptile groups, and by the awareness that genomic diversity in Reptiles is substantially greater than that of mammals. Reptiles also demonstrate immune components with an apparently higher activity than other vertebrates. Their ability to resist serious injuries makes them interesting models to elucidate mechanisms within the defense system. In the same way, interesting studies were performed to propose immune components to be used as indicators of toxics exposure. Environmental contaminants can significantly affect many reptiles. However, these species are often excluded from toxicology studies and ecological risk assessments, even though they are important elements of ecosystems and show similar sensitivity to that reported for birds and mammals. Genotoxicity, immunotoxicity and oxidative stress biomarkers provide promising alternatives for measuring the effects of different 2 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. compounds in reptile species, serving as early-warning signals of populations environmentally exposed. Keywords: Model organisms, crocodiles, lizards, turtles, immune system, population genetics, genetic toxicology GENERAL INTRODUCTION To identify an organism as a study model may present limitations. Sometimes, to study a species or a species group of interest, scientist must identify alternative species that still allow for addressing particular questions of interest and experimentation. In every research topic, researchers have to be careful in the selection of animals to be used. This is so important that often the solution to a physiological or pathological problem proposed depends only on the selection of the most appropriate subject of experience, which makes the result more clear, indicative and demonstrative. Alternatives to animal testing are primarily based on biochemical assays or experiments with cells cultures, typically far more sophisticated and specific than traditional approaches but all fail towards the main end goal, to produce information about the more reliable biological effects to transfer the results to make inferences in their natural habitats. Thus, animal models for experimental purposes have countless requirements, some of the more important are: to be reliable as an assessment tool, to allow replication of the results obtained, to show detectable manifestations of the process, predictable effects and the possibility to extrapolate results to other species in experimental situations or make inferences to populations in natural environments. Reptiles are underutilized vertebrate models in the study of most research fields, including genetics, immunology and toxicology. Their unique physiology, indefinite growth, and increasing fecundity across the adult female lifespan motivate the study of how physiology at the mechanistic level, life history at the organismal level, and natural selection at the evolutionary timescale define lifespan in this diverse taxonomic group. They constitute an “ancient” group with physiological mechanisms as convoluted as its behavior, and with particular adaptations that probably explain the evolutionary success of this group over millions of years and its resulting diversity. The way in which reptiles react physiologically and behaviorally in their environments, or under controlled conditions, change in front of any potentially stressful situation, positioning them as interesting models for research. Some studies suggests that reptiles have unusual means of coping with normal energetic stresses (e.g., thermal variation) as well as stresses thought to induce the flight stress response (e.g., predation). Unlike most of the species used in these study areas, reptiles do not typically exhibit short generation times, do not produce many offspring in a short time, and they are usually difficult to keep in captivity for breeding requirements and space. Ironically, the characteristics that make reptiles appear difficult to study actually present scientists with a singular opportunity to create new and ecologically meaningful paradigms of animal models to study environmental and relevant topics. In this chapter, we present different examples of the use of reptile species, including crocodiles, lizards, and turtles, as animal models across different research fields. We specifically present several examples for their utility in genetics, immunology and toxicology. Reptiles Across Research Fields 3 GENETICS OF REPTILES: FOOTPRINTS OF LIVING FOSSILS Introduction In the past two decades genetic studies in reptiles, as well as population genetics in several vertebrates groups, advanced quickly through the use of molecular tools. In particular, reptiles constitute a group of great interest to study genetics because of the conflicts arising constantly in their taxonomy. Besides, they have intrinsic characteristics such as longevity, poor dispersal capability and strong dependence on environmental conditions linked to major problems of species conservation. These features, combined with the limited information available in many of their biological aspects become a focus of attention of many interdisciplinary conservation programs. The study of reptilian genomes is essential if we are to understand the patterns of genomic evolution across amniotes. A better knowledge of several unique biological attributes of this group could contribute significantly to the understanding of basic evolutionary biology and molecular mechanisms of various species, including human health and diseases. Recent technical advances in DNA sequencing in addition to the use of random molecular markers (RAPD- Random Amplified Polymorphic DNA and ISSR: Inter-Simple Sequence Repeat) and specific markers (microsatellite loci and mitochondrial genes) have helped to start exploring the genomes of a variety of species of reptiles, applying the results of these studies to management and conservation programs. In collaborative group, we have focused genetic studies on Argentinean species belonging to different reptilian orders: Crocodylia (crocodiles), Testudinae (turtles) and Squamata (lizards). Crocodilians are one of the most fascinating living vertebrate groups because of their complex biology and behavior, large size and longevity, hierarchy in the zoological scale and successful management systems and conservation applied to their populations. On the other hand, their taxonomic classification is still being argued, and there is lack of data about many biological issues. This is a key group within Reptilian, and crocodilian genome drafts would provide insights into ancestral reptilian and amniotes genomes (St John et al. 2012). In addition to their ecological, sociological and economic significance, crocodilians have genomes that will be useful sources of data for biological and biomedical research. Crocodilians represent important research organisms for diverse fields that include evolution and phylogenetics (Brochu 1997, Li et al. 2007, Hrbek et al. 2008), sex determination (Lang and Andrews 1994, Pieau et al. 1999, Piña et al. 2003), hybridization (Ray et al. 2004, Weaver et al. 2008) and population genetics (Davis et al. 2001, 2002, Wu et al. 2002, Farias et al. 2004, Amavet et al. 2007, 2008, 2009, 2012). Also, recent progress in the development of genomic resources for studies in crocodilians has given impetus to comparative genomics aimed at understanding the evolution and structure of the reptilian genome (St John et al. 2012). We have been working for almost twenty years in genetic studies in the two species of caimans living in Argentina: Caiman latirostris and Caiman yacare. Initially, due to lack of background in Genetics in our regional species, we started our work in a basic discipline within genetic studies: Cytogenetics. Later, due to the fact that it was never reported the existence of hybrids between these species, although they coexist in some areas of the 4 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. country, we considered necessary to further study genetic in these species using molecular tools, which can provide a deeper level of analysis. We started population genetic analyses in C. latirostris using RAPD markers (Amavet et al. 2007). RAPD technique is recognized for their utility in carrying out initial screenings in many loci because of the minimal amounts of prior knowledge about sequences that is required and the chance to distinguish several organisms simultaneously (Lynch and Milligan 1994). We complement the study of genetic variability with a study of quantitative phenotypic traits. This study was possible because of the advantage of being able to obtain morphometric measurements in addition to the genetic material in siblings (members of the same nest) due to the presence of animals in breeding sites with a reliable dial. In addition, the feature of low vagility of these species (i.e. their movements over the territory are not linked to reproduction) motivates them to build nests in the same places always and this enables a representative sampling of the geographical area of nests (Amavet et al. 2009). In parallel, due to the development of specific microsatellite markers for this species (Zucoloto et al. 2002) we studied families (mother and their hatchlings) to analyze the mating system of this species. Data on reproductive behavior of C. latirostris are limited because mating occurs in the water between groups of males and females (which are not easily distinguishable) and unambiguous observations are not often possible (Lang 1989). After building her nest and lay their eggs, the C. latirostris female has a defense behavior of their hatchlings staying on or near the nest site, so that female can be sampled as the mother of this brood more likely. Thus, microsatellite markers are useful in helping to understand mating patterns of C. latirostris because they allow detecting individual compounds genotypes (Amavet et al. 2008). Turtles have been around for nearly 300 million years, but the order of the adaptive advantages became risk factors for them and today represents one of the most affected groups of animals. Their habitats are being intensively degraded, fragmented and contaminated, they suffer overexploitation of poachers, are affected by the introduction of diseases, pests, and exotic species, and by climate change and catastrophic events. Therefore, the data can be gathered about the population and ecological status of these species, as input of information from Genetics area is invaluable to address conservation measures. Our aim is focus on the study of Trachemys dorbigni and Phrynops hilarii, two freshwater turtle’s species widespread in the southern part of the American continent. There were no recent records in Argentina that refer to population parameters, ranges and conservation status of these species, and although they are not considered threatened or endangered, there are reasons to believe that the situation could change. For many years, T. dorbigni (Testudinata, Emydidae) was subject to ongoing taxonomic discussion. Initially it was named Emys dorbigni (Dumeril and Bibron 1835), then adopted different names and is now Trachemys dorbigni (Seidel 1989). For a long time this species was considered a subspecies of Trachemys scripta (Moll and Legler 1971), but on the other hand, several authors considered this as a distinct species (Bickham et al. 2007). Due to limited cytogenetic data of this species in Argentina, as it is subject to pet trade, being confused by the color of his head with a native species of the United States, T . scripta elegans (Cleiton and Giuliano-Caetano 2008), we undertook a characterization of the karyotype of T. dorbigni, providing the basic knowledge of the biology of this native species and allowing the genetic differentiation of individuals exposed to this method of exploitation. Added, due to the lack of studies of genetic variation in these turtle species we proposed methodologies of RAPD and ISSR to initiate these analyses in our country. Reptiles Across Research Fields 5 Research Methods Comparative cytogenetic studies between C. latirostris and C. yacare (Amavet et al. 2003) were performed using extraction of blood from the spinal vein (Tourn et al. 1993) and culture of whole blood to obtain analyzable metaphases. These techniques were adapted specifically for C. latirostris and C. yacare, taking into account previous work in other reptiles and considering the body temperature of these species. The preparations were analyzed by traditional Giemsa staining in addition to C and NOR banding. In turtles karyotypic analyses, we employed fourteen specimens that were bled from the cervical dorsal sinus (Owens and Ruiz 1980) (Figure 1). To obtain metaphases, other technique of whole blood culture was used (Salas et al. 2013). Preparations were stained with conventional Giemsa staining and then treated for C and NOR banding. The morphological classification of the karyotype was corroborated by average arm ratio and centromeric index. In population genetic analyses we employed 233 RAPD markers using 8 primers to obtain data about population structure and genetic variation in 40 specimens from four populations of C. latirostris in Santa Fe province. The PCR products were analyzed by means electrophoresis on polyacrylamide gels (Figure 2). To carry out the morphometric study, eleven allometric measurements were obtained in 160 animals from the same populations within 48 h after birth (Amavet et al. 2009). In T. dorbigni and P. hilarii variability analyses we used RAPD and ISSR methodologies, and employed bioinformatics programs (Genalex and TFPGA) to initiate studies comparing genetic variability and diversity within and between species. We selected three RAPD and four ISSR primers for genetic analysis, and the amplified products of these markers were evaluated by electrophoresis in agarose and polyacrylamide gels (Guidetti 2013). Figure 1. Blood extraction to an adult specimen of T. dorbigni from the cervical dorsal sinus. 6 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. Figure 2. RAPD bands of visualized on a 4% polyacrylamide gel stained with silver nitrate solution. EEE, CSA, EDP, and AES: samples of individuals from four different populations of Caiman latirostris. The vertical arrow on the left shows the direction of electrophoresis. L: Ladder pGem (Promega) (Amavet et al., 2009). Mating system studies were performed by means the sampling of C. latirostris families including the nest-guarding female and the hatchlings of each nest. DNA samples were amplified using four microsatellite primers and PCR products were analyzed on polyacrylamide gels (Amavet et al. 2012). Results and Discussion In karyotypic analyses of caimans we observed that the studied species shared cytogenetic characteristics such as chromosome number (2n=42), formulae and absence of distinguishable sexual chromosomes (Figure 3), between them and with available data about the genus Caiman (Cohen and Gans 1970, Lui et al. 1994). In spite the opinion of Cohen and Gans (1970) it was possible to suggest the occurrence of microchromosomes in these two species. This suggestion was based on the analysis of the two smallest chromosomes of the complement that are very short, with almost indistinguishable shape. Surprisingly, besides the chromosome number and morphology were equal between C. latirostris and C. yacare preparations, so were their C and NOR banding patterns very similar. The obtained results in turtle karyotypes showed a diploid chromosome number of 2n = 50. Chromosomes were classified according to their morphology and this classification was corroborated by average arm ratio and centromeric index (Figure 4). No morphological differences were observed between the karyotypes of males and females. Reptiles Across Research Fields 7 Figure 3. Karyotypes of (a) Caiman yacare and (b) Caiman latirostris. Giemsa staining. T: telocentrics chromosomes; M-SM metacentrics-submetacentrics chromosomes; m: microchromosomes (Amavet et al., 2003). Figure 4. Karyotype of T. dorbigni (M/SM: metacentrics/submetacentrics chromosomes; A/T: acrocentrics/telocentrics chromosomes; MICRO: microchromosomes). Our classification and banding results were different from that published by Cleiton and Giuliano-Caetano (2008) and Martinez et al. (2009). This might be due to different classification criteria and banding methodology used (as these authors did not perform a full 8 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. description of the protocol used) or to real differences between the analyzed karyotypes: Martinez et al. (2009) studied only 4 specimens in Argentina, while Cleiton and GiulianoCaetano (2008) worked with nine specimens sampled in Brazil. We believe that the data are insufficient to compare karyotypes as may be different chromosomal population and / or premises between individuals. In the population genetic analysis of C. latirostris, our results suggested low to intermediate levels of mean heterozygosity (H), mean number of alleles per locus (A), and percentage of loci polymorphic (P). Furthermore, the structure values of gene flow (Nm) and FST seem to show low levels of gene flow and some population subdivision (Amavet et al. 2009). In the phenotypic study we found that some head measurements (WN: maximal width of external nares and IOW: minimal interorbital width) had the highest contribution to morphological differences among populations. This fact could support the role of these traits in reproductive or feeding behavior. Estimated genetic differentiation value (FST) among populations was higher than quantitative trait differentiation value (QST), suggesting a higher contribution of neutral than adaptive loci to the genetic differentiation among populations. Besides, the high heritability (h2) estimates for some traits indicate great potential to improve them in management plans. In particular, TTL (total length), SVL (Snout–vent length), and BM (Body mass) are traits taken into account in the growing of animals in nurseries for commercial use in ranching programs. Owing to their high heritability values their response to directional selection is expectedly high (Amavet et al. 2009). Variability indexes (Heterozygosity and polymorphism levels, Shannon information index, numbers and effective alleles) in turtles were slightly higher in P. hilarii for both types of markers. RAPD and ISSR molecular markers were used for the first time in our country to analyze potentially useful future temporal and spatial distribution of genetic diversity in both species extending working scales. The obtained results in the parentage analysis in twelve C. latirostris families showed that each candidate mother was successfully assigned to their corresponding nest. In the paternity analysis, more than one father was detected in two nests, which could be explained by capacity of storage sperm, proposed in females of a related species. The behavior of multipaternity could contribute to maintain viable populations of C. latirostris, since the maintenance of genetic variability within populations could help increase their capacity to respond to selective pressure (Amavet et al. 2012). We are currently extending these studies to the other caiman living in Argentina, C. yacare using mentioned techniques. TOXICOLOGY: REPTILES AS SENTINELS OF ENVIRONMENTAL CONTAMINATION: EFFECT OF PHYSICAL AND CHEMICAL AGENTS Introduction Since the early 1990s, the concern about the impacts of xenobiotics on biodiversity has been increasing. Wildlife, people, and entire ecosystems around the world are threatened by agents that can endanger survival and physiology of the organisms. Even though there is considerable and growing scientific evidence of damage to wildlife from chemical exposures, researchers are only just beginning to answer the questions about the effects of chronic low- Reptiles Across Research Fields 9 level exposure, mixtures of chemicals, and interactions between chemicals and other physiological and environmental factors (Brown 2003). Chronic exposure to chemicals, even at very low concentrations, may interfere with development and growth, hematological and physiological parameters and genetic stability of organisms (Glusczak et al. 2007). It has become increasingly obvious in recent years that environmental contaminants can significantly affect many species of reptiles (Brisbin et al. 1998). However, these animals are often excluded from environmental contamination studies and ecological risk assessments, even though they are important elements of the ecosystem. They are likely to show any effects associated with environmental contamination (Brisbin et al. 1998) and regarding their biological and ecological characteristics, they can be exposed to contaminants in all life stages. Contaminants accumulated by females can be transferred to the embryos through the egg yolk, affecting embryo development in ovo. In the same way, embryos might be directly affected by contaminants that pass through the eggshell from the surrounding environment during incubation. Juveniles or adults may be exposed to contaminant by the food web, water and sediments typical of the natural environment where they live (Hall and Henry 1992). One of the main challenges of the Environmental Toxicology is to relate the presence of a chemical in the environment with a valid prediction of the resultant hazards for those organisms potentially receptors. Alterations or malfunction of organism normal vital functions can be identified using biomarkers of effect, considered as biologic responses (biochemical, histological, physiological, and morphological changes) to an environmental xenobiotic at the individual level or below, which demonstrates a departure from normal status. Changes at the level of population, community, or ecosystem are not included in this definition, although they are the ultimate concern of ecologists when applying the biomarker concept (IPCS 1993). Of immediate interest and relevance are non-destructive assays that provide a measure of toxic effect in vertebrate species and that can be used in both laboratory and parallel field studies. Non-destructive assays can be performed on blood, skin, excreta, and eggs of reptiles. Also, non-destructive sampling is generally desirable, as it permits serial sampling of individuals and minimizes negative effects on the population under investigation. Although reptiles have shown to be excellent models for studies of association between chemical or physical agents and genetic damage (Sparling et al. 2006, Poletta et al. 2009, 2011a, Capriglione et al. 2011, Schaumburg et al. 2012), they remain to be the group of vertebrates less studied in genetic toxicology. Genotoxic biomarkers, especially nondestructive short term tests, provide a particularly promising alternative of increasing interest and relevance for measuring the potential effects in vertebrate species in both, laboratory and field studies. Among the variety of short term tests developed for detecting DNA damage, the Comet Assay (CA) and the Micronucleus (MN) test are often used since they are fast, convenient and of easy application, particularly interesting as methods to undertake in vivo monitoring studies (Schmid 1975, Singh et al. 1988). Other biomarkers widely used for the study of genetic damage are Chromosome Aberration and Sister Chromatid Exchange. Genotoxicity and Oxidative Stress Induced by Pesticides Many reptile species are being affected as much of their habitat are being intensively degraded, fragmented and contaminated. We have focused our studies on five different reptile 10 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. species living in Argentina. Four of them were previously presented in the first part of this chapter considering Genetic studies: the two crocodilian species C. latirostris and C. yacare, and the two freshwater turtles P. hilarii and T. dorbigni. Toxicology studies include besides, the tegu lizard (Tupinambis merianae), one of the two native species of the genus Tupinambis (Teiidae, Squamata) living in Argentina (Yanosky and Mercolli 1992). All these species share part of their geographic distribution with the same environmental problem. Since the introduction of transgenic crops, and the implementation of non-tillage system (Paruelo et al. 2006, Aizen et al. 2009) there was a fast and continuous expansion of cultivated areas into new regions, leading to deforestation, drainage of wetlands to obtain more lands for agriculture, and contamination of natural environments through increasing pesticides application. The expansion of agricultural frontiers, mainly with soy, onto natural ecosystems resulted in many areas of the natural domain of these reptiles to become closer to areas with high agricultural activity. As a result, wild populations of these species have become continuously exposed to pesticides discharge. In addition, their breeding season occur in the same period of the year of maximum pesticide application (from October to April) as a requirement of agricultural system success. Therefore, a potential contamination risk exists for these reptile species. A great number of studies had demonstrated genotoxic effects of pesticides in vivo in different organisms such as fishes, amphibians, and mammals including humans. However, studies on reptile species were rather scarce and in certain aspects they did not even exist when we began working on this some years ago. In an effort to evaluate the potential risk associated to pesticide exposure undergoing in the natural environments of C. latirostris, different in vivo toxicological studies have been carried out during the last years. We conducted an extended evaluation in different instances including laboratory controlled condition experiments, field-like experiments and a direct evaluation of environmentally exposed populations. Analysis was done in various C. latirostris life stages including embryos, hatchlings and adults, considering different ways of exposure, and through several endpoints (Poletta et al. 2008, 2009, 2011a, 2011b). One of the main mechanisms indicated for pesticide toxicity is Reactive oxygen species (ROS) production and the corresponding oxidative damage may be one of the major causes of impairments in organisms. Studies showed that Roundup® (glyphosate-based formulation) exposure may induce oxidative damage at different tissues leading to lipid peroxidation, alteration of antioxidant capacity and oxidative DNA damage. This was demonstrated for different species of fish (Glusczak et al. 2007, Ballesteros et al. 2009, Modesto and Martinez 2010, Cattaneo et al. 2011) and amphibians (Costa et al. 2008) but no studies were conducted for reptiles. Some authors suggest that ectothermic vertebrates such as reptiles have a low antioxidant capacity that would lead to an unfavorable balance between prooxidants and antioxidants (Voituron et al. 2006). As it happened with genotoxicity assays, there was no previous data on the application of any oxidative stress technique in C. latirostris and only a few reports were found in other reptile species as indicators of natural stressors like extreme cold (Valdivia et al. 2006, Voituron et al. 2006). In crocodilians, studies reported basal values of lipid peroxidation in C. yacare (Furtado Filho et al. 2007) or modifications in the level of TBARS and GST in Alligator mississipiensis from contaminated environments related to heavy metals (Gunderson et al. 2004, Lance et al. 2006). All these studies were applied in tissues from kidney, muscles, gonads or liver, so that animals have to be sacrificed or samples obtained from animals recently dead. Reptiles Across Research Fields 11 Considering our first results in genotoxicity induced by different pesticides in C. latirostris and information reported by other authors about oxidative stress and alterations in antioxidant defense systems in different species, we postulate that these parameters would be good early markers of pesticide effects in wild populations of reptile species. Genotoxicity Induced by Ultraviolet Radiation Basking by ectothermic vertebrates is thought to have evolved for thermoregulation (Allen et al. 1994, Johnson et al. 2008). However, another beneficial effect of sunlight exposure, specifically the UVB component, includes endogenous production of vitamin D. In spite of many positive effects of UV on most organisms, overexposure of animals can generate alterations in DNA directly and indirectly, inducing important mutagenic and cytotoxic lesions. DNA damage induced by UV-A/UV-B exposure occurs mainly by direct excitation of the nitrogenous bases, leading basically to thymine dimers mutations and chromosome fragmentation (Cadet et al. 2005). Alterations most frequently induced are cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pirimidone photoproducts (64PPs) (Pfeifer et al. 2005). Other different photoproducts generated in lesser proportion are purine dimers and pyrimidine monoaducts (Caldwell et al. 1998). UVR can also induced alterations in DNA indirectly through photosensitization reactions, promoting reactive oxygen species (ROS) that oxidate the bases, leading later to DNA breaks (Cadet et al. 2005, Häder and Sinha 2005). As a result of ozone layer reduction in some areas of the world, there has been an increase in UVR incidence on ecosystems, representing a potential hazard to living organisms, including reptiles (Wang and Wang 1999). Also, in caiman captive breeding systems and management programs the influence of natural or artificial light on animal growth has not been studied enough (Príncipe 2007). The change in natural sunlight conditions in these systems can produce alterations in the production of vitamin D3, so additional use of full spectrum and low intensity lamps has been implemented for normal development and organic function under these conditions (Frye 1991, Seufer 1991). Considering these two different situations, we conducted a study to evaluate genotoxic effects of UVA-B/visible light on broad-snouted caiman (C. latirostris), using the MN test as a biomarker. Research Methods Considering that no previous report of the application of genotoxicity biomarkers existed for any of the mentioned species, at the beginning we had to standardize the CA and the MN test, introducing different modifications to the protocols for their application in erythrocytes of C. latirostris (Figures 5 and 6). In the same way, we determined MN and damage index (CA) baseline values in this species, in order to establish its suitability as a sentinel organism for genotoxic monitoring of environmental pollutants (Poletta et al. 2008). The same was made later for C. yacare (Poletta et al. 2010), T. merianae (Schaumburg et al., 2012), T. dorbigni and P. hilarii (Boned et al., 2011), as no report of genotoxicity biomarkers was found in the literature for any of these species. Therefore, we optimized the MN test and CA for these species, in order to determine the baseline values of DNA damage and to propose 12 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. them too as sentinel species to characterize genotoxic effects of different environmental contaminants. Figure 5. C. latirostris erythrocytes stained with fluorescent dye (acridine orange), showing a micronucleus (arrow) (400X). Figure 6. C. latirostris comet images from erythrocytes stained with fluorescent dye (ethidium bromide) showing DNA damage (400X). Reptiles Across Research Fields 13 Then, we conducted two consecutive experiments under laboratory controlled conditions for the evaluation of RU effects. A total of 174 eggs were used receiving different concentrations of Roundup® (RU; glyphosate based formulation): from 50 to 1750 µg/egg at the beginning of the embryonic stage, and then compared with a negative control group (NC) (Poletta et al. 2009). We evaluate embryonic and post-natal development and genotoxicity, analysing size of the animals at birth and later (3 and 12 months) and applying the MN test and CA in erythrocytes of the newborn caimans. Similar experiments were done for the active principle glyphosate (GLY) in order to determine the contribution of the active ingredient to the toxicity of the formulation previously observed. We conducted the same experiment described previously, using GLY concentrations equivalent to those determined as effective for the formulation, considering that the percentage of GLY included in Roundup® Full II formulation is 66.2%. We analyzed genotoxic effects (CA and MN test), development, enzymatic and metabolic endpoints of AST, ALT, LDH, CK, total protein (TP) and albumin (ALB), using commercial kits (Wiener Lab®, Rosario, Argentina) (Poletta et al. 2011b). The effects of immersion-exposure to Roundup® on hatchlings were studied through an experiment maintaining animals during two months in water with concentration similar to those applied at crops. The amount of formulation added to the solution was reduced through time, following a degradation curve for glyphosate in water previously determined (López González et al. 2013). After exposure, once again we analyzed genotoxic, enzymatic and metabolic endpoints. Then, we aimed to model real environmental conditions more closely through a field-like experiment simulating the environmental exposure that a caiman nest can receive in neighbouring croplands habitats, using pesticide practises and concentrations commonly applied in agriculture. Artificial caiman nests were built, eggs placed inside and the area spread with RU formulation or a mixture of endosulfan, cypermethrin and glyphosate formulations. Then we analysed genotoxicity, developmental effects, enzymatic and biochemical determinations in newborn caimans. At present, we are conducting studies to apply oxidative damage biomarkers, together with genotoxic techniques, for the evaluation of wild caiman populations environmentally exposed to pesticides, with the advantage that bleeding causes any damage to the animals. Besides, we are working on standardization of Sister Chromatid Exchange and Chromosome Aberration test to be applied in C. latirostris as new tools for the evaluation of genotoxic effects in this species. Concerning the evaluation of gentoxic effect of UVR, juvenile caimans were maintained under three different treatments: total darkness, 8 and 16 hours of exposure to artificial UV/visible light, during three months. MN test was applied before and after the experiment and the difference in MN frequency was determined (Schaumburg et al. 2010). Results and Discussion MN and CA baseline values for C. latirostris and C. yacare were found to be independent of the clutch of origin, sex and size of the animals, showing that they are quite stable among juvenile caimans, and demonstrating thereby the suitability of these techniques as accurate biomonitoring tools for the evaluation of genotoxic agents. Baseline MN levels 14 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. were comparable to those reported for mammal and bird species considered good sentinel organisms for genotoxic evaluation (Zúñiga González et al. 2000). Then, we observed similar results for T. merianae with the MN test (Figure 7) and CA (Schaumburg et al. 2012) and for P. hilarii with the MN test (Boned et al. 2011). These were the first references to the application of genotoxic techniques in all these species and the first report of the CA for all reptiles (Poletta et al. 2008). These results are very useful as reference values for future in vivo studies to assess the genotoxicity of different agents. Besides, the possibility to take blood samples without causing any damage to these animals (Figures 1 and 8) implies a great advantage for future studies on these native species. Considering that T. merianae is an endemic species to South America, together with the existence of a sustainable use program on it in Argentina (Proyecto Iguana), and the demonstrated sensitivity for the application of MN test and CA, the tegu lizard is being used now as a model for research on the in vivo effect of different pesticides potentially affecting wild populations in their natural geographic distribution (Schaumburg et al. 2012). In experiments under laboratory controlled conditions, we demonstrated genotoxic effect through the MN test and CA in C. latirostris neonates after in ovo exposure to the Glyphosate-based formulation Roundup® (RU; 66.2 % glyphosate), at 500 µg/egg and higher concentrations applied directly on the eggshell (by topication). A concentration-dependent effect was observed, showing an increment in DNA damage as RU concentrations increased (Poletta et al., 2009). Data on the size of animals demonstrated that no effect at the moment of hatching, but the increment in growth of animals exposed to the herbicide formulation was less in comparison to the controls in the first months of caiman life, showing detrimental effect on post-natal development of the caimans exposed in ovo. Figure 7. T. merianae micronucleated erythrocytes (arrow) stained with Giemsa (1000X) (Schaumburg et al., 2012). Reptiles Across Research Fields 15 Figure 8. Blood extraction to an adult of T. dorbigni from the caudal vein. Data of caiman exposure to the active principle alone demonstrated an increase in DNA damage at all concentration of GLY tested, an increase in the enzymatic activity of CK, AST, LDH and ALP and a decrease in TP, as well as a lower weight of the exposed animals at three months of life compared to NC. These results would indicate that the contribution of the active ingredient to the toxicity of the formulation Roundup® previously observed (Poletta et al, 2009) is also relevant. Similar results were obtained in genotoxicity and growth of neonates maintained during two months in water (in a semi immersion system) with concentration of RU similar to those applied at crops (López González et al. 2013). However, no significant enzymatic alterations were observed, even though ChE showed less activity in both treatments compared to control (Poletta et al. 2011b). These results showed that hatchlings remaining in small water bodies near agricultural areas during their first months of life are also under risk, as they can receive pesticide residues by run off from different surroundings. In field-like experiments again we found biochemical alterations, genotoxicity and effect on growth in caimans exposed to the mixture of glyphosate (66.2 %), endosulfan (35 %) and cypermethrin (25 %) formulations, as well as in those exposed to the single formulation Roundup® (Poletta et al. 2011a). The pesticides were tested as the complex commercial mixtures due to the fact that this is the form in which they are routinely applied in agriculture and introduced into the environment. Environmental factors that lead to a period of reduced growth can be particularly important during the juvenile period, when animals growth at a maximum rate. Growth reductions at this moment would result in a longer period of risk of size-dependent mortality (Mitchelmore et al. 2005). In natural environments this situation could be worse by repeated pesticide exposures and unfavourable conditions produced by cold in reptiles that include poor nutritional state on animals (Larriera et al. 2008). 16 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. Figure 9. Increase in MN frequency (final-basal MN values) in different treatments (mean±S.D.) TD: total darkness, T8: 8 h UV/visible light exposure, T16: 16h UV/visible light exposure. Number of animals per treatment: 24. *Significantly different respect to TD treatment (Mann–Whitney U-test) (Schaumburg et al., 2010). Caimans can be exposed to pesticides as embryos and neonates (December-March), later, these animals can receive new successive exposures at 8-12 months old, moment in which extensive fumigations start again (November-March) (Paruelo et al. 2006). Under these conditions, it is difficult to ensure that animals can recover at short term from genetic damage, enzymatic and metabolic disorders, and growth retardation. During the last year, we have successfully adapted techniques to detect oxidative damage to lipids (TBARS), DNA (CA modified with lesion-specific enzymes, Azqueta et al. 2009) and antioxidant enzymatic (CAT, SOD) and non-enzymatic (GSH/GSSG) systems for their application in C. latirostris blood. In experiments evaluating the effect of UVR on DNA damage of C. latirostris, we observed a significant increase in MN frequency in all treatments compared to basal values (before experiment), but there were differences in the increase depending on treatment. Animals exposed to UV radiation (UVR) presented a higher increment in MN frequency compared to total darkness treatment (Figure 9). These results provide relevant information about the possible harmful effects generated by sub-chronic exposure to UVR in zoos, reptile hobbyist and breeding programs, as well as the deleterious consequences of increased UV environmental impact on wild species as the broad-snouted caiman. IMMUNOLOGY: THE REPTILIAN IMMUNE SYSTEM AND ITS COMPONENTS Introduction From protozoa to humans, organisms have immune defence strategies that ensure the ability to react against foreign microorganisms and molecules, to counter any attempt to alter the homeostatic balance (Cooper 2002). Immune responses have evolved from innate Reptiles Across Research Fields 17 mechanisms to the most elaborated adaptive to recognize and discriminate between self and non-self as central aspect to the integrity of the host. All invertebrates and vertebrates studied have shown a broad range of immune responses that can recognize and eliminate foreign material. Animals are exposed daily to millions of potential pathogens through contact, ingestion, and inhalation (Hancock and Scott 2000). Immunity to infection is mediated by two general systems, innate (or natural) and acquired (or adaptive). Such mechanisms have evolved and diversified in response to many factors, including the environments in which organisms live, body complexity, distinct physiology, and lifespan (Zarkadis et al. 2001). This multiplicity of mechanism is a consequence of the importance of the task to be accomplished and there are alternatives when some of the mechanisms do not work properly. Due to the lack of ability of the innate immunity systems to recognize specifically pathogens and to provide specific responses, organisms develop adaptive immune responses. Dendritic cells and macrophages ingest microorganisms and present antigens derived therefrom to T lymphocytes. This triggers a clonal expansion of T cells specific for each antigen (Ulevitch et al. 2004). In turn, the cytokines and chemokines of T cell regulate B cell proliferation and antibody production, as well as the activity of cells of innate immunity systems. The intercommunications between innate and acquired facilitate a robust and flexible immune response. The adaptive immune system, with its randomly generated and vast antigen-receptor diversity, allows organisms the prospect of evading microbial insults (Flajnik and Du Pasquier 2004). In contrast to adaptive immunity, which is restricted to vertebrates, innate immunity is more ancient and is used by invertebrates such as insects and echinoderms, as well as by higher animals (Song et al. 2000). Components of the innate immune system are markedly conserved between insects and mammals, indicating a common ancestral origin for this branch of immunity (Hoffmann et al. 1999). The importance of knowledge of the immune system from different phylogenetic positions is crucial to understanding the evolution of the system and the relationship with other factors. The deepening of such knowledge may be useful, by exploiting natural resources in a differential or alternative for the production of pharmaceuticals, biological model generation, production of nutritional supplements and application of resources to prevent or solve problems environment. Reptiles have been shown to have immune components with an apparently higher activity than others animals, even humans. The ability to resist infection with serious injuries in places with high concentrations of pathogen microorganisms, without signs of illness, makes them interesting models for the elucidation of those mechanisms underlying immune responses. Immunological effector mechanisms necessary for the efficient control of an infectious agent are dependent on the distinct routes. Some reptiles components involved in these routes of the defense system were identified and characterized for our and other research groups. Amongst them, it can be mentioned serum complement cascades (Merchant et al. 2003, Siroski et al. 2009), dipeptidilpeptidase (Merchant et al. 2009b, Siroski et al. 2011), white blood cell (Siroski 2011, Latorre et al. 2013) phospholiphases (Merchant et al. 2009a, 2011, Siroski et al. 2013) and chitinases (Siroski et al. in consideration) and they are being detailed in this chapter. As a result of the findings of molecular research, the evolutionary origin of the complement system found that it is older than what was known (Nonaka and Kimura 2006), which emerged at least 600-700 million years ago, long before the appearance of 18 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. immunoglobulins (Sunyer et al. 1998). The serum complement is an important element of the innate immune defense of animals against infectious agents. It appears to be highly conserved in vertebrates, although research on reptiles and amphibians is scarce. The complement system consists of a group of plasma proteins that play critical roles in host defense by interacting with components of both the innate and adaptive immune systems. It represents an important arm of the innate system in vertebrates and invertebrates (Song et al. 2000), which can be sequentially activated in a multi-step cascade reaction by classical, alternative, and lectin pathways (Götze and Müller-Eberhard 1976, Loos 1982). Studies showed that the complement system, as part of the innate mechanism of poikilothermic vertebrates, is more diverse than that of higher vertebrates, so a broader range of antigens can be recognized (Sunyer et al. 1998). Poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of homeothermic vertebrates (Zarkadis et al. 2001). In some ectothermic species, the serum complement system is present in multiple forms, and researchers have hypothesized that this serum complement diversity has been used as an evolutionary strategy to expand innate immune recognition capabilities (Sunyer et al. 1998). Peptidases Membrane peptidases are a group of ectoenzymes with a broad range of functions. Their importance in protein metabolism has been well-documented, especially in peptide degradation and amino acid scavenging (Scharpé and De Meester 2001). Dipeptidyl peptidase IV (CD26/DPPIV) can be considered a multifunctional protein because it exerts different functions depending on cell type and intracellular (mainly in the membrane of cells) or extracellular (enzymatically active plasma isoform) locations (Boonacker and Van Noorden 2003). Based on its multiple functions, DPPIV has been proposed as a diagnostic or prognostic marker for various tumors, hematological malignancies, viral infections, as well as immunological, inflammatory, and psychoneuroendocrine disorders (Lambeir et al. 2003). The in vivo expression of DPPIV in epithelial, endothelial and lymphoid cells is compatible with its role as a physiological regulator of a number of peptides that serve as biochemical reporters between, and within, the immune and neuroendocrine systems (Ludwig et al. 2002). DPPIV, which is identical to the lymphocyte surface glycoprotein CD26, is unique among these peptidases due to its ability to liberate Xaa-Pro and, less efficiently, Xaa-Ala dipeptides from the N-terminus of regulatory peptides (Boonacker et al. 2002). It has an important role in immune function as a co-stimulatory molecule in T cell activation and as a regulator of the functional effect of selected biological factors. Its well-characterized protease enzymatic functionality is related to the activity of many bioactive peptides, with broad substrate specificity for which at least 62 natural substrates have been identified (Chen 2006). Based on its multiple functions, DPPIV has been proposed as a diagnostic or prognostic marker for immunological and inflammatory disorders. Few studies have been focused on reptilian DPPIV and most of them are limited to snakes. DPPIV substrate specificity, susceptibility to inhibitors, and optimum pH of the partially purified enzyme was investigated in venoms glands (Ogawaa et al. 2006), seasonal variation of peptidase activity was studied in the reproductive tract (Marinho et al. 2009), and the taxonomic distribution of DPPIV activity was examined in venoms of 59 ophidian taxa Reptiles Across Research Fields 19 (Aird 2008). Recently, works in crocodilians detected and characterized DPPIV activities in the plasma and whole blood of the American alligator (Alligator mississippiensis; Merchant et al. 2009), and activities of the soluble form of DPPIV in C. latirostris and C. yacare plasma (Siroski et al. 2012). These activities were much higher compared with murine in vitro activity (Kubota et al. 1992), pig (Faidley et al. 2006) and human serum (Lefebvre et al. 2002, McKillop et al. 2008). Based on the DPPIV immunological functions previously noted, this could be related to the efficiency of the crocodilian immune system (Merchant et al. 2003, 2004, 2005b, Siroski et al. 2009), showing another advantage of this successful group of ancient vertebrates. The variations found in plasma DPPIV activities between these crocodilians could be one of the reasons for different susceptibility to infection observed in these species. Phospholipases The recognition of pathogens stimulates the synthesis of inflammation mediators called eicosanoids. These mediators are derived from arachidonic acid (AA), a polyunsaturated omega-6 fatty acid that organisms can incorporate directly through the diet, or synthesize from linoleic acid (Balsinde and Winstead 2002). The AA never circulates in the free acid form, but is esterified to the sn-position of membrane glycerophospholipids. Therefore, prior to the synthesis of eicosanoids, the AA has to be released from phospholipids. The enzymes involved in that release are phospholipases A2 (PLA2) (Figure 10) (Astudillo et al. 2009). Figure 10. Schematic diagram of a phospholipid (i.e. phosphatidylcholine) hydrolysis caused by various phospholipases. PLA1, phospholipase A1; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; R1/R2, variable fatty acid residues (Budnik and Mukhopadhyay, 2002; Siroski et al., 2013). 20 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. Consequently, PLA2s are important enzymes involved in signaling, as they regulate the generation of specific types of secondary lipids messengers with significant roles in innate immune response. Although messenger pathways that mediate the production of eicosanoids by cells involved in innate immunity are not well characterized, all of these groups have been implicated in various types of lipid metabolism and in the progression of several diseases. Given the clinical importance of PLA2 enzymes, they are of interest for pharmaceutical industry and biotechnology, regarding the development of selective and potent inhibitors of each of these phospholipases (Burke and Denis 2009). A number of agents that exert effects on cellular receptors of the innate immune system emit a series of signals leading to an increased activity of PLA2. The bactericidal effect is based on the hydrolysis of the phospholipid component of bacterial cell membrane by PLA2 (Nevalainen et al. 2009), which perturbs membrane trafficking and modulates intracellular bacterial growth (Mansueto et al. 2012). Phospholipase A2 enzymes are toxic components detected and characterized in the venoms of some reptiles, including most venomous snakes (Kini 2003), Heloderma lizards (Vandermeers et al. 1991) and members of the Varanidae family (Fry et al. 2006). Recently, PLA2 has also been described in crocodilian plasma. Merchant et al. (2009) demonstrated its presence in A. mississippiensis, and Nevalainen et al. (2009) in Crocodylus siamensis, C. porosus and C. siamensis hybrids with C. porosus. These enzymes have also been reported in the plasma of C. niloticus, Mecistops cataphractus, and Osteolaumus tetraspis (Merchant et al. 2011), and by our group in the two caiman species living in Argentina, C. yacare and C. latirostris (Siroski et al. 2013). Chitinases Chitinases enzymes, which are essential for maintaining normal life cycle functions such as morphogenesis of arthropods (Merzendorfer and Zimoch 2003) or cell division and sporulation of yeast and other fungi. Chitinolytic enzymes have also been found in organisms that do not contain chitin polymers themselves, such as viruses, bacteria, plants and animals (Badariottia et al. 2007). Indeed, chitinases have also an important role in parasite invasion of chitinous hosts. Several studies have demonstrated that chitinases are also produced constitutively, or inducibly, as pathogen resistant proteins; thus vertebrates synthesize chitinases to defend themselves against chitin-containing pathogens such as protozoa, fungi, insects and nematodes (Renkema et al. 1995, Gooday 1999). Chitotriosidase enzyme glycosyl hydrolase, is one of the main proteins secreted by activated macrophages (Hollack et al. 1994). Macrophages overloaded with the enzyme accumulated in lysosomal material (lipids) were shown to secrete chitotriosidase (CHT). In contrast, low levels of CHT have been associated with susceptibility for infection by chitin containing parasites (Bussink et al. 2006). In fact, many studies were performed to detect chitotriosidase activities towards chitincontaining pathogens both in vitro and in vivo (van Eijk et al. 2005). Due to the possible role of CHT enzymes in the generation of the reptiles innate immune system, the presence of this enzyme in caiman plasma, and characterize its activity under different biochemical conditions. There are few studies of chitinase activity in other reptiles. The genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta (Jeuniaux 1961, 1962, 1963); and only two of New World lizards, Anolis carolinensis (Jeuniaux 1962, Reptiles Across Research Fields 21 1963) and Sceloporus undulatus garmani (Marsh et al. 2001) have been reported to secrete chitinase. These studies have shown that some vertebrates produce chitinase in the whole digestive tube with the aim of metabolizing as chitin as a food source (Karasov 1989). Thus, the production of chitinase would allow them to utilize the chemical potential energy in their food more efficiently, but it is not necessarily related to chitotriosidase. Based on the importance and functions of CHT enzyme, and the variation of these parameters as useful tools to distinguish normal and diseased conditions, and the high concentrations detected in caiman plasma, it should be of interest to continue increasing the knowledge about their properties and homologies. The chitotriosidase enzyme levels should be evaluated as a possible application in the veterinary clinic as a biomarker of individual health status, as well as in the study of immune phylogenetic mechanisms. Immune Mechanisms as Biomarkers Components of reptilian immune system mentioned were used as indicators of exposure to physical and chemical agents (ultraviolet radiation, Siroski et al. 2011, pesticides, Latorre et al. in press). All these features may converge in alter the antimicrobial properties detected in different crocodilians tissues (Shaharabany et al. 1999, Merchant et al. 2003, Siroski et al. 2009). Immunotoxixity of Ultraviolet Radiation Reptilian husbandry is important to hobbyists and zoos, where many species are raised as components of conservation projects, as well as in farming ventures that raise them for pets, food, and raw materials. Reptiles are also used as laboratory models in bioscience research (Brames 2007). In conscientious husbandry programs, keepers must replicate the UV spectrum required for the photochemical process involved in the synthesis of vitamin D. UVB radiation is involved directly in vitamin D synthesis, which acts to regulate calcium and phosphorus absorption, both implied directly in the metabolism related to bone growth and maintenance (Lian et al. 1999). If the correct UV range is not provided, calcium absorption will not be optimal (Brames 2007) no matter what kind of diet, temperature, etc., is supplied. Many reptile keepers and managers supply UV light to animals in order to offer or complement UV radiation (UVR) with the aim to mimic sunlight in the wild. Reptilian husbandry lamps contain three kinds of light spectrum: visible, UVA, and UVB. With respect to the visible light, it has been demonstrated to cause no suppression of the immune response (Kubasova et al. 1995), but instead, may directly modulate immune function; on the contrary, UVR has been shown to produce negative effects on the immune system (Roberts 1995). Ilyas (1986) and Jeevan and Kripke (1993) emphasize that damage to the immune system due to UVB could have far-reaching effects for the health of populations. The immunosuppressive properties of UVR are of major biological and clinical relevance (Schwarz 2005). More research has been focused on the effects of UV irradiation on cellular than on humoral immune system (Hersey et al. 1983). However, Artyukhov et al. (2007) established that UV light can modulate the activity of the serum complement system. 22 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. Few efforts were made to investigate the effects of UV exposure on wildlife. Despite the fact that serum complement system is a relevant component of innate immune response in reptilians; some studies on reptiles have showed that sunlight is more convenient than artificial light to obtain a better growth of animals under captivity. Sunlight provides the suitable quantity and quality of UVR for organism to develop vital process (Ferguson et al. 2005, Karsten et al. 2009). Our study revealed that hemolytic activity decreased when caimans were exposed to different time periods of artificial UVR and total darkness (Siroski et al. 2011) and lowest activity was measured in serum from animals exposed to longer periods of UVR. The reduction in serum complement system activity at long exposure per day of artificial UV/visible light demonstrates a UVA-B dose-dependent response, which might affect the immune system of captive herpetofauna. Similar reports were obtained in mice using experimental skin lesions, in which long UVR is the major factor responsible for this pathology (Natali et al. 2005). The benefit of artificial light as a UV supplement for captive herpetofauna depends on appropriate photoperiod and UV wavelength exposure (Caliman Filadelfi et al. 2005). UVR can cause innate immune activity alteration, which could possibly lead to the impaired response of exposed individuals to infections, increasing their incidence and making them more lethal (Leaf 1993).The artificial UV should be considered a potential hazard if it is not properly managed and this should be taken into account by those dedicated to reptilian husbandry and maintenance facilities design. Sunlight exposure at normal photoperiod seems to be the better alternative for providing proper amount and quality of UVR to caimans in captivity. Anyway, if artificial UV light is required, it should be combined with shaded area in order they can self-regulate exposure (Siroski et al. 2011). Immunotoxicity of Pesticides The reptilian immune response is profoundly affected by ecological factors, including population dynamics, stress, nutritional state, environmental temperature, seasonal variations, age, and infectious pathogens. The dramatic effects of seasonal changes and related steroid fluctuations have been the topic of many studies on the reptilian immune system (Zapata et al. 1992). Reptilians, due to their occurrence in a wide variety of habitats, their position at a higher level of the food chain, permanence in aquatic bodies, wide geographical distribution, longevity, and fairly restricted home ranges, are especially vulnerable to pesticide exposure (Campbell 2003, Beldoménico et al. 2007). Different studies have shown that many pesticides are immunotoxic as they can alter the structure of some components of the immune system and, in turn, generate a lower host resistance to antigens/infectious agents (Christin et al. 2003, Dorucu and Girgin 2001, Mansour 2004). A first study made by our group demonstrated that newborn C. latirostris exposed to glyphosate (Roundup®, RU) showed a decrease in the response of their complement system and altered leukocyte counts. Therefore, the activity of the immune system can be used as an indicator of toxicities induced by pesticides and, potentially, by other environmental factors (Siroski 2011). In a second study, we exposed two groups of caimans during two months to different concentrations of RU (taking into account the Reptiles Across Research Fields 23 concentration recommended for its application in the field), while one group was maintained as control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. Animals were measured and weighed at the beginning and end of the experiment, and blood samples taken to determine total and differential white blood cell (WBC) counts as well as total protein concentration (TPC), and for performing protein electrophoresis. The results showed that, compared against control hosts, there was a decrease in WBC counts, a higher percentage of heterophils, a higher TPC (with a low percentage of F2 protein fraction), and a negative effect on growth in the young caimans exposed to RU. These results demonstrate that in vivo exposure to RU induced alterations in the selected immune parameters, plasma proteins, and growth of caimans, thereby providing relevant information about the effects of this type of pesticide in this important species in the Argentinian wetlands (Latorre et al., in press). Traditional hematological parameters may provide information about the general state of an individual, where an increase in the heterophil/lymphocyte (H/L) index is a common response to stress caused by different factors, especially in birds and reptiles (Morici et al. 1997). Plasma proteins help maintain circulating fluid volume and assist in the inactivation of toxic compounds and defense against pathogens. Alterations in total protein levels occur during pathological conditions, including exposure to xenobiotic agents. As such, determination of total protein (levels and profiles) commonly used as an end-point of overall health of an organism. The total protein levels were similar to those reported by other studies in wild and they consider this an interesting end-point for further investigations in ongoing and future studies, i.e. to determine the particular components affected and potential consequences. Finally, environmental factors that lead to a period of reduced growth can be particularly important during the first months of life when animals grow at a maximum rate to attain a size at which certain predators can be avoided. Thus, reductions in growth rates can serve as a biomarker of stressful environmental conditions, although mechanisms responsible for the reductions may not be immediately evident (Mitchelmore et al. 2005). The importance of knowledge of the immune system from different phylogenetic positions is crucial to understanding the evolution of the system and the relationship with other factors. The deepening of such knowledge may be useful, by exploiting natural resources in a differential or alternative for the production of pharmaceuticals, biological model generation, nutritional supplements production and application of resources to prevent or solve environment problems. Research Methods The susceptibilities of microorganisms to antibacterial substances are often determined in the lab by measuring the inhibitory capacity of such substances. In this case, a standard in vitro bactericidal assay that measured the antibacterial activities of C. latirostris, G. gallus, and human plasma against Escherichia coli (ATCC 11105) was made at different time intervals. E. coli bacteria were incubated with human and animal plasma. The E. coli colonies were suspended in a sterile saline solution and mixing, with plasma sample. At different times (0, 1, 3, and 6 h), then they were spread on a nutrient agar surface in Petri dishes and incubated overnight at 37°C. Plates containing 30-300 colony forming units (CFUs) were utilized for analyses. 24 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. The sheep red blood cell (SRBC) hemolysis assay was developed to detect complementsystem activity. These assays were used herein to detect and characterize the serum complement activity of C. latirostris. Fresh SRBCs were obtained from heparinized whole blood collected from Merino sheep (Ovis aries) and it was washed with phosphate-buffered saline (PBS, pH 7.4) several times until the supernatant was clear, and then a 2% SRBC (v/v) solution was prepared. To characterize the C. latirostris complement system, we used the SRBC hemolysis assay. This assay is based on the hemolytic disruption of SRBCs by means of the immunological proteins of the serum. Frequently, it is used to evaluate complement systems in clinical laboratories (Kirschfink and Mollnes 2003), and it was adapted to crocodilian serum by Merchant et al. (2006). The hemolytic assay was performed to detect complement-system activity of broad-snouted caiman serum, including 2 classical inactivators of the serum complement, heat and ethylenediamine tetraacetic acid (EDTA). Another assay was performed to evaluate the effects of temperature, plasma concentration and the time of plasma exposure on the complement system of broad-snouted caiman serum. To characterize caiman DPPIV activity, we used the assay adapted from American alligator plasma and whole blood by Merchant et al. (2009). This assay is based on the effects of DPPIV on the hydrolysis of Ala-Pro-AFC. Assay buffer to initiate the reaction (100 mM Tris HCl, ph 7.4) and stop buffer to finished it (100 mM Tris HCl, ph 7.4, 15 mm EDTA), were used. DPPIV requires divalent cations (Ca2+ and Mg2+) to exert its functions. Based on its activity as chelant of divalent cations, stop buffer includes EDTA as a common protease inhibitor (Ghersi et al., 2006). The effects of temperature on C. latirostris and C. yacare DPPIV plasma activity, mixture incubation was carried out at different temperatures (5◦C to 40◦C, at 5◦C intervals). To study the concentration-dependence of DPPIV plasma activity, different volumes of caiman plasma. Kinetic parameters of DPPIV plasma activity were determined at different time intervals. The presence of DPPIV activity resulted in cleavage of AFC from the dipeptide, producing large increases in fluorescent intensities that were measured spectrofluorimetrically. PLA2 determination assay was developed to measure the secretory PLA2 enzyme activity in plasma. It is used fluorescent BODIPY® to label a fatty acid that binds occupies the sn-2 position of phospholipids. Bacterial cultures add these macromolecules as component of the outer membrane during growth and proliferation. When bacteria are exposed to the activity of the PLA2 enzyme, the labeled sn-2 fatty acid is hydrolyzed from the membrane and released into the assay buffer. After a brief centrifugation, the pellet formed contains bacteria with the labeled fatty acid in the membrane, while the supernatant has free labeled fatty acid that can be measured by spectrofluorometry. The effects of temperature, concentration, and time dependence on Caiman PLA2 enzyme activity plasma dependence assay activity, were evaluated. Results and Discussion The likelihood that microorganisms cause illness in crocodilians increases under stressful conditions (Shotts et al. 1972, Brisbin 1982, Franklin et al. 2003). Such stressful conditions include extreme temperatures, capture and restraint, increased population density, an inappropriate diet, etc. Crocodilians and others reptiles are exposed to such conditions more frequently when they are in captivity than when they are in the wild. Reptiles Across Research Fields 25 Many studies have been carried out to detect antimicrobial activities in a number of products derived from animal and vegetable sources (Marshall and Arenas 2003). Previous work on immunity in ectotherms was intended to compare their resistance to bacterial pathogens to those of humans and other mammals (Manning and Turner 1976, Merchant et al. 2003). Shaharabany et al. (1999) were the first to demonstrate antibacterial activities in tissues of Crocodylus niloticus and a variety of wild and domestic birds. Recent studies showed antimicrobial serum activity in other crocodilians (Merchant et al. 2003, 2004, 2005a, b, Merchant and Britton 2006). In this study, antibacterial activity against E. coli ATCC 11105 was detected in all plasma samples, but different sensitivities were shown depending on the species and time. C. latirostris plasma exhibited time- dependent inhibition of E. coli. At the same time, the caiman antibacterial activity was consistently superior to those of human and hen plasma. Only caiman plasma had completely inhibited E. coli growth at 6 h of exposure, thus showing rapid bactericidal activity. Human plasma exhibited less of an effect than hen plasma on bacteria viability, except at 3 h of exposure, at which time, there was no significant difference. The antibacterial assays used in this work were a simple but valid method for studying plasma antimicrobial activity independent of bactericidal origin. Many researchers reported the presence of complement system serum in reptiles (Koppenheffer 1987, Sunyer et al. 1998). Mastellos et al. (2004) described the complement system as a phylogenetically conserved arm of innate immunity. Carey et al. (1999) included the complement system as a part of the innate defense mechanisms in urodele and anuran amphibians together with antimicrobial peptides, natural killer cells, and phagocytic cells. The incubation of broad-snouted caiman serum with SRBCs resulted in hemolytic activity determined by an increase in the optical density at 540 nm due to the release of hemoglobin by disrupted erythrocytes. Observations of similar findings in normal human (Morgan 2008), American alligator (Merchant et al. 2005b), and freshwater and saltwater crocodile serum (Merchant and Britton, 2006) strongly suggest definite physiological significance of the serum complement system in broad-snouted caiman serum in terms of protection against various pathogens. The hemolytic activity of C. latirostris serum toward SRBCs was characterized in terms of dependence on the concentration, temperature, and kinetics. The hemolysis of SRBCs by broad-snouted caiman serum in vitro depends on the temperature at which it is incubated. The complement system was implicated in the pathogenesis of several disorders. Frequently, hemolytic assays are used in clinical laboratories to evaluate the complement system and identify human patients with complement deficiencies. The relationship of the hemolytic activity of C. latirostris toward non-sensitized SRBCs was generated on the basis of the data obtained from simultaneous serum titrations carried out with 11 different serum concentrations. The incubation of broad-snouted caiman serum with SRBCs showed a time-dependent reaction. When caiman serum was exposed to SRBCs, hemolysis steadily occurred, with measurable activity at 2 min which increased to a maximum of 86% SRBC hemolysis at 60 min. These observations can be considered a rapid response of the immunological properties of caiman serum to avoid any challenge from microbial infection. The serum complement system in some ectothermic species is present in multiple forms, and researchers hypothesized that this complement diversity is used by the species as a strategy to expand its innate immune recognition capabilities (Sunyer et al. 1998). From our 26 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. results, we could not distinguish if the hemolytic activities were due to classical or alternative pathways. This study indicated that the mechanisms of serum complement activities for diverse crocodilian species studied are similar. The foregoing data highlight the significant contribution of the complement system to immunological activities of this species. Crocodilians represent an extremely successful group of organisms that have changed little for millions of years. The serum complement system could be a very important innate immune component in the resistance to attack by micro-organisms. DPPIV activity showed a significant positive correlation based on the data obtained from titrations of different plasma concentrations. Peptidase activity showed concentrationdependent activity for both C. latirostris and C. yacare plasma. These activities were much higher compared with murine in vitro activity (Kubota et al. 1992), and pig (Faidley et al. 2006) and human serum (Lefebvre et al. 2002, McKillop et al. 2008). Similar results were found in American alligator (Merchant et al. 2009) but the values detected in whole blood were higher than in plasma at all titrations, which was attributed to the presence of DPPIV on the surface of T-cells (Mentlein et al. 1984). Previous studies indicated that environmental temperature plays a fundamental role in the physiology of ectothermic vertebrates, including antibody formation and immune response (Klesius 1990), and specifically in crocodilians, affecting sex determination and clutch size (Simoncini et al. 2008), as well as components of innate immune system (Merchant et al. 2005a, Siroski et al. 2010). Crocodilians prefer to maintain body temperatures within a narrow range of 28-33 ºC by using thermal gradients in their natural environment: sunshine and shade, warm surface and cold deep waters (Huchzermeyer 2002). Temperature influenced DPPIV activities in caiman plasma, and this influence seems to be more pronounced in C. latirostris than in C. yacare. At low temperatures, both species showed very low activities and coincide with the fact that ectothermic vertebrates’ metabolisms slow down to a minimum (Johnson et al. 2008). Maximum DPPIV activities were detected at those temperatures in which crocodilian metabolic processes, including immune responses, are optimal, as demonstrated by Glassman and Bennett (1978). The kinetics of caiman plasma DPPIV activity was similar to that observed in vivo in mice (Kubota et al. 1992), and the values detected in this study were similar to those found in American alligator (Merchant et al. 2009). According to Merchant et al. (2009), the increment in caiman DPPIV activity with time could be due to the enzymatic activity requiring a certain time to achieve its catalytic efficiency by the same level of protein. There is much evidence for a role of DPPIV in the regulation of the immune response which focuses on the putative role of the catalytic domain (Kahne et al. 1999, Ludwig et al. 2002) among other functions. The results from this study demonstrate and characterize plasma DPPIV activity in two species of caiman, and show that they are similar to that of American alligator (Merchant et al. 2009), probably due to their common phylogenetic origin. Based on the DPPIV immunological functions previously noted, this could be related to the efficiency of the crocodilian immune system (Merchant et al. 2003, 2004, 2005b, Siroski et al. 2009), showing another advantage of this successful group of ancient vertebrates. Using the spectrofluorometric technique described, we detected PLA2 activity in plasma of C. latirostris and C. yacare. Recently, Merchant et al. (2009) conducted a similar assay adding different proportions of bacteria without the fluorescent marker to demonstrate the specificity of the reaction. Given a fixed amount of A. mississippiensis serum, a reduction in fluorescent activity was observed when the amount of unlabeled bacteria in the solution Reptiles Across Research Fields 27 increased. The catalytic activity of PLA2 competed with labeled and unlabeled bacteria, reducing the amount of fluorescent product formed depending on the concentration of labeled substrate. Another study found the presence of PLA2 in the serum of two species of crocodiles, C. siamensis and C. porosus (Nevalainen et al. 2009). The enzymatic activity was dependent on the incubation temperature during the reaction assay. At low tem peratures (from 5 to 15°C), the enzyme activity in the plasma of C. latirostris was higher than in C. yacare. This difference may be attributable to the greater climatic tolerance of C. latirostris (Siroski et al., 2004). At low temperatures (less to 20°C), the plasma PLA2 activity of C. latirostris was higher than that of C. yacare. These results could be related to the average temperature range in the latitudes at which these species live. C. latirostris has a wider geographical distribution in South America than C. yacare. Both are distributed in northern Argentina, but the range occupied by C. latirostris extends much farther south than C. yacare. In this range, there are important differences with respect to latitudinal temperatures. The average temperature during the coldest months (June-August) in the southern limit for the range of the C. yacare population (30°S latitude) (Verdade 1998), is about 17°C. In the case of C. latirostris, the southern distribution limit occurs at 32°S (Verdade 1998), where the average temperature during the same months is approximately 10°C (Servicio Meteorológico Nacional 2010). Although these data are not sufficient to make any kind of statement, we consider them, in addition to those previously made in our laboratory (Siroski et al. 2011) and future studies related, will allow us to elucidate if these enzyme activities may be the result of an adaptation of the immune system, along with other physiological activities, at these temperatures. The effect of volumes of caiman plasma with constant amounts of the labeled bacteria solution was associated with increased PLA2 activity. At the very beginning, small amounts of plasma produced a significant increase in PLA2 activity, higher in C. latirostris plasma, which repre- sents approximately 40% of the maximum observed. These results are consistent with similar findings in other species of crocodilians (Merchant et al. 2009, Nevalainen et al. 2009) but none of these studies detected whether the plasma contained high amounts of PLA2 or increased activity when it appears in moderate amounts. This ability gives this technique an advantage, as reproducible results with very low variations can be obtained with only small volumes of plasma. In addition, future studies of PLA2 activities time and concentration dependence at low reaction temperature (15°C, approximately) would be performed to contribute the controversial idea about the physiological activities adapted to geographical factors. Another important feature of PLA2 in caiman plasma is the reaction rate. Within five minutes of the exposure of bacteria to plasma, high activity was demonstrated for both species, but was higher in C. latirostris plasma at all times studied. These results coincide with those observed in similar studies made with dipeptidyl peptidase enzymes (DPPIV) (Siroski et al. 2011). CONCLUDING REMARK Throughout this chapter we have presented examples of how reptiles are interesting models for research in genetics, toxicology and immunology, giving valuable information concerning phylogenetic, biological model generation, application of resources to prevent or 28 Gisela L. Poletta, Pablo A. Siroski, Patricia S. Amavet et al. solve environment problems, alternative exploitation of natural resources for the production of pharmaceuticals and nutritional supplements, among others. Besides, information generated through all these studies is extremely useful for the optimization of the different farming systems and the sustainable use of these reptiles, allowing the responsible use of natural resources and environment. Studies of reptiles’ natural populations are scarce, and in our country have been mostly produced by this collaborative group, strengthen an emerging research discipline related to national productive systems. Furthermore, all this work responds to the need for conservation of native biodiversity, framed in national and international agreements, thus constituting a priority line of research and development in our region. ACKNOWLEDGMENTS All the studies presented in this chapter are supported by Consejo Nacional de Investigaciones Científicas y Técnicas - CONICET (PIP 2010-105 to PSA and PIP 2009-112 to MSM), Agencia Nacional de Promoción Científica y Tecnológica – ANPCyT (PICT 2010469 to PSA and PICT 2011-1349 to GLP), Universidad Nacional del Litoral (UNL-Sta. Fe, Argentina), and Proyecto Yacaré and Yacarés Santafesinos (Gob. Sta. Fe / MUPCN). We are especially grateful to Alejandro Larriera and all the members of ‘Proyecto Yacaré’. We would like to thank particularly to Lucía Fernández, Agustina Latorre, Evelyn López González, Ma. Josefina Boned, Laura G. Schaumburg, Ma. 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