Immuno-endocrine alterations and oxidative stress induced by commercial formulations of pesticides in the tegu lizard (Salvator merianae).

Recent research has shown the risk of environmental contamination by pesticides in Argentina. Salvator merianae is considered as "sentinel species" for monitoring their effects. We intended to investigate growth, immunological and endocrine functions, and oxidative stress in S. merianae exposed to two commercial brands of the herbicide glyphosate: Roundup and Panzer Gold, as well as, to two insecticides: chlorpyrifos, and cypermethrin. Our results showed a higher increase in growth in animals exposed to Roundup and chlorpyrifos and a lower increase in those exposed to Panzer Gold. Higher total leukocyte and lymphocyte count values were observed in lizards exposed to Panzer Gold. The heterophils/lymphocytes ratio decreased in all treated animals. Regarding lobularity index, natural antibodies titers, and complement system activity values, lizards exposed to cypermethrin had higher values. Low corticosterone levels were observed in animals exposed to Roundup. Our results reveal different toxic effects of pesticides contributing to the knowledge of the pesticides' potential deleterious impact in the habitat of S. merianae.

A New Species of Sprentascaris (Nematoda: Raphidascarididae) in Hypostomus commersoni (Pisces: Loricariidae) from Argentina.

PURPOSE: To report a new species: Sprentascaris pampayensis, a Raphidascarididae parasite from the Neotropical region. METHODS: During a parasitological survey, specimens were collected of a new species of Sprentascaris from Hypostomus commersoni Valenciennes from urban lakes in Santa Fe City, Santa Fe Province, in the Pampean region of Argentina. Morphological and morphometric descriptions, and molecular analyses were performed. RESULTS: The new species, S. pampayensis n. sp., is characterized by having slightly extended lateral wings in both sexes, oral structure with three well-developed lips with double papillae, ventrolateral lips with an amphidial pore. Triangular buccal opening with three well-developed lips with flattened extensions at their bases, which end in a pointed manner towards the back. Males have 17 pairs of preanal papillae, 1 pair of adanal papillae, 3 pairs of postanal papillae and 1odd median postanal papilla located between the first and second pairs, unusual shape of caudal papillae with the central tubular part conspicuously elevated. Females have pre-equatorial vulva, smooth-shelled eggs, and mature embryonated eggs. DISCUSSION: Sprentascaris pampayensis n. sp. differs morphologically and genetically from S. manherti Petter and Cassone, 1984, S. lanfrediae Melo, Santos, Giese, Santos and Santos, 2011 and S. andersoni Malta, Paiva, Elisei, Tavares and Pereira, 2018. Morphological and morphometric comparison also distinguishes it from S. pimelodi Petter and Cassone, 1984, S. hypostomi Petter and Cassone, 1984, S. marano Ramallo, 2009, and S. saltaensis Ailán-Choke, Ramallo and Davies, 2017. Sprentascaris pampayensis n. sp. is a new parasitic nematode of Hypostomus commersoni.

Effects of glyphosate, cypermethrin, and chlorpyrifos on hematological parameters of the tegu lizard (Salvator merianae) in different embryo stages.

Different studies have demonstrated effects of pesticides during embryo development in vertebrates and stage-dependent effects, but there is no information concerning this for Salvator merianae. We evaluated the effects of the herbicides Glyphosate Roundup (GLY-RU) and Glyphosate Panzer Gold (GLY-PZ); and the insecticides Chlorpyrifos (CPF) and Cypermethrin (CYP), and their complex mixtures, at different concentrations in hematological parameters of S. merianae embryos at two different development stages. The analyzed parameters were Total and Differential White Blood Cells Count, Heterophils/Lymphocytes index (H/L), Lobularity index, and Natural Antibodies (Nabs titres), as well as growth, embryo mortality and birth delay. Heterophils decreased in the intermediate concentrations tested of CYP and GLY-RU, in animals exposed at 33-days development. Lymphocytes increased in the intermediate concentration tested of GLY-RU, and the H/L index decreased in the maximum concentration tested of GLY-RU. NAbs titres increased in those animals exposed to the maximum CYP concentration tested. However, animals exposed at 3/5-days development showed no differences among treatments in most of the analyzed parameters, suggesting a stage-dependent response. Nevertheless, those animals exposed to GLY-PZ showed lower Nabs titres in relation to negative control. These results suggest effects on different hematological parameters related to the immune system of S. merianae, according to the used pesticide (herbicide or insecticide), its concentration and commercial formulation (GLY-RU or GLY-PZ), and the stages of development of the exposed animals. Our results reveal the importance of carrying out studies that evaluate the effects of permanent exposure of living beings and their environments to these toxics.

Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu).

Several geographical areas where Salvator merianae is distributed in Argentina are included in regions with agricultural activity and exposed to pesticide formulations. Some pesticides could affect defense mechanisms being able alter structures of some components of immune and endocrine systems. To assess the potential effects of pesticides in this reptile under seminatural conditions, on the immune system and endocrine responses in S. merianae we analyzed several blood parameters. Total (TWBCC), differential (DWBCC) white blood cells count, heterophils/lymphocytes index (H/L), lobularity index (LI), natural antibodies (NAbs) titres, complement system (CS), and corticosterone concentration were analyzed in animals exposed to a mixture of cypermethrin (25%), glyphosate (66.2%) and chlorpyrifos (48%) formulations. In addition, body size was considered in these analyzes. TWBCC and NAbs revealed lower values in organisms exposed to pesticides respect to a control indicating a possible immunosuppression effect. Besides, the LI showed a greater number of lobes in organism exposed demonstrating symptoms of chronic infection. In addition, we observed a reduced growth in these animals possibly related to a less energy investment in body mass to maintain an active defense against pesticides. Finally, we found high levels of plasma corticosterone in animals exposed to mix formulation that could demonstrate neuroendocrine axis activation. Other parameters like DWBCC, H/L index and activity of CS showed no differences in treated animals respect to control group, which could indicate low sensibility of these parameters to the concentration of pesticides used. Our results provide evidence of the toxic effects of pesticides on different immune system parameters, but also a trade-off among these parameters, corticosterone levels and growth. In this way, we can conclude that the formulated pesticides applied widely and constantly in the areas occupied by S. merianae, would be affecting its immune and endocrine systems and therefore its ability to defend against external agents. This kind of studies is of great interest to know the possible responses of wild species to anthropogenic disturbances such as pesticide contamination.

Analysis of morphological variability and heritability in the head of the Argentine Black and White Tegu (Salvator merianae): undisturbed vs. disturbed environments.

The heterogeneity of biotic and abiotic factors influencing fitness produce selective pressures that promote local adaptation and divergence among different populations of the same species. In order for adaptations to be maintained through evolutionary time, heritable genetic variation controlling the expression of the morphological features under selection is necessary. Here we compare morphological shape variability and size of the cephalic region of Salvator merianae specimens from undisturbed environments to those of individuals from disturbed environments, and estimated heritability for shape and size using geometric morphometric and quantitative genetics tools. The results of these analyzes indicated that there are statistically significant differences in shape and size between populations from the two environments. Possibly, one of the main determinants of cephalic shape and size is adaptation to the characteristics of the environment and to the trophic niche. Individuals from disturbed environments have a cephalic region with less shape variation and also have a larger centroid size when compared to individuals from undisturbed environments. The high heritability values obtained for shape and size in dorsal view and right side view indicate that these phenotypic characters have a great capacity to respond to the selection pressures to which they are subjected. Data obtained here could be used as an important tool when establishing guidelines for plans for the sustainable use and conservation of S. merianae and other species living in disturbed areas.

Baseline values of immunologic parameters in the lizard Salvator merianae (Teiidae, Squamata).

The genus Salvator is widely distributed throughout South America. In Argentina, the species most abundant widely distributed is Salvator merianae. Particularly in Santa Fe province, the area occupied by populations of these lizards overlaps with areas where agriculture was extended. With the aim of established baseline values for four immunologic biomarkers widely used, 36 tegu lizards were evaluated tacking into account different age classes and both sexes. Total leukocyte counts were not different between age classes. Of the leucocytes count, eosinophils levels were higher in neonates compared with juvenile and adults; nevertheless, the heterophils group was the most prevalent leukocyte in the peripheral blood in all age classes. Lymphocytes, monocytes, heterophils, azurophils and basophils levels did not differ with age. Natural antibodies titres were higher in the adults compared with neonates and juveniles lizards. Lastly, complement system activity was low in neonates compared with juveniles and adults. Statistical analysis within each age group showed that gender was not a factor in the outcomes. Based on the results, we concluded that S. merianae demonstrated age (but not gender) related differences in the immune parameters analyzed. Having established baseline values for these four widely-used immunologic biomarkers, ongoing studies will seek to optimize the use of the S. merianae model in future research.

Mating System of Caiman yacare (Reptilia: Alligatoridae) Described from Microsatellite Genotypes.

The yacare caiman (Caiman yacare) is a reptile from South America and 1 of the 2 crocodilian species present in Argentina. The degradation of their natural habitat and strong hunting pressure led to a sharp numerical decline of wild populations. Nowadays, C. yacare is included in Appendix II of CITES, and ranching practices in some areas in Argentina are helping hatching success. In this context, it is important to better understand the population structure and mating system of the species. To do this, we amplified 10 microsatellite markers (SSRs) in 148 individuals of 13 C. yacare nests. All of the markers were polymorphic with 2­12 alleles per locus, with allelic sizes ranging between 154 and 400 bp and medium levels of polymorphism (Ho = 0.152­0.551 and He = 0.221­0.621). We were able to determine the maternal genotype in 9 out of 13 nests. In 6 of them we found more than 1 paternal genotype, with a maximum of 3 fathers for a single nest. This study is the first to provide evidence of multiple paternity behavior. These findings will be useful to improve management and conservation strategies for the species.

FullSSR: Microsatellite Finder and Primer Designer.

Microsatellites are genomic sequences comprised of tandem repeats of short nucleotide motifs widely used as molecular markers in population genetics. FullSSR is a new bioinformatic tool for microsatellite (SSR) loci detection and primer design using genomic data from NGS assay. The software was tested with 2000 sequences of Oryza sativa shotgun sequencing project from the National Center of Biotechnology Information Trace Archive and with partial genome sequencing with ROCHE 454® from Caiman latirostris, Salvator merianae, Aegla platensis, and Zilchiopsis collastinensis. FullSSR performance was compared against other similar SSR search programs. The results of the use of this kind of approach depend on the parameters set by the user. In addition, results can be affected by the analyzed sequences because of differences among the genomes. FullSSR simplifies the detection of SSRs and primer design on a big data set. The command line interface of FullSSR was intended to be used as part of genomic analysis tools pipeline; however, it can be used as a stand-alone program because the results are easily interpreted for a nonexpert user.

Phenotypic variability and heritability of the cephalic region of Caiman latirostris.

The study of the cephalic shape of crocodilian is relevant in the fields of ecology, systematics, evolution, and conservation. Therefore, the integration of geometric analysis within quantitative genetics allows the evaluation of the inheritable shape components. In this study, the dorsal cephalic region of 210 Caiman latirostris hatchlings was analyzed from seven populations in Santa Fe, Argentina, to detect intra-, and inter-population phenotypic variability, and to determine the heritability of biological shape and size, using newly available geometric morphometric tools. The principal component analysis showed two configurations of cephalic shape that could be related to sexual dimorphism. In the canonical variate analysis, Procrustes distances between groups indicated that there are differences in shape among populations. Furthermore, the method of partial least squares indicated a covariation between cephalic shape and environmental variables. Regarding to CS of the skull we found significant differences among populations, moreover the partial least squares was also significant. Estimates of the heritability of shape and size were high, indicating that the components of these features are susceptible to the selection.

Genetic and morphometric variability in Caiman latirostris (broad-snouted caiman), reptilia, Alligatoridae.

Caiman latirostris (broad-snouted caiman) is a crocodilian species from Argentina subject of management plans. The goal of this study was estimating the distribution of genetic variability using RAPD markers and quantitative traits in wild populations of C. latirostris from Santa Fe province, Argentina. We sampled animals from four populations to obtain DNA and morphometric measurements. Eight RAPD primers were used and PCR products were analyzed on 4% polyacrylamide gels stained with silver nitrate. Eleven allometric measurements were obtained in animals within 48 hr after birth. We were able to reveal a relatively high number of variable markers in the studied populations. Our estimates of polymorphism and heterozygosity are higher than recorded values in other crocodilians using isozymes, the studied populations showed low levels of gene flow and some population subdivision. The study of quantitative traits conducted by nested analysis of variance and principal component analysis indicated higher levels of variance among nests within populations than among populations. We found that some head measurements have 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 (F(ST)) among populations was higher than quantitative trait differentiation value (Q(ST)), suggesting a higher contribution of neutral than adaptive loci to the genetic differentiation among populations. Quantitative traits are probably more related with fitness and the differentiation among populations remained relatively lower. The high heritability estimated for some traits indicates great potential to improve them in management plans.

Microsatellite DNA markers applied to detection of multiple paternity in Caiman latirostris in Santa Fe, Argentina.

Detecting multiple paternity in wild populations of the broad-snouted caiman (Caiman latirostris) has important implications for conservation efforts. We have applied microsatellite markers to examine genetic variation in C. latirostris and also have provided the first data concerning detection of multiple paternity in wild populations of this species. Blood samples from four nest-guarding C. latirostris females and their hatchlings were obtained from Santa Fe Province, Argentina. Amplified products were analyzed by electrophoresis on 10% polyacrylamide gels and visualized with silver staining. Four out of the eight markers tested reliably amplified and yielded useful data. Using polyacrylamide gels with silver staining provides high enough resolution to obtain individual genotypes. In order to assess the presence or absence of more than two parents in each nest, we used the single locus Minimum Method, and applied Cervus 3.0 and Gerud 2.0 software in parentage analyses. Our results indicate more than one father in at least two families. This behavior could be the consequence of high habitat variability in the area where our population was sampled. The ability to understand mating systems is important for maintaining viable populations of exploited taxa like C. latirostris.