Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Venom phenotype conservation suggests integrated specialization in a lizard-eating snake.

Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.

Reconsidering reproductive patterns in a model dissociated species, the red-sided garter snake: Sex-specific and seasonal changes in gonadal steroidogenic gene expression.

Sex steroid hormones are powerful regulators of reproductive behavior and physiology in vertebrates, and steroidogenesis has distinct sex- and season-specific patterns ultimately dictated by the expression of key enzymes. Most comparative endocrinology studies, however, focus only on circulating levels of sex steroids to determine their temporal association with life-history events in what are termed associated reproductive patterns. The red-sided garter snake (Thamnophis sirtalis parietalis) is a notable exception; this species exhibits maximal sex behavior decoupled from maximal sex steroid production and gametogenesis in what is termed a dissociated reproductive pattern. And while this is true for male red-sided garter snakes and their production of testosterone, females have maximal estradiol production during peak breeding (spring) but only immediately after mating. Here, we demonstrate that expression of ovarian aromatase (conversion of androgens to estrogens) matches the established seasonal hormone pattern in females. Additionally, steroidogenic gene expression in the ovary is broadly reduced if not suppressed compared to the testis throughout the active year. Bizarrely, male red-sided garter snakes demonstrate an unexplained pattern of steroidogenic gene expression in the testis. StAR (import of cholesterol to steroidogenesis) is maximally expressed in spring, yet Hsd17b3 expression (conversion of androstenedione to testosterone) is highest in summer, with the latter matching the established summer peak in male testosterone. The function of elevated StAR in spring is unknown, but our results suggest a decoupling between maximal StAR expression and testosterone biosynthesis (Hsd17b3 expression). We also purport that the reproductive pattern binary should be reassessed given its lack of fit for many vertebrate species that demonstrate seasonal, mixed patterns of (a)synchrony between circulating sex hormones and reproductive behavior.

Oxidative stress and behavioral responses of moorish geckos (Tarentola mauritanica) submitted to the presence of an introduced potential predator (Hemorrhois hippocrepis).

Stressful situations induce an increase in the production of reactive oxygen species (ROS) which can lead to molecular damage and alteration of cell function. The introduction of new potential predators induces physiological stress in native fauna. However, behavioral responses have been reported in preys, demonstrating an induction of the defenses against alien species. Behavioral and antioxidant enzyme responses in the moorish gecko, Tarentola mauritanica, against the invasive predator horseshoe whip snake (Hemorrhois hippocrepis) were assessed. Behavior was recorded and a tissue sample from the tail was collected after placing the gecko in a terrarium with previous absence or presence of the snake in 'Control' and 'H. hippocrepis' groups, respectively. Fifteen behavioral variables were examined, including tongue flick (TF) and locomotion patterns. Antioxidant enzyme activities -catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR)-, and the levels of reduced (GSH) and oxidized glutathione (GSSG), glutathione/glutathione disulfide ratio (GSH/GSSG) and malondialdehyde (MDA) concentrations were measured in the tissue sampled. Geckos exposed to the snake's odor showed a higher number of TF, longer amounts of time remaining motionless or moving in slow motion and they spent less time on the ground in comparison to the 'Control' group. The presence of the snake produced a significant increase in the activities of CAT, SOD and GR and a decrease in the GSH/GSSG ratio in T. mauritanica individuals exposed to the snake's scent. Thus, both behavioral responses and oxidative stress biomarkers clearly showed that T. mauritanica is able to recognize H. hippocrepis as a potential predator, despite being a recently introduced snake at the Balearic Islands.

The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms.

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey's physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure-activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey's physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.

Divergent Specialization of Simple Venom Gene Profiles among Rear-Fanged Snake Genera (Helicops and Leptodeira, Dipsadinae, Colubridae).

Many venomous animals express toxins that show extraordinary levels of variation both within and among species. In snakes, most studies of venom variation focus on front-fanged species in the families Viperidae and Elapidae, even though rear-fanged snakes in other families vary along the same ecological axes important to venom evolution. Here we characterized venom gland transcriptomes from 19 snakes across two dipsadine rear-fanged genera (Leptodeira and Helicops, Colubridae) and two front-fanged genera (Bothrops, Viperidae; Micrurus, Elapidae). We compared patterns of composition, variation, and diversity in venom transcripts within and among all four genera. Venom gland transcriptomes of rear-fanged Helicops and Leptodeira and front-fanged Micrurus are each dominated by expression of single toxin families (C-type lectins, snake venom metalloproteinase, and phospholipase A2, respectively), unlike highly diverse front-fanged Bothrops venoms. In addition, expression patterns of congeners are much more similar to each other than they are to species from other genera. These results illustrate the repeatability of simple venom profiles in rear-fanged snakes and the potential for relatively constrained venom composition within genera.

Cytotoxicity and inflammation induced by Philodryas patagoniensis venom.

The Green racer Philodryas patagoniensis is a snake species from South America and accidents with this genus are often neglected. Therefore, this study aimed to evaluate the toxicological, cytotoxic, and inflammatory potential of P. patagoniensis venom (PpV). The experimental model Artemia salina was used to determine toxicity through the median lethal dose (LD50). Cell viability and genotoxicity were evaluated in human mononuclear cells using the Trypan blue test and the Comet assay, respectively. To assess inflammation, mice had the ventral surface of the right hind paw injected with saline, formalin, and three different concentrations of venom (1, 1.5, and 2 µg. 50 µL-1). LD50 in A. salina was 461 µg. mL-1. PpV caused a significant increase in cell death and genotoxicity in human mononuclear cells at two concentrations (575 and 1150 µg. mL-1). PpV shown also to be a strong agent causing nociception in mice. Paw edema totaled four days at 1.5 µg. 50 µL-1. The hyperalgesia caused by the venom had a long duration in mice, lasting eight days at all concentrations evaluated. Thus, we evaluated for the first time the toxicological potential of PpV in A. salina model and in leukocytes. We concluded that systemic oxidative stress, which we infer to be in the genesis of cytotoxicity and genotoxicity observed in vitro, and the inflammatory process are part of the pathways that trigger the venom damage cascades. Relevant data for both scientific research and clinical medicine. Nonetheless, studies are needed to elucidate these mechanisms.

Role of Melatonin in Temperature-Induced Activation of the Neuroendocrine Reproductive Axis in Garter Snakes.

An animal's ability to respond optimally to changing environmental conditions is paramount to successfully reproducing and thus maximizing fitness. Studies on photoperiod-induced changes in neural thyroid hormone metabolism have conclusively linked environmental cues to the neuroendocrine reproductive axis of birds and mammals. Whether this conserved mechanism also transduces changes in environmental temperature, however, has not been fully addressed. We investigated whether the hormone melatonin mediates the effects of low-temperature dormancy on thyroid hormone metabolism within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis parietalis). To address this question, we used immunohistochemistry to assess changes in thyroid-stimulating hormone (TSH) in the infundibulum of the pituitary and deiodinase 3 (Dio3) and gonadotropin-releasing hormone (GnRH) in the hypothalamus. We also asked if changes in TSH, Dio3, and/or GnRH immunoreactivity are associated with changes in male courtship behavior. In contrast to our predictions, 6 weeks of dormancy at 4°C significantly decreased the number of TSH-labeled cells in the infundibulum. It is possible that the observed decrease in TSH is related to the release of snakes from temperature refractoriness, but this idea needs further testing. Treatment of snakes with the melatonin precursor 5-hydroxytryptophan during dormancy at 4°C both reversed the temperature-induced change in TSH immunoreactivity and disrupted the temporal pattern of male courtship behavior. These results suggest that TSH cells within the infundibulum are both modulated by temperature and sensitive to changes in melatonin. As predicted, male snakes hibernated at an elevated temperature of 12°C for 6 weeks and treated with vehicle showed no change in TSH-, Dio3-, or GnRH-immunoreactive cell number. Treatment of snakes with the melatonin receptor antagonist luzindole was not sufficient in rescuing the effects of dormancy at 12°C on TSH immunoreactivity or courtship behavior. However, luzindole-treated snakes showed a significant increase in GnRH-immunoreactive cell number, suggesting that melatonin exerts an inhibitory effect on GnRH in garter snakes. In summary, our results provide critical insights into the mechanisms that mediate the effects of temperature on reproductive physiology and behavior.

New Insights Into Dietary Toxin Metabolism: Diversity in the Ability of the Natricine Snake Rhabdophis tigrinus to Convert Toad-Derived Bufadienolides.

The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.

Architecture of the Pancreatic Islets and Endocrine Cell Arrangement in the Embryonic Pancreas of the Grass Snake (Natrix natrix L.). Immunocytochemical Studies and 3D Reconstructions.

During the early developmental stages of grass snakes, within the differentiating pancreas, cords of endocrine cells are formed. They differentiate into agglomerates of large islets flanked throughout subsequent developmental stages by small groups of endocrine cells forming islets. The islets are located within the cephalic part of the dorsal pancreas. At the end of the embryonic period, the pancreatic islet agglomerates branch off, and as a result of their remodeling, surround the splenic "bulb". The stage of pancreatic endocrine ring formation is the first step in formation of intrasplenic islets characteristics for the adult specimens of the grass snake. The arrangement of endocrine cells within islets changes during pancreas differentiation. Initially, the core of islets formed from B and D cells is surrounded by a cluster of A cells. Subsequently, A, B, and D endocrine cells are mixed throughout the islets. Before grass snake hatching, A and B endocrine cells are intermingled within the islets, but D cells are arranged centrally. Moreover, the pancreatic polypeptide (PP) cells are not found within the embryonic pancreas of the grass snake. Variation in the proportions of different cell types, depending on the part of the pancreas, may affect the islet function-a higher proportion of glucagon cells is beneficial for insulin secretion.

Duvernoy's Gland Transcriptomics of the Plains Black-Headed Snake, Tantilla nigriceps (Squamata, Colubridae): Unearthing the Venom of Small Rear-Fanged Snakes.

The venoms of small rear-fanged snakes (RFS) remain largely unexplored, despite increased recognition of their importance in understanding venom evolution more broadly. Sequencing the transcriptome of venom-producing glands has greatly increased the ability of researchers to examine and characterize the toxin repertoire of small taxa with low venom yields. Here, we use RNA-seq to characterize the Duvernoy's gland transcriptome of the Plains Black-headed Snake, Tantilla nigriceps, a small, semi-fossorial colubrid that feeds on a variety of potentially dangerous arthropods including centipedes and spiders. We generated transcriptomes of six individuals from three localities in order to both characterize the toxin expression of this species for the first time, and to look for initial evidence of venom variation in the species. Three toxin families-three-finger neurotoxins (3FTxs), cysteine-rich secretory proteins (CRISPs), and snake venom metalloproteinases (SVMPIIIs)-dominated the transcriptome of T. nigriceps; 3FTx themselves were the dominant toxin family in most individuals, accounting for as much as 86.4% of an individual's toxin expression. Variation in toxin expression between individuals was also noted, with two specimens exhibiting higher relative expression of c-type lectins than any other sample (8.7-11.9% compared to <1%), and another expressed CRISPs higher than any other toxin. This study provides the first Duvernoy's gland transcriptomes of any species of Tantilla, and one of the few transcriptomic studies of RFS not predicated on a single individual. This initial characterization demonstrates the need for further study of toxin expression variation in this species, as well as the need for further exploration of small RFS venoms.

First insights into the biochemical and toxicological characterization of venom from the Banded Cat-eyed Snake Leptodeira annulata pulchriceps.

With the aim to widen the current knowledge of toxinological implications of bites from rear-fanged snakes and biological roles of their venoms, this study focuses on the biochemical composition and toxic effects of the venom of Leptodeira annulata pulchriceps from Argentina. We analyzed the protein composition by electrophoresis and mass spectrometry, and enzymatic properties by quantitative assays on different substrates. Additionally, we evaluated local and systemic toxicity in mice, and tested its cross-reactivity with elapid and viperid antivenoms used in Argentina. This venom showed features reminiscent of venoms from snakes of Bothrops genus, containing components ranging from ~17 to 75 kDa, which are mainly tissue-damaging toxins such as proteinases. Although showing low lethality to mice (LD50 = 20 µg/g body weight), prominent hemorrhage developed locally in mice intramuscularly and intradermally injected with the venom, and the minimum hemorrhagic dose was found to be 12.7 µg/mouse. This study is the first comprehensive investigation of the venom of L. a. pulchriceps, and sheds new light on differences between this and those of the other two subspecies of L. annulata. Additionally, the study provides new insights into the venom components of "colubrid" snakes, advocating for considering bites from this rich diversity of snakes as a public health problem that needs to be addressed worldwide.

Genome mapping of a LYST mutation in corn snakes indicates that vertebrate chromatophore vesicles are lysosome-related organelles.

Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the corn snake Pantherophis guttatus The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in corn snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender corn snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.

Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between fast- and slow-growing king ratsnakes (Elaphe carinata).

In some countries, snakes are important protein sources in human diets, and their economic value depends predominantly on their muscle production, including in the king ratsnake (Elaphe carinata). Muscle growth in the king ratsnake clearly differs among individuals. To date, few potential molecular mechanisms underlying these differences in muscle growth and development have been reported. Here, we integrated mRNA and miRNA expression profiles to screen for genes, pathways, and predicted miRNA-mRNA networks associated with muscle growth and development in fast-growing and slow-growing King ratsnakes. Six hundred eight differentially expressed genes (DEGs) were identified, 48 of which were associated with muscle growth. The 37 genes upregulated in fast-growing individuals (FGIs) may be related to the promotion of muscle growth, whereas the 11 upregulated genes in slow-growing individuals (SGIs) may be related to the inhibition of muscle growth. Seven DEGs were enriched in the PI3K-AKT-MTOR signaling pathway, which appears to promote muscle growth in FGIs. Eleven DEGs were enriched in the ubiquitin-proteasome pathway, which appears to inhibit muscle growth in SGIs. It may interpret why muscle growth differences. Furthermore, 698 miRNA were identified, including 125 novel miRNAs. 63 differentially expressed miRNA (DEMs) were screened, and 950 negative miRNA-mRNA interactions with the 63 DEMs and 608 DEGs were predicted. The miRNA-targeted genes were enriched in pathways related to muscle growth, protein synthesis, and protein degradation. Therefore, in addition to the identified DEGs, miRNAs may play important roles in the differential regulation of muscle growth in FGIs and SGIs of the king ratsnake.

Cellular Turnover: A Potential Metabolic Rate-Driven Mechanism to Mitigate Accumulation of DNA Damage.

Oxidative stress, the imbalance of reactive oxygen species and antioxidant capacity, may cause damage to biomolecules pivotal for cellular processes (e.g., DNA). This may impair physiological performance and, therefore, drive life-history variation and aging rate. Because aerobic metabolism is supposed to be the main source of such oxidative risk, the rate of oxygen consumption should be positively associated with the level of damage and/or antioxidants. Empirical support for such relationships remains unclear, and recent considerations suggest even a negative relationship between metabolic rate and oxidative stress. We investigated the relationship between standard metabolic rate (SMR), antioxidants, and damage in blood plasma and erythrocytes for 35 grass snakes (Natrix natrix). Reactive oxygen metabolites (dROMs) and nonenzymatic antioxidants were assessed in plasma, while two measures of DNA damage and the capacity to neutralize H2O2 were measured in erythrocytes. Plasma antioxidants showed no correlation to SMR, and the level of dROMs was positively related to SMR. A negative relationship between antioxidant capacity and SMR was found in erythrocytes, but no association of SMR with either measure of DNA damage was detected. No increase in DNA damage, despite lower antioxidant capacity at high SMR, indicates an upregulation in other defense mechanisms (e.g., damage repair and/or removal). Indeed, we observed a higher frequency of immature red blood cells in individuals with higher SMR, which indicates that highly metabolic individuals had increased erythrocyte turnover, a mechanism of damage removal. Such DNA protection through upregulated cellular turnover might explain the negligible senescence observed in some ectotherm taxa.

Investigating the relationship between corticosterone and glucose in a reptile.

The glucocorticoid hormone corticosterone (CORT) has classically been used in ecophysiological studies as a proxy for stress and energy mobilization, but rarely are CORT and the energy metabolites themselves concurrently measured. To examine CORT's role in mobilizing glucose in a wild reptile, we conducted two studies. The first study measured natural baseline and stress-induced blood-borne CORT and glucose levels in snakes during spring emergence and again when snakes return to the denning sites in autumn. The second study manipulated the hypothalamic-pituitary-adrenal (HPA) axis in male snakes in the autumn by taking a baseline blood sample, then subjecting individuals to one of five treatments (no injection, saline, CORT, adrenocorticotropin hormone and metyrapone). Subsequent samples were taken at 30 and 60 min. In both studies, we found that glucose levels do increase with acute stress, but that the relationship was not directly related to CORT elevation. In the second study, we found that none of the HPA axis manipulations directly affected blood glucose levels, further indicating that CORT may play a complex but not direct role in glucose mobilization in snakes. This study highlights the need for testing mechanisms in wild organisms by combining in situ observations with manipulative studies.

Occurrence, biomagnification and maternal transfer of legacy and emerging organophosphorus flame retardants and plasticizers in water snake from an e-waste site.

Water snake and small common carp samples collected from a Chinese pond polluted with electronic waste (e-waste) were analyzed for organophosphorus flame retardants (PFRs), PFR metabolites, and plasticizers to investigate their occurrence, biomagnification, and maternal transfer in ovoviviparous species. Mean concentrations of total PFRs, PFR metabolites, and plasticizers were 2.2-16, 1.3-2.8 and 151-1320 ng/g wet weight (ww), respectively in analyzed organisms. Metabolites of PFRs were found in the same order of magnitude as or even higher than their parent compounds, indicating the importance of monitoring metabolites to evaluate the internal exposure of PFRs in organisms. Biomagnification factors (BMFs) were below 1 for all targeted chemicals and negatively correlated with metabolite/parent ratios (MPRs), suggesting a biodilution driven by metabolism. The lipid normalized concentrations were lower in eggs than in muscle for most of targeted chemicals. The maternal transfer potential was significantly and positively correlated with log KOW (p < 0.05) when log KOW was below 6.

Effect of hypothermia on the functional activity of liver mitochondria of grass snake (Natrix natrix): inhibition of succinate-fueled respiration and K+ transport, ROS-induced activation of mitochondrial permeability transition.

The article considers the comparative analysis of the functional activity of mitochondria isolated from the liver of grass snakes, Natrix natrix (Linnaeus, 1758) that were kept at different temperatures (23-26 °C and 4-5 °C). It was found that liver mitochondria of hypothermia-exposed grass snakes are characterized by weak coupling of oxidative phosphorylation as compared to mitochondria of active animals which is caused by inhibition of succinate-fuelled respiration in ADP-stimulated state, as well as by activation of basal non-phosphorylating rate. Inhibition of mitochondrial respiration in hibernating animals is associated with a decrease in the activity of the respiratory chain complexes of organelles. A significant decrease in the rate of K+ transport in the liver mitochondria of hibernating animals has been established. Under these conditions, a decrease in the calcium capacity of the organelles was also revealed, which indicates a decrease in the resistance of the mitochondria of hibernating animals to the induction of the Ca2+-dependent mitochondrial pore. All these changes in the functional activity of mitochondria are observed on the background of increasing H2O2 production as well as increasing the proportion of polyunsaturated fatty acids in phospholipid composition of mitochondrial membranes, which are the targets of reactive oxygen species. It can lead to increased formation of lipid peroxides and activation of destructive processes associated with the induction of Ca2+-dependent mitochondrial pore.

Facultative mobilization of eggshell calcium promotes embryonic growth in an oviparous snake.

The mineralized eggshell of Reptilia was a major innovation in the evolution of the amniotic egg. Inorganic components strengthen the eggshell and are a potential source of nutrients to developing embryos. Embryos of oviparous reptiles do extract calcium from eggshells but vary interspecifically in exploitation of this resource. The pattern of embryonic calcium nutrition of the corn snake, Pantherophis guttatus, is similar to a diversity of squamate species: embryos obtain most calcium from yolk, yet also mobilize calcium from the eggshell. We tested the hypothesis that embryonic development is not dependent on eggshell calcium by manipulating calcium availability. We peeled away the outer calcareous layer of the eggshell of recently oviposited eggs; control eggs were left intact. Eggs were sampled periodically and calcium content of egg compartments (embryo, yolk, eggshell) was measured. We also analyzed skeletal development and size of hatchlings. There was no difference in survivorship or length of incubation between treatments. However, hatchlings from intact eggs contained more calcium and were larger in mass and length than siblings from peeled eggs. There were no observable differences in ossification but hatchlings from intact eggs had larger skeletal elements (skull, vertebrae). Our results indicate that mobilization of eggshell calcium is not a requirement for embryonic development of P. guttatus and that embryos augment yolk calcium by extracting calcium from the eggshell. This pattern of embryonic calcium nutrition would favor embryos with a greater capacity to mobilize calcium from the eggshell by promoting growth and thereby potentially enhancing hatchling fitness.