Through the Looking Glass: Revisiting Smoking and Health.

January 11, 2024, marked the 60th anniversary of the initial U.S. Surgeon General report "Smoking and Health," which definitively linked cigarette smoking and lung cancer.1 Similar to the old Virginia Slims slogan "you've come a long way, baby," smoking rates have diminished greatly since the release of that report.2 However, smoking still represents the leading cause of preventable deaths.3 Numerous countries have enacted policies aimed at decreasing conventional cigarette use, such as including warning labels on tobacco products, limiting advertising, and imposing bans on particular products. Such measures have contributed to significant reductions in cigarette use.

Coinfection by Mycobacterium marinum and Mycolicibacterium fortuitum in a captive adult diamondback water snake causing disseminated mycobacteriosis with acute cutaneous ulceration.

An adult male captive diamondback water snake (Nerodia rhombifer) was found dead after a 1-d history of lethargy and cutaneous ulcers. The snake had eaten 2 sunfish (Mola spp.) 5 d before death. Gross examination revealed white-to-tan nodules in the lung and liver and segmental intestinal impactions with digested fish. Histopathology confirmed disseminated granulomas with numerous intrahistiocytic acid-fast bacteria in the skin, skeletal muscle, lung, liver, and intestines. Mycobacterium marinum and Mycolicibacterium fortuitum were identified by culture of the hepatic granuloma, followed by PCR and rpoB gene sequencing. To our knowledge, this is the first description of M. marinum and M. fortuitum coinfection in this species. Although M. fortuitum has been isolated from reptiles, lesions associated with its presence in tissues have not been described previously. Interestingly, the mineralization within granulomas that we observed in our case is not reported in mycobacterial infection in reptiles, whereas this finding is common in mammals.

Health Assessment of Free-Ranging Eastern Indigo Snakes (Drymarchon couperi) from Hydrologic Restoration Construction Sites in South Florida, USA.

There is a paucity of information regarding the health status of free-ranging eastern indigo snakes (EIS; Drymarchon couperi) in heavily modified and developing landscapes. As a component of regional Florida Everglades restoration efforts, several areas occupied by EIS are being converted from agricultural lands to reservoirs. From 2020 to 2022, 28 EIS were opportunistically captured at two of these sites and brought into captivity to join a captive breeding colony; however, 11 snakes died within 5 mo of capture. Health assessments were performed on 28 individuals and included hematology and plasma biochemistry analysis, as well as screening for pesticide contaminant levels, parasites, and other pathogens. Overall, the presence of pathogens was relatively high, suggesting immunosuppression secondary to stress: 25/28 (89.4%) Kalicephalus sp.; 12/28 (42.9%) Raillietiella orientalis; 11/28 (39.2%) Ochetosoma validum; 7/28 (25.0%) Cryptosporidium serpentis; 3/28 (10.7%) snake adenovirus 1; and 1/28 (3.6%) Ferlavirus genotype C. Stress may have been caused by physical displacement, habitat modification, and noise pollution. These potential stressors (including the presence of remnant harmful chemicals from previous land use and the impacts on this federally threatened species) should be considered further when making restoration or construction decisions.

Melanophoroma in a Lichtenstein's green racer snake.

Melanophoroma is a neoplasm of reptilian pigment cells, considered uncommon and part of a group of neoplasms called chromatophoromas. The objective of this work was to describe a case of melanophoroma in a free-living Lichtenstein's green racer snake (Philodryas olfersii), presenting with an ulcerative nodular neoformation in the integument of the head region. In the neurologic evaluation, a proprioceptive deficit was observed. Ultrasound, X-ray, and mass cytology examinations were performed. Radiographic and ultrasound findings of the tumor indicated infiltrative behavior, and cytology indicated a presumptive diagnosis of a melanocytic neoplasm. Based on the results of the examinations and the patient's clinical condition, euthanasia was chosen. At necropsy, there was a nodule measuring 4.5 × 2.5 × 2.0 cm, with a blackish-colored cut surface, mottled by gray areas, which infiltrated soft tissue and bone, and extended dorsally from the mouth to the cervical musculature. On microscopy, a non-delimited, non-encapsulated, and invasive neoplastic proliferation was observed, with moderate cellularity, which was predominantly composed of fusiform cells with distinct borders and foci of epithelioid cells. The cells had ample cytoplasm, which had a brown to black (melanocytic) granular pigment compatible with a melanophoroma. To the best of the authors' knowledge, this is a unique description of a melanophoroma in P. olfersii.

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.

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

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.

Morganella sp. infection in scent gland of corn snake (Pantherophis guttatus) - case report / Infecção por Morganella sp. em glândula de cheiro de corn snake (Pantherophis guttatus) - relato de caso

The corn snake (Pantherophis guttatus) is a non-venomous snake from the Colubridae family. The pair of scent glands is indispensable in the physiology of reproduction and defense of the species. This structure is located caudal to the cloaca. It is responsible for releasing a thick material based on lipids containing pheromones that perform functions associated with animal behavior and survival. This work aims to report infection by Morganella sp. in a scent gland in a female corn snake, evidencing the diagnosis and treatment. An increase in volume was seen in the final third, proximal to the cloaca, firm consistency, immobile, and approximately 4 cm long and painful to touch. Samples of the material were collected for bacterial culture and antibiogram examination, being positive for bacteria of the genus Morganella. Based on the result of the antibiogram, it was possible to determine the most appropriate therapeutic protocol, with the referral to perform the surgical procedure to remove the tissue compromised by the infection, remaining stable until the removal of the stitches 40 days after the procedure, with total surgical wound healing.(AU)

A corn snake (Pantherophis guttatus) é uma serpente não peçonhenta, da família Colubridae. Na fisiologia da reprodução e defesa da espécie, o par de glândulas de cheiro é indispensável. Essa estrutura está localizada caudal à cloaca e é responsável pela liberação de um material espesso à base de lipídeos, contendo feromônios que exercem funções associadas ao comportamento animal e à sua sobrevivência. O objetivo desse trabalho é relatar uma infecção por Morganella sp. em glândula de cheiro em uma fêmea de corn snake, evidenciando o diagnóstico e tratamento. Observou-se um aumento de volume no terço final, proximal a cloaca, de consistência firme, imóvel e com aproximadamente 4 cm de comprimento e sensibilidade dolorosa ao toque. Foram coletadas amostras do material para cultura bacteriana e exame de antibiograma, sendo positivo para bactérias do gênero Morganella. Com base no resultado do antibiograma, foi possível determinar o protocolo terapêutico mais adequado, como o encaminhamento para realização do procedimento cirúrgico para remoção do tecido comprometido pela infecção, se mantendo estável até a remoção dos pontos, 40 dias após o procedimento, com total cicatrização da ferida cirúrgica.(AU)

The molecular basis of venom resistance in the non-venomous snake Sinonatrix annularis.

Various snake species and snake predators have natural neutralization against snake toxins, which their antidotal abilities are commonly attributed to the intrinsic inhibitors produced by the liver, e.g., phospholipase A2 inhibitor (PLI) and metalloproteinase inhibitor (SVMPI). Sinonatrix annularis was found to possess broad-spectrum neutralization to different snake venoms in our lab. Although the anti-venom compound PLIγ has been previously characterized in our laboratory, the mechanism of resistance of S. annularis to snake venoms remains obscure. In this research, a venom affinity chromatography was constructed by immobilizing D. acutus venom to NHS-agarose beads and applied for antitoxins mining from S. annularis. The binding capacity of the venom column was validated using a self-prepared rabbit antivenom against D. acutus. Serum and liver homogenate of S. annularis were then applied to the column, the bound components were profiled using SDS-PAGE and mass spectrometry. PLIs, snake venom metalloproteins inhibitor (SVMPI), small serum protein (SSP), heat shock proteins, etc were identified. To identify their toxin targets in D. acutus venom, a reverse separation was conducted by coupling the fractionated S. annularis serum proteins to NHS-agarose beads. Fifteen toxins of five families were captured and identified as follows: PLA2s, metalloproteinases, cysteine-rich secretory proteins, snake venom serine proteinases, and C-type lectins. These discoveries increased our understanding of the capacity and mechanism of the natural neutralization of S. annularis to snake venom. These natural inhibitors are medically significant due to their powerful and broad antidotal activities, which may provide alternative and promising drug candidates for snakebite treatment.

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.

Decreased attractivity in female garter snakes treated with an aromatase inhibitor.

Most experimental studies on sexual signal regulation via hormone manipulation have focused on male signals, yet female signals demonstrate substantial phenotypic variation and hormone-dependent expression. Female red-sided garter snakes (Thamnophis sirtalis parietalis) produce a skin-based sex pheromone used by males in mate selection. The principle female sex steroid, 17 ß-estradiol, controls pheromone production in snakes, but studies manipulating female garter snakes have produced conflicting results, relied on behavioral tests with males in the laboratory, and did not quantify pheromone expression. Because aromatase is the terminal enzyme in estradiol biosynthesis, we hypothesized that female garter snakes rely on aromatase to ultimately control pheromone production during the annual cycle of this species. To test this, we used a known pharmacological inhibitor of aromatase, fadrozole (FAD). Wild-caught female garter snakes were chronically treated via subcutaneous injections of either FAD (1.0 mg kg-1 ) or saline (control) for six months in the laboratory during the active period of the annual cycle then hibernated. In two separate field bioassays the next spring at the den site, FAD females received approximately 50% less courtship from wild, sexually active male garter snakes compared to SHAM females. Pheromone analysis revealed that four of the largest, unsaturated methyl ketones were specifically downregulated in FAD females, indicating that aromatase action is a crucial, permissive step in the maintenance of female attractivity.

Trans-seasonal activation of the neuroendocrine reproductive axis: Low-temperature winter dormancy modulates gonadotropin-releasing hormone neurons in garter snakes.

All animals use external cues from the environment to accurately time life-history events. How the brain decodes environmental stimuli to effect changes in physiology and behavior, however, is poorly understood, particularly with regard to supplementary environmental cues such as temperature. We asked if low-temperature dormancy alters the synthesis and/or release of gonadotropin-releasing hormone (GnRH). We used the well-studied red-sided garter snake (Thamnophis sirtalis) for this study, as low-temperature exposure is both necessary and sufficient to induce reproduction in northern populations of this species. Snakes were collected from the field and hibernated at 4°C or 10°C in complete darkness for up to 16 weeks. In males, increasing duration of low-temperature dormancy significantly increased GnRH-immunoreactive cell number and GnRH soma size (a proxy for relative cell activity) in the forebrain. These changes mirrored those in male reproductive behavior (reported previously) and plasma androgen concentrations. The changes in GnRH cell area observed in males were specific to the neuroendocrine population of cells in the medial preoptic area; soma size in the rostral GnRH cells did not change. Finally, temperature-induced changes in GnRH were sexually dimorphic: neither hibernation temperature nor the duration of winter dormancy significantly modulated GnRH cell number or soma size in females, despite the fact that plasma estradiol and corticosterone increased significantly in response to both. These data demonstrate that the neuroendocrine GnRH system is sensitive to environmental temperature and suggest that GnRH neurons play a conserved but trans-seasonal role in mediating changes in reproductive physiology and behavior in dissociated breeders.

Temperature-dependence of metabolism and fuel selection from cells to whole organisms.

Temperature affects nearly every aspect of how organisms interact with and are constrained by their environment. Measures of organismal energetics, such as metabolic rate, are highly temperature-dependent and governed through temperature effects on rates of biochemical reactions. Characterizing the relationships among levels of biological organization can lend insight into how temperature affects whole-organism function. We tested the temperature dependence of cellular oxygen consumption and its relationship to whole-animal metabolic rate in garter snakes (Thamnophis elegans). Additionally, we tested whether thermal responses were linked to shifts in the fuel source oxidized to support metabolism with the use of carbon stable isotopes. Our results demonstrate temperature dependence of metabolic rates across levels of biological organization. Cellular (basal, adenosine triphosphate-linked) and whole-animal rates of respiration increased with temperature but were not correlated within or among individuals, suggesting that variation in whole-animal metabolic rates is not due simply to variation at the cellular level, but rather other interacting factors across scales of biological organization. Counter to trends observed during fasting, elevated temperature did not alter fuel selection (i.e., natural-abundance stable carbon isotope composition in breath, δ13 Cbreath ). This consistency suggests the maintenance and oxidation of a single fuel source supporting metabolism across a broad range of metabolic demands.

RETROSPECTIVE REVIEW OF OPHIDIOMYCOSIS (OPHIDIOMYCES OPHIODIICOLA) AT THE SMITHSONIAN'S NATIONAL ZOOLOGICAL PARK (1983-2017).

A retrospective review of systemic or localized mycotic infections in captive snakes confirmed via biopsy or necropsy from 1983 to 2017 was performed at the Smithsonian's National Zoological Park. Quantitative polymerase chain reaction (qPCR) confirmed infection with Ophidiomyces ophiodiicola (Oo) in 36.8% (n = 14) of the 38 mycotic infections. Infections with Oo were evenly distributed over the 35-y period and lacked a sex predilection. There was a period prevalence of 4.5% of completed snake necropsy or biopsy cases that were Oo positive. Species affected included green anaconda (Eunectes murinus, n = 4), garden tree boa (Corallus hortulanus, n = 1), false water cobra (Hydrodynastes gigas, n = 5), yellow anaconda (Eunectes notaeus, n = 1), eastern milksnake (Lampropeltis triangulum, n = 1), Brazilian rainbow boa (Epicrates cenchria cenchria, n = 1), and eastern diamondback rattlesnake (Crotalus adamanteus, n = 1). Histopathology demonstrated one or more of the following: heterophilic to necrotizing epidermitis with or without granulomatous dermatitis (n = 12), granulomatous pneumonia (n = 5), granulomatous endophthalmitis (n = 1), and subcutaneous-intramuscular fungal granuloma (n = 1). This study documents the presence of ophidiomycosis in a captive collection for almost 40 years, despite current literature designating it a recently emerging pathogen.

COELOMIC STEATITIS IN TENTACLED SNAKES (ERPETON TENTACULATUM).

A retrospective study revealed seven cases of coelomic steatitis in adult tentacled snakes (Erpeton tentaculatum), including two males and five females, between May 2014 and August 2020. Common clinical signs included death after unusual floating, generalized weakness, inappetence, reduced body condition, coelomic distension, and reproductive pathology in females. Hematology of one specimen revealed marked monocytosis and lymphocytosis with mild heterophilia (chronic and active inflammation). Gross examination identified variable degrees of intracoelomic fat necrosis in all snakes. Consistent histopathologic features included necrotic adipocytes, lipid saponification, lipofuscin/ceroid deposition, granulomatous inflammation, and multinucleated giant cells (Langhans type). Three females exhibited intralesional yolk fluid associated with periovarian steatitis. Hepatic lipidosis was the second most frequent pathologic finding. Thawed frozen lesser sand eels (Ammodytes tobianus) were fed during this period, stored in vacuum-sealed or opened packets at -18°C (frozen). After the death of the last specimen, vitamin E concentrations and peroxide values of the diet were analyzed. For the sealed and opened frozen batches, respectively, vitamin E concentrations were 0.71 and 0.49 mg/100 g (compared with 4 to 8 mg/100 g in average, fresh, raw mixed eel species samples) and peroxide values were 62.5 and 48.6 meq/kg (exceeding the acceptable peroxide values of 8 meq/kg for fish oils). This case study represents the first report of coelomic steatitis in tentacled snakes of unconfirmed etiology but with a putative association with feeding a long-term frozen-stored sand eel diet containing low vitamin E concentrations and fish oils with high peroxide values at time of analysis.

Testing the febrile response of snakes inoculated with Ophidiomyces ophidiicola, the causative agent of snake fungal disease.

Snake Fungal Disease (SFD) negatively impacts wild snake populations in the eastern United States and Europe. Ophidiomyces ophidiicola causes SFD and manifests clinically by the formation of heterophilic granulomas around the mouth and eyes, weight loss, impaired vision, and sometimes death. Field observations have documented early seasonal basking behaviors in severely infected snakes, potentially suggesting induction of a behavioral febrile response to combat the mycosis. This study tested the hypothesis that snakes inoculated with Ophidiomyces ophidiicola would seek elevated basking temperatures to control body temperature and behaviorally induce a febrile response. Eastern ribbon snakes (Thamnophis saurita, n = 29) were experimentally or sham inoculated with O. ophidiicola. Seven days after inoculation, snakes were tested on a thermal gradient and the internal body temperature and substrate temperature of each snake was recorded over time. Quantitative PCR was used when snakes arrived, during pre-inoculation, and post-inoculation to test snakes for the presence of O. ophidiicola. Some snakes arrived with O. ophidiicola and were subsequently inoculated, allowing for an assessment of secondary exposure effects. Snake thermoregulatory behavior was compared between 1) O. ophidiicola inoculated vs. sham inoculated treatments, 2) infected vs. disease negative groups, and 3) disease naïve vs. pre-exposed immune response categories. Neither internal nor substrate temperatures differed among initially prescribed, and qPCR recovered disease states, although infected snakes tended to reach a preferred body temperature faster than disease negative snakes. Snakes experiencing their first exposure (disease naïve) sought higher substrate temperatures than snakes experiencing their second exposure (pre-exposed). Here, we recover no evidence for behaviorally induced fever in snakes with SFD but do elucidate a febrile immune response associated with secondary exposure.

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.

Parasitological findings in the invasive California kingsnake (Lampropeltis californiae) in Gran Canaria, Spain.

The California kingsnake (Lampropeltis californiae), native to North America, is a significant threat to the conservation of endemic species in the Spanish Macaronesian island of Gran Canaria. However, its role disseminating potential invasive parasites, such as zoonotic pentastomids, has not been proven. Among its parasitic fauna, only protistans have been documented, in contrast to other Lampropeltis spp., which are known to carry pentastomids. Thus, a parasitological study is urgently required. Between 2016 and 2018, a total of 108 snakes were necropsied and stool samples examined. A single snake was infested with Ophionyssus natricis, and another individual with Serpentirhabdias sp. Only this latter snake presented gross lesions, characterized by granulomatous pneumonia. No Pentastomida were found. By contrast, almost the entire population (98.5%) was infested with larval helminths (three different nematode and two cestode species), characterized by granulomatous gastrointestinal serositis. This suggests the snake poses a 'dead end' host for local parasites. Based on these findings, snakes in Gran Canaria carry potential zoonotic mites, which along with Serpentirhabdias sp. could represent a threat to endemic lizards. The presence of metazoan parasites and their lesions are reported for the first time in the California kingsnake.

Description of a New Species Hepatozoon quagliattus sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting the Sleep Snake, Dipsas mikanii (Squamata: Colubridae: Dipsadinae) from Goiás State, Brazil.

PURPOSE: Species of Hepatozoon Miller, 1908 (Hepatozoidae) are blood protozoans with a cosmopolitan distribution and are reported to parasitize a range of vertebrate hosts including mammals, birds, reptiles, and amphibians. The present study aimed to describe a new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) found infecting the sleep snake Dipsas mikanii (Schlegel, 1837) (Squamata: Colubridae: Dipsadinae). METHODS: The snake was collected in 2017 at the municipality of Britânia, Goiás State, Brazil. Blood smears were made in order to find blood gametocytes and PCR was performed targeting the 18S rRNA gene. RESULTS: Microscopy screening of blood smears revealed the presence of intraerythrocytic gamont stages of Hepatozoon sp. in the peripheral blood with a parasitemia of 0.25%. Furthermore, meronts and monozoic cysts were observed in histological sections of the liver from the infected individual. The interspecific divergence of 18S rRNA sequences fragments isolated from D. mikanii had differences (2.39-11.3%) as compared to other sequences of species of Hepatozoon from snakes. CONCLUSIONS: Based on morphological and molecular data, a new species of Hepatozoon infecting D. mikanii from Brazil is described.