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Reptiles and amphibians are largely present in many environments, including domestic areas when they are kept as pet animals. They often harbor zoonotic pathogens, which can pose a serious risk of infection for humans, mainly immunocompromised individuals, the elderly, children, and pregnant women. Several studies have been carried out to verify the role of cold-blooded animals in the epidemiology of some bacteria, mainly Salmonella, whereas scarce attention has been focused on these animals as a source of staphylococci. These bacteria are often antimicrobial-resistant and they act as opportunistic pathogens, which can cause relevant infections in humans and animals, both domestic and wild. Asymptomatic reptiles and amphibians often harbor staphylococcal strains, such as Staphylococcus aureus and coagulase-negative Staphylococcus spp.; however, these bacteria have been associated with clinical conditions that usually appear in animals under stress conditions. In all cases, greater attention should also be focused on staphylococci in cold-blooded animals due to their implications in human and veterinary medicine.

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In the last years, lipid physiology has become an important research target for systems biology applied to the field of ecotoxicology. Lipids are not only essential components of biological membranes, but also participate in extra and intracellular signaling processes and as signal transducers and amplifiers of regulatory cascades. Particularly in sauropsids, lipids are the main source of energy for reproduction, growth, and embryonic development. In nature, organisms are exposed to different stressors, such as parasites, diseases and environmental contaminants, which interact with lipid signaling and metabolic pathways, disrupting lipid homeostasis. The system biology approach applied to ecotoxicological studies is crucial to evaluate metabolic regulation under environmental stress produced by xenobiotics. In this review, we cover information of molecular mechanisms that contribute to lipid metabolism homeostasis in sauropsids, specifically in crocodilian species. We focus on the role of lipid metabolism as a powerful source of energy and its importance during oocyte maturation, which has been increasingly recognized in many species, but information is still scarce in crocodiles. Finally, we highlight priorities for future research on the influence of environmental stressors on lipid metabolism, their potential effect on the reproductive system and thus on the offspring, and their implications on crocodilians conservation.

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Pharmacokinetics studies of anesthetic agents are important for understanding of the pharmacology and metabolism of anesthetic agents in reptilians. This study was designed to examine the pharmacokinetic and pharmacodynamic properties of intravenous dextroketamine alone or combined with midazolam in Caiman crocodilus. Eight caimans were anesthetized with dextroketamine (10 mg/kg; group D) or dextroketamine and midazolam (10 and 0.5 mg/kg respectively; group DM) into the occipital venous sinus. The pharmacokinetic parameters were calculated by HPLC using a non-compartmental modeling. Serial blood samples were collected at baseline and within 15 and 30 min, and 11.5, 2, 4, 8, 12, 24 and 48 h of drug administration. Sedation status over time differed between groups. All animals in group D (8/8; 100%) showed signs of light sedation at t10. Half (4/8; 50%) of these caimans did not progress to deeper levels of sedation. In spite of light sedation at t10, animals in group DM were deeply sedated within 13.13 ± 7.04 min of anesthetic agent injection. The area under the plasma concentration-time curve (AUC0-48) and half-life of dextroketamine changed significantly after combination with midazolam. Even without significant changes in clearance, the almost two-fold increase in the half-life of dextroketamine suggests a slower rate of elimination.

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The evolution of nest site use and nest architecture in the non-avian ancestors of birds remains poorly understood because nest structures do not preserve well as fossils. Nevertheless, the evidence suggests that the earliest dinosaurs probably buried eggs below ground and covered them with soil so that heat from the substrate fuelled embryo development, while some later dinosaurs laid partially exposed clutches where adults incubated them and protected them from predators and parasites. The nests of euornithine birds-the precursors to modern birds-were probably partially open and the neornithine birds-or modern birds-were probably the first to build fully exposed nests. The shift towards smaller, open cup nests has been accompanied by shifts in reproductive traits, with female birds having one functioning ovary in contrast to the two ovaries of crocodilians and many non-avian dinosaurs. The evolutionary trend among extant birds and their ancestors has been toward the evolution of greater cognitive abilities to construct in a wider diversity of sites and providing more care for significantly fewer, increasingly altricial, offspring. The highly derived passerines reflect this pattern with many species building small, architecturally complex nests in open sites and investing significant care into altricial young. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

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(1) Background: Pancuronium bromide is a neuromuscular blocker used for immobilizing crocodiles that can be reversed with neostigmine. A recommended drug dose has only been established for saltwater crocodiles (Crocodylus porosus), mostly based on trials in juveniles and subadults. After trialing a dose recommendation in a small cohort of nine Nile crocodiles (Crocodylus niloticus), we developed and applied a new dose recommendation for large adult Nile crocodiles. (2) Methods: we trialed and adapted a pancuronium bromide (Pavulon 4 mg/2 mL) dose in Nile crocodiles originally established for saltwater crocodiles and applied the new dose for the immobilization of 32 Nile crocodiles destined for transport. Reversal was achieved with neostigmine (Stigmine 0.5 mg/mL). (3) Results: Nine crocodiles were included in the trial phase; the induction time was highly variable (average: 70 min; range: 20-143 min), and the recovery time was prolonged (average: 22 h; range: 50 min-5 days), especially in large animals after reversal with neostigmine. Based on these results, we established a dose-independent recommendation (3 mg pancuronium bromide and 2.5 mg neostigmine) for animals weighing ≥ 270 kg (TL ≥ ~3.8 m). When applied to 32 adult male crocodiles (BW range: 270-460 kg; TL range: 3.76-4.48 m), the shortest induction time was ~20 min and the longest ~45 min. (4) Conclusions: Pancuronium bromide and its antidote, neostigmine, are effective for the immobilization and reversal of adult male Nile crocodiles (TL ≥ 3.8 m or BW ≥ 270 kg) when given in a weight-independent fashion.

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The visual ecology of early mammals remains poorly resolved. Studies of ancestral photopigments suggest an ancient transition from nocturnal to more crepuscular conditions. By contrast, the phenotypic shifts following the split of monotremes and therians-which lost their SWS1 and SWS2 opsins, respectively-are less clear. To address this, we obtained new phenotypic data on the photopigments of extant and ancestral monotremes. We then generated functional data for another vertebrate group that shares the same photopigment repertoire as monotremes: the crocodilians. By characterizing resurrected ancient pigments, we show that the ancestral monotreme underwent a dramatic acceleration in its rhodopsin retinal release rate. Moreover, this change was likely mediated by three residue replacements, two of which also arose on the ancestral branch of crocodilians, which exhibit similarly accelerated retinal release. Despite this parallelism in retinal release, we detected minimal to moderate changes in the spectral tuning of cone visual pigments in these groups. Our results imply that ancestral forms of monotremes and crocodilians independently underwent niche expansion to encompass quickly changing light conditions. This scenario-which accords with reported crepuscular activity in extant monotremes-may help account for their loss of the ultraviolet-sensitive SWS1 pigment but retention of the blue-sensitive SWS2.

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Lead (Pb) exposure is a widespread wildlife conservation threat, but impacts on reptile populations remain poorly documented. In this study, we examined Pb exposure and accumulation in a wild population of Nile crocodiles (Crocodylus niloticus) at Lake St Lucia, South Africa. Recreational angling has occurred in the area since the 1930s and incidental ingestion of Pb fishing weights has previously been identified as a major source of Pb poisoning in the local crocodile population. In 2019, we sampled blood and tail fat tissues from wild (n = 22) and captive (n = 3) crocodiles at Lake St Lucia to investigate potential impacts of chronic Pb exposure on crocodilian health. Lead was detected in blood samples of all wild crocodiles, although concentrations varied widely between individuals (86-13,100 ng ml-1). The incidence of Pb poisoning was higher in male crocodiles, with mean blood lead (BPb) concentrations in males (3780 ± 4690 ng ml-1) significantly (p < 0.001) higher compared to females (266 ± 230 ng ml-1). Blood Pb concentrations were correlated with concentrations measured in tail fat tissue (n.d - 4175 ng g-1 wet wt.). Although most of the crocodiles sampled appeared to be in good physical condition, highly elevated BPb concentrations (>6000 ng ml-1) were associated with markedly suppressed packed cell volumes (4.6-10.8%) and severe deterioration in tooth condition. These findings suggest that anaemia and tooth loss may be clinical signs of long-term environmental exposure to Pb. Although previously undocumented in crocodilians, these symptoms are consistent with Pb poisoning observed in birds and mammals, and suggest that crocodilians may be more susceptible to the long-term toxic effects of Pb than previously thought. In light of these findings, we suggest that the impact of accumulated Pb on crocodilian fitness, reproduction and mortality requires urgent attention.

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ABSTRACT The objective of the study was to determine the morphological characteristics of peripheral blood cells (erythrocytes, leukocytes, thrombocytes) and the leukocyte differential count (heterophils, eosinophils, basophils, lymphocytes, monocytes, azurophils) of captive Morelet's crocodiles (Crocodylus moreletii) from Veracruz, Mexico. Peripheral blood from 80 apparently healthy farmed crocodiles (39 subadults [19 females, 20 males] and 41 adults [18 females, 23 males]) was examined for morphology through stained blood smears and manual count was used for the leukocyte differential. Blood was collected during the non-breeding (n = 42) and breeding (n = 38) seasons. Blood examination indicated similar morphological characteristics of blood cells in subadult and adult individuals and in females and males in both seasons. Erythrocytes were the largest blood cells and lymphocytes the smallest. The leukocyte differential count showed that lymphocytes were the most abundant leukocytes and basophils the least numerous. The percentages of some leukocytes showed difference by season (non-breeding and breeding) in subadult and adult males (p < 0.05) and by size (subadults and adults) in males and females but only in the non-breeding season (p < 0.05). The leukocytes that showed the greatest variation were lymphocytes, heterophils and eosinophils. The knowledge of blood cell morphology and the leukocyte differential count in healthy farmed Morelet's crocodiles will allow the accurate diagnosis of some diseases of captive and wild individuals.

RESUMEN El objetivo del estudio fue determinar las características morfológicas de células sanguíneas periféricas (eritrocitos, leucocitos, trombocitos) y el conteo diferencial de leucocitos (heterófilos, eosinófilos, basófilos, linfocitos, monocitos, azurófilos) del cocodrilo de Morelet (Crocodylus moreletii) mantenido en cautiverio en Veracruz, México. Se examinó sangre periférica de 80 cocodrilos aparentemente sanos criados en granja (39 subadultos [19 hembras, 20 machos] y 41 adultos [18 hembras, 23 machos]) mediante frotis sanguíneos teñidos para determinar la morfología celular y mediante conteo manual para el diferencial de leucocitos. La sangre se recolectó durante las temporadas no reproductiva (n = 42) y reproductiva (n = 38). El examen sanguíneo indicó similar morfología de células sanguíneas en individuos subadultos y adultos, así como en hembras y machos, en ambas temporadas. Los eritrocitos fueron las células sanguíneas más grandes y los linfocitos las más pequeñas. El conteo diferencial de leucocitos mostró que los linfocitos fueron los leucocitos más abundantes y los basófilos los menos numerosos. Los porcentajes de algunos leucocitos mostraron diferencia por época (no reproductiva y reproductiva) en machos subadultos y adultos (p < 0,05) y por talla (subadultos y adultos) en machos y hembras sólo en temporada no reproductiva (p < 0,05). Los leucocitos que tuvieron la mayor variación fueron linfocitos, heterófilos y eosinófilos. El conocer la morfología de las células sanguíneas y el conteo diferencial de leucocitos en individuos sanos de cocodrilo de Morelet criados en granja permitirá diagnosticar con precisión algunas enfermedades de individuos manejados en cautiverio y también de individuos silvestres.

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Next-generation sequencing has allowed us to explore new methods, where comparative and population genomics can be used simultaneously. Keeping this in mind, we surveyed and analyzed the frequency and distribution of microsatellites in the Indian gharial (Gavialis gangeticus) and compared it with American alligator (Alligator mississippiensis) and saltwater crocodile (Crocodylus porosus) to enrich them with genomic resources. The Indian gharial has a low frequency, relative abundance (RA), and relative density (RD) of microsatellites as compared to other crocodilians. RA and RD were positively correlated with the GC content of genomic and transcriptomic sequences. The genomic sequences were dominated by dinucleotide repeats, whereas the transcriptomic sequences had an excess of trinucleotide repeats. Motif conservation studies among the three crocodilians revealed conservation of 69.2% of motifs. Species-specific unique motifs identified in this study could be used as molecular probes for species identification. A total of 67,311 primers were designed in all three species to enrich the crocodilians with genomic resources. The genomic resources developed in this study could accelerate diversity analysis within its individuals to design a proper mating plan to reduce inbreeding stress and further improve the species.

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Caiman latirostris is one of the two species of the order Crocodylia that inhabit Argentina and is considered a species of vital ecological and economic importance in the north-east of Argentina. In this region, pesticides are the most common contaminants in natural environments and wild caiman populations are subject to this contamination constantly. The aim of this study was to evaluate the effects the main pesticides used in the region: glyphosate (GLY), cypermethrin (CYP) and chlorpyrifos (CPF) -based formulations, as well as the mixture of them, on C. latirostris juveniles under semi-controlled condition of exposure (ex-situ) during 75 days. One hundred yearling caimans (10-month-old) were equally distributed into five experimental groups (20 animals per group): a negative control (NC -tap water), GLY 2% (Roundup® Full II formulation -RU), CYP 0.12% (Atanor® formulation), CPF 0.8% (Lorsban® formulation), and a mixture of the three pesticides (Mx3: GLY 2% + CYP 0.12% + CPF 0.8%). We applied early warning biomarkers to detect damage induced by these chemicals in peripheral blood: activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), analysis of lipid peroxidation (LPO) by the thiobarbituric acid reactive substances (TBARS), DNA damage and specific base oxidation through the standard and modified comet assay (CA), chromosome damage by micronucleus (MN) test and other nuclear abnormalities (NAs), hematological and growth parameters. Results showed a statistically significant increase in MN and NAs frequency, DNA damage, with an important contribution of base oxidation for all exposed groups compared to the NC. Total white blood cells count (TWBCC), and growth parameters showed effects mainly at the Mx3. The principal component analysis (PCA) demonstrated more sensitivity for biomarkers associated to genetic damage, including base oxidation to DNA than LPO, antioxidant enzyme modulation, immunotoxicity or growth parameters, to detect pesticides effects, applied under conditions similar to that found in natural environments.

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PURPOSE: The species of the genus Trypanosoma are carried and transmitted by horseflies parasitizing a high diversity of vertebrates. In the Coastal Plain of Rio Grande do Sul, southern Brazil, Tabanus triangulum is the most abundant species and, similarly to the other species of horseflies, there is little knowledge about its vector competence. Therefore, this study aimed to screen the field-collected T. triangulum for the presence of Trypanosoma, to estimate infectivity. METHODS: Horseflies were sampled by the Malaise trap in the forest fragments at the coastal plain and DNA was extracted from whole body flies. The Polymerase Chain Reaction was performed. RESULTS: Horseflies presented amplification of 18S ribosomal gene-specific of Trypanosoma species. DNA sequencing and phylogenetic analysis positioned the strains in the Kaiowa clade with Trypanosoma kaiowa, associated with the crocodilian clade of Trypanosoma. CONCLUSION: This study represents the first report of the presence of the Tr. kaiowa in T. triangulum and the expansion of the parasite's range further south in South America.

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The Proterodiplostomidae Dubois, 1936 is a family of digeneans within the superfamily Diplostomoidea Poirier, 1886. Members of the family are distributed mostly in the tropics and subtropics, primarily in crocodilians, although some species are known from other reptiles. Despite their broad geographical distribution, the knowledge of proterodiplostomid diversity remains limited, mostly because a number of potential host species and regions of the world have not been sufficiently explored for these parasites. In this study, we use morphological and molecular data to describe four new genera (Afroproterodiplostomum gen. nov., Dungalabatrema gen. nov., Australiadiplostomum gen. nov. and Nattererodiplostomum gen. nov.) and five new species (Afroproterodiplostomum ingwenyae sp. nov., Australiadiplostomum blairi sp. nov., Dungalabatrema kostadinovae sp. nov., Dungalabatrema snyderi sp. nov. and Pseudoneodiplostomum angustus sp. nov.) of proterodiplostomids from crocodilians in Australia, South Africa and South America. Nattererodiplostomum gen. nov. has been established upon re-evaluation of the status of Proterodiplostomum medusae (Dubois, 1936) from caimans in Brazil using combined morphological and molecular evidence. Only a few previous studies provided DNA sequence data of proterodiplostomids. We generated partial 28S rDNA and cytochrome c subunit (cox1) mtDNA for three previously undescribed proterodiplostomids collected from Crocodylus spp. in Australia and South Africa. The newly generated 28S sequences were used to examine phylogenetic affinities of these taxa. All three newly sequenced proterodiplostomid species appeared in the phylogenetic tree in a strongly supported monophyletic clade comprising exclusively parasites of Crocodylus.

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Crocodiles are remarkable animals that have the ability to endure extremely harsh conditions and can survive up to a 100 years while being exposed to noxious agents that are detrimental to Homo sapiens. Besides their immunity, we postulate that the microbial gut flora of crocodiles may produce substances with protective effects. In this study, we isolated and characterized selected bacteria colonizing the gastrointestinal tract of Crocodylusporosus and demonstrated their inhibitory effects against three different cancerous cell lineages. Using liquid chromatography-mass spectrometry, several molecules were identified. For the first time, we report partial analyses of crocodile's gut bacterial molecules.

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Environmental contaminants affect ecosystems worldwide and have deleterious effects on biota. Non-essential mercury (Hg) and lead (Pb) concentrations are well documented in some taxa and are described to cause multiple detrimental effects on human and wildlife. Additionally, essential selenium (Se) is known to be toxic at high concentrations but, at lower concentrations, Se can protect organisms against Hg toxicity. Crocodilians are known to bioaccumulate contaminants. However, the effects of these contaminants on physiological processes remain poorly studied. In the present study, we quantified Hg, Pb and Se concentrations in spectacled caimans (Caiman crocodilus) and investigated the effects of these contaminants on several physiological processes linked to osmoregulatory, hepatic, endocrine and renal functions measured through blood parameters in 23 individuals. Mercury was related to disruption of osmoregulation (sodium levels), hepatic function (alkaline phosphatase levels) and endocrine processes (corticosterone levels). Lead was related to disruption of hepatic functions (glucose and alanine aminotransferase levels). Selenium was not related to any parameters, but the Se:Hg molar ratio was positively related to the Na+ and corticosterone concentrations, suggesting a potential protective effect against Hg toxicity. Overall, our results suggest that Hg and Pb alter physiological mechanisms in wild caimans and highlight the need to thoroughly investigate the consequences of trace element contamination in crocodilians.

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All crocodilians exhibit temperature-dependent sex determination without sex chromosomes. This temperature dependency can be overridden by exposure to estrogen via estrogen receptor 1. Thus, the sex ratio of crocodilian species is vulnerable to estrogenic xenobiotics. Multiple investigations of the mechanism and effects of xenobiotics in crocodilian species have been conducted since the early 1990s. This review focuses on the impact of xenobiotics on sex determination rather than gonadal functions in crocodilians. The thermosensitive and estrogen-sensitive periods that commit the bipotential gonad to develop as an ovary end by stages 24.5 and 25.3, respectively. In contrast, it is ambiguous when the estrogen-sensitive stage begins for ovarian development, although the thermosensitive period for ovarian development initiates around developmental stage 15 at an extreme female-producing temperature of 30°C. To accurately assess the effect of xenoestrogens on sex ratio in crocodilians, it is critical to collect eggs before the sex-determining period and to incubate them under precisely controlled temperatures. A well-studied system of xenobiotic effects on crocodilians is Lake Apopka (FL, USA), an EPA superfund clean-up site heavily contaminated with Dieldrin, Endrin, and p,p'-DDE. The sum of estimated estrogenicity of xenobiotics measured in Lake Apopka was insufficient to activate the estrogen receptor 1 of Alligator mississippiensis, which is an essential receptor to induce ovarian development. Although juvenile A. mississippiensis showed gonadal alterations in sex hormone production and histology, the environmentally relevant concentration of xenobiotics in Lake Apopka was unlikely to alter the sex ratio of A. mississippiensis. Experimental exposure to xenobiotics such as 17α-ethynylestradiol, p,p'-dichlorodiphenyldichloroethylene, and 2,3,7,8-tetrachlorodibenzodioxin at environmentally relevant concentrations in ovo induced more female offspring in A. mississippiensis as compared with the control group. Bisphenol-A, atrazine, 2,4-dichlorophenoxyacetic acid, endosulfan, and Corexit did not alter the sex ratio of A. mississippiensis or Caiman latirostris under the tested conditions. Egg-incubation temperature has pronounced effects on estrogen sensitivity in crocodilian sex determination. Therefore, crocodilians are vulnerable to xenobiotic contamination and climate change in the wild. It is vital to further investigate the detailed mechanism and effects of environmental xenobiotics in crocodilian sex determination to mitigate their effect on sex ratio and conserve this ancient lineage.

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Arterial wall tension increases with luminal radius and arterial pressure. Hence, as body mass (Mb) increases, associated increases in radius induces larger tension. Thus, it could be predicted that high tension would increase the potential for rupture of the arterial wall. Studies on mammals have focused on systemic arteries and have shown that arterial wall thickness increases with Mb and normalizes tension. Reptiles are good models to study scaling because some species exhibit large body size range associated with growth, thus, allowing for ontogenetic comparisons. We used post hatch American alligators, Alligator mississippiensis, ranging from 0.12 to 6.80 kg (~ 60-fold) to investigate how both the right aortic arch (RAo) and the left pulmonary artery (LPA) change with Mb. We tested two possibilities: (i) wall thickness increases with Mb and normalizes wall tension, such that stress (stress = tension/thickness) remains unchanged; (ii) collagen content scales with Mb and increases arterial strength. We measured heart rate and systolic and mean pressures from both systemic and pulmonary circulations in anesthetized animals. Once stabilized alligators were injected with adrenaline to induce a physiologically relevant increase in pressure. Heart rate decreased and systemic pressures increased with Mb; pulmonary pressures remained unchanged. Both the RAo and LPA were fixed under physiological hydrostatic pressures and displayed larger radius, wall tension and thickness as Mb increased, thus, stress was independent from Mb; relative collagen content was unchanged. We conclude that increased wall thickness normalizes tension and reduces the chances of arterial walls rupturing in large alligators.

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Mercury (Hg) is a contaminant that is impacting ecosystems worldwide. Its toxicity is threatening wildlife and human populations, leading to the necessity of identifying the most affected ecosystems. Therefore, it is essential to identify pertinent bioindicator organisms to monitor Hg contamination. In this study, we determined the stable carbon (δ13C) and nitrogen (δ15N) isotope ratios in the red blood cells (RBCs), and the total Hg concentration in total blood of 72 Melanosuchus niger in French Guiana. The goals of our study were to assess the level of Hg contamination in total blood of Black caimans and to further investigate the influence of individual traits (i.e., sex, size/age, diet) on Hg concentrations. Mercury concentration in total blood of Black caimans ranged from 0.572 to 3.408 µg g-1 dw (mean ± SD is 1.284 ± 0.672 µg g-1 dw) and was positively correlated to individual body size and trophic position (δ15N). We did not find any sexual or seasonal effects on Hg concentrations in the blood. The use of blood of M. niger is relevant to determine Hg concentrations within the population and suggests that this species can be used as a bioindicator for environmental contamination. In addition, our results emphasize trophic position as a major source of Hg variation and further suggest that it is essential to take trophic position (δ15N) into account for future studies.

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The deleterious effects of mercury (Hg) contamination are well documented in humans and wildlife. Chronic exposure via diet and maternal transfer are two pathways which increase the toxicological risk for wild populations. However, few studies examined the physiological impact of Hg in crocodilians. We investigated the Hg contamination in neonate Smooth-fronted Caimans, Paleosuchus trigonatus, and the use of keratinized tissues and blood to evaluate maternal transfer. Between November 2017 and February 2020, we sampled 38 neonates from 4 distinct nests. Mercury concentration was measured in claws, scutes and total blood. Highest Hg concentrations were found in claws. Strong inter-nest variations (Hg ranging from 0.17 ± 0.02 to 0.66 ± 0.07 µg.g-1 dw) presumably reflect maternal transfer. Reduced body size in neonates characterized by elevated Hg concentrations suggests an influence of Hg during embryonic development. We emphasize the use of claws as an alternative to egg collection to investigate maternal transfer in crocodilians. Our results demonstrated the need of further investigation of the impact of Hg contamination in the first life stages of crocodilians.

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OBJECTIVE: To develop a technique for performing the mandibular nerve block in Nile crocodiles. STUDY DESIGN: Experimental cadaveric study. ANIMALS: A total of 16 juvenile Nile crocodile heads. METHODS: To study the course of the mandibular nerve, one head was dissected. Computed tomography (CT) examination was performed in two heads to identify useful landmarks. Thereafter, a hypodermic needle was inserted through the external mandibular fenestra of 17 hemimandibles (13 heads), and a mixture of methylene blue and iohexol was injected. Injection volumes were 0.5 (n = 7) and 1.0 mL (n = 10) for hemimandibles < 15 and ≥ 15 cm long, respectively. Iohexol spread and nerve staining with methylene blue were assessed with CT and anatomical dissection, respectively. Data were analysed with one-sample t test or Mann-Whitney U test. Significance was set at p < 0.05. RESULTS: Both anatomical dissection and imaging confirmed the external mandibular fenestra as a useful anatomical landmark for needle insertion. The CT images acquired after needle positioning confirmed that its tip was located on the medial bony mandibular surface formed by the fusion of the angular and coronoid bones in 100% cases. In all the hemimandibles, the rostrocaudal spread of contrast was > 23 mm. The length of the stained mandibular nerve in the temporal region and of the stained medial branch of the mandibular nerve, as well as the dorsoventral and mediolateral spread of iohexol, was greater in group 1.0 than in group 0.5 (p < 0.001). The caudal spread of iohexol was greater in group 1.0 than in group 0.5 (p = 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: The technique developed in this study is feasible. Both injection volumes resulted in staining of the mandibular nerve. The spread of contrast in the anatomical region of interest may result in successful sensory block.

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The State of Mexico has a unique combination of geographic characteristics and topography that promotes a high biodiversity. Unfortunately, continued human population growth of the metropolitan areas of Mexico City and Toluca have degraded the environment of the State of Mexico, which threatened its wildlife. An updated checklist of the amphibians and reptiles of the State of Mexico is provided and their conservation status summarized. The State of Mexico has 49 species of amphibians and 101 species of reptiles. The majority of the amphibians (73.5%) and reptiles (70.3%) found in the State of Mexico are endemic to Mexico. Of the amphibian and reptile species in the State of Mexico, 20.1% are IUCN listed (i.e., Vulnerable, Near Threatened, or Endangered), 18.4% are placed in a protected category by SEMARNAT (excluding NL and Pr, this last category is equivalent to the LC category of IUCN), and 34.9% are categorized as high risk by the EVS. The importance of forested habitats for the protected amphibians and reptiles in the State of Mexico suggest that management of these habitats to maintain or expand them needs to be considered.