Gastrolith ingestion is a known inherent behaviour in crocodiles. However, the interpretation of coelomic radiographs in crocodilians is challenging due to the limited informative literature in this field. Dorsoventral coelomic radiographs of 49 juvenile saltwater crocodiles (Crocodylus porosus) were acquired twice, with an interval of 4 months. Any gastroliths present were evaluated and classified as sand, small, medium and large, relative to the size of the crocodile, by comparing the gastrolith size to the length of the 11th dorsal vertebral body. A total of 1971 gastroliths were recorded during the two examinations (mean of 21 per crocodile, range 0‒98). Small gastroliths were the most commonly recorded (94%). There was no significant correlation between weight gain and gastrolith numbers in either examination (p = 0.506 and 0.2034). There was a significant reduction in small gastroliths (p = 0.034) and a significant increase in medium gastroliths (p = 0.023) between examinations. All radiographs were performed on a cohort of juvenile crocodiles at the same collection. Orthogonal lateral radiographic projections were not performed due to practical and ethical limitations. Large numbers of relatively small gastroliths are likely a normal radiographic finding in juvenile crocodiles. As crocodiles increase in size, ingestion and retention of medium/large gastroliths may reduce body buoyancy to aid in increasing dive times and hunting of larger prey.
Crocodilians are well endowed with multiple cutaneous receptors and specializations, such as integumentary sensory organs (ISOs), which provide formidable mechanical sensitivity despite their protected shield. We investigated the free intraepidermal nerve terminals, focusing on the desmosomes, transitional desmosomes (TDs), corneodesmosomes (CD), and the tonofibril (TF) network that potentially act as force transducers to activate the mechanoreceptors. Two Cuban crocodiles (Crocodylus rhombifer) were analyzed using light and transmission electron microscopy (TEM) after glutaraldehyde fixation and decalcification. Discoid nerve terminals were richly enclosed by an epidermal force-transmitting system (e.g., pressure and vibration) through a rigid network of diverse desmosomes and CDs. TDs were anchored to keratinocyte's cytoskeletons via a dense meshwork of intermediate filaments or TFs, creating a continuous, mechanically-linked web connecting nerve terminals in the epidermis to the stratum corneum. The cutaneous receptors were innervated by myelinated and unmyelinated neural complexes surrounded by thin-walled mesothelial cells. Here, we describe for the first time the ultrastructure of TDs in the crocodile skin with diverse expression of CDs that may focus and amplify force via a tonofibril system "hugging" the receptor. Corneocytes, granular keratinocytes, and nerve endings function as a single integrated system. Thereby, mechanical strain is gathered from a relatively large area of the epidermis and concentrated onto the small surface of the discoid receptor. This may ensure that any deformation of the surrounding corneocytes is efficiently and reliably transferred to the nerve membrane, allowing the crocodile to detect very subtle stimuli. The crocodile system appears to have a far more structured and specialized adaptation for high-fidelity mechanosensation than that of humans.
The intestine of a large adult (male) Nile crocodile, Crocodylus niloticus (Laurenti, 1768) (Crocodilia: Crocodylidae) from a site (18°08'24.5''S, 21°40'58.4''E) on the Kavango River (Namibia) was infected by Ngubuvangandu francoisjacobsi Dutton and Bullard n. gen., n. sp. (Digenea: Liolopidae). The new genus and species differs from all other liolopids by the combination of having a linguiform body that is ∼3 times longer than wide, a weakly muscular ventral sucker (with pre- and post-ventral sucker distances equal), lobed testes that nearly span the intercecal space and that occupy the posterior one-third of the body, a posterior testis that occupies the space between the tips of the posterior ceca, an ovary abutting the anterior testis, a vitellarium that extends anterior to the ventral sucker (not reaching cecal bifurcation), and a uterus that traverses the intercecal space immediately anterior to the anterior testis. We herein reassign two previously named crocodilian liolopids formerly of Dracovermis Brooks and Overstreet, 1978 to the new genus: Ngubuvangandu brayii (Baylis, 1940) Dutton and Bullard, 2024 (infecting the west African slender snouted crocodile, Mecistops cataphractus Cuvier, 1825 in the Congo River) and Ngubuvangandu rudolphii (Tubangui and Masilungan, 1936) Dutton and Bullard, 2024 (infecting the saltwater crocodile, Crocodylus porosus Schneider, 1801 in the Philippines). The phylogenetic analysis recovered the new genus sister to Liolope Cohn, 1902. That clade was sister to Harmotrema Nicoll, 1914, with Dracovermis sister to the clade and Paraharmotrema Dutton and Bullard, 2022, sister to all liolopids analyzed. This result demonstrates that the crocodilian liolopids are paraphyletic, rejecting the notion that natural groups of liolopids can be defined by the definitive host they infect (i.e., no evidence of phylogenetic host specificity of liolopid genera/lineages). This is the first liolopid described from the Nile crocodile and only the second liolopid species described from an African crocodilian.
The isolation of fibroblast-like cells from crocodile skeletal muscle provides a valuable platform for studies in comparative physiology, reptilian biology, regenerative medicine, and tissue engineering. In this article, we present an optimized protocol for isolating and characterizing fibroblast-like cells derived from the embryonic skeletal muscle of the Siamese crocodile (Crocodylus siamensis). The procedure improved cell yields and viability while maintaining phenotypic and genetic stability. Dorsal and tail skeletal muscle tissue was cultured in flasks pre-coated with collagen. The cells attached and began migrating from the explants within one day. Optimal culture conditions were achieved using Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 or Minimum Essential Medium Alpha supplemented with 10% fetal bovine serum, 2% crocodile serum, and growth-promoting factors, incubated at 28 °C without CO2. These conditions supported a shorter population doubling time and enhanced cell proliferation. The established cells displayed a spindle-shaped fibroblastic morphology, expressed the fibroblast-specific marker fibronectin, and maintained a stable karyotype through passage 20. The cell line remained stable and proliferative for at least 30 passages under standard culture conditions. Our study established the first standardized fibroblast-like cell line derived from C. siamensis, thus providing a foundation for future studies in reptilian physiology, cytogenetics, and regenerative biotechnology.
The West African dwarf crocodile (Osteolaemus tetraspis), classified as Vulnerable by the IUCN and listed under CITES Appendix I, is increasingly threatened by unregulated trade and habitat degradation. This study investigated the trade dynamics of the species across Delta and Edo States, Nigeria, focusing on market structure, trade routes, seasonal trends, and size-class composition. Market surveys, semi-structured interviews, and ecological observations were employed to examine trade dynamics of the West African dwarf crocodile from 2019 to 2021 identified nine key markets, with Ugbenu emerging as the primary hub, recording 1,818 individuals over 105 market days. Trade was dominated by mid-sized, sexually mature crocodiles (101-120 cm), while juveniles (0-20 cm and 21-40 cm) were scarcely represented, suggesting depletion of older cohorts. The trade is economically motivated and gendered, with middlewomen controlling over 80% of market flow, sourcing from remote riverine communities. Hunting methods included wire traps, baited hooks, and burrow excavation, raise ecological and welfare concerns. Findings highlight unsustainable exploitation and underscore the need for urgent conservation measures, including legal enforcement, harvest regulations, and community-based monitoring aligned with CITES and IUCN guidelines.
Crocodilians rarely develop cancer despite long lifespans and continuous exposure to environmental carcinogens, suggesting robust natural anti-tumor defense mechanisms. We investigated the anti-cancer activity of sera derived from the phylogenetically related species-alligators, crocodiles, and chickens, and studied their underlying immune mechanisms. The anti-tumor activity of alligator serum was tested in murine models of melanoma and lymphoma. Alligator serum (AS) and its (NH4)2SO4-precipitated fraction (ASa) showed rapid and potent cytotoxicity toward multiple murine and human cancer cell lines while sparing non-malignant human cells. Importantly, ASa attenuated melanoma and lymphoma tumor growth in mice. Electrophysiological analyses in PN71 cancer cells treated with ASa revealed rapid membrane depolarization and formation of high-conductance pores consistent with Complement-mediated membrane attack complex (MAC) activity. Proteomic analyses identified the Complement component C5 as a major protein enriched in active fractions, implicating the Complement system in cancer cell killing. Based on phylogenetic similarity of C5, crocodile and chicken sera exhibit alligator-like comparable anti-cancer activity. Mechanistic studies in chicken serum showed that the anti-cancer activity depends on Ca2+ and Mg2+ ions, terminal Complement components (C5-C8), and IgM antibodies that initiate Complement activation. Immunodepletion of IgM from CSa significantly reduced cytotoxicity, whereas purified chicken IgM activated human Complement to induce cancer cell death. These findings identify a conserved IgM-Complement immune mechanism capable of selectively targeting malignant cells. The evolutionary conservation and cross-species functionality of this pathway highlight its potential as a bio-inspired strategy for developing novel Complement-based cancer immunotherapies.
Florfenicol (FFC) is widely used to treat bacterial infections in veterinary medicine; however, its pharmacokinetic characteristics in reptiles remain limited. This study investigated the pharmacokinetic profiles of FFC after intramuscular (IM) injection at doses of 20 or 30 mg/kg body weight (b.w.) in freshwater crocodiles (Crocodylus siamensis). A sample of 10 healthy crocodiles was randomly divided into two groups (n = 5 for each group) according to a parallel study design. Blood samples were obtained from pre-dose to 168 h post-administration. Plasma FFC concentrations were quantified using high-performance liquid chromatography with diode array detection (HPLC-DAD) and analyzed by non-compartmental analysis. The mean maximum plasma concentrations of FFC were 4.05 µg/mL and 6.11 µg/mL for the 20 and 30 mg/kg b.w. doses, respectively. The mean elimination half-lives of FFC were long but not significantly different (51 h). The average plasma protein binding was 37.15%. Based on the pharmacokinetics/pharmacodynamics (PK/PD) index, a single dose of FFC via IM elicited plasma concentrations above the MIC90 values reported for several susceptible bacterial pathogens. Consequently, both dose levels provided plasma exposure consistent with previously reported reference MIC values. However, further PK/PD and multiple-dose investigations are needed to refine species-specific dosage regimens.
Crocodylus niloticus and Crocodylus siamensis are high-value aquaculture species. C. niloticus is large-bodied but less abundant, while C. siamensis grows fast but is small-sized. Their hybrids combine parental advantages, yet relevant research is scarce. This study compared the histological and transcriptomic characteristics of the caudal muscle across the three taxa. HE staining indicated that C. niloticus had significantly larger myofiber diameters (p < 0.05); C. siamensis had the smallest, and the myofiber density of hybrids was much closer to that of C. siamensis. Masson's trichrome staining indicated that C. niloticus had the thickest collagen fibers (p < 0.05), C. siamensis the thinnest, and hybrids exhibited highly similar histological traits to C. siamensis. C. niloticus had higher LDH and SDH activities in caudal muscles, whereas the hybrid crocodile indicated the highest CK activity. Transcriptomic analysis identified numerous differentially expressed genes (DEGs), which were enriched in growth, muscle metabolism, and energy allocation pathways via GO/KEGG annotations. PPI analysis screened 24 hub genes related to energy metabolism. This study systematically reveals caudal muscle differences, providing insights into growth-related molecular mechanisms and theoretical support for crocodile artificial breeding.
Entamoeba protozoa are known to cause enteric and sometimes systemic disease in both domestic and non-domestic animals. In amphibians, Entamoeba spp. are primarily documented as incidental gastrointestinal inhabitants, with rare reports of clinically significant entamoebiasis. In the last decade, mortality outbreaks caused by a recently described protozoan, Entamoeba sp. CT1, have been documented in free-ranging anurans in Australia and managed anurans in the USA associated with gastroenteritis and substantial morbidity and mortality. In 2023, a small population of Anderson's crocodile newts Echinotriton andersoni in managed care at a zoological institution experienced a disease outbreak resulting in the death or euthanasia of 4/13 (31%) newts over an approximately 1 mo period. Clinical signs included dehydration, poor skin quality, and lethargy. Postmortem examination of 3 newts revealed marked, proliferative, and necrotizing inflammation of the intestinal tract, urinary bladder, and cloaca with intramucosal amoebic protozoa. The gonadal ducts (2/3 newts) were also variably affected. Entamoeba PCR and Sanger sequencing of formalin-fixed paraffin-embedded intestinal tissue from all examined newts amplified DNA with 100% sequence identity to the recently identified Entamoeba sp. CT1. However, unlike reports of Entamoeba sp. CT1 in anurans, lesions in the crocodile newts were not restricted to the gastrointestinal tract. This is the first report of Entamoeba sp. CT1 causing significant clinical disease in caudates, emphasizing its importance as an amphibian pathogen with the potential to cause disease in managed and free-ranging amphibian populations.
Crocodile blood is a rich and valuable source of bioactive compounds derived from natural products. Crocodile blood powder (CP) has garnered significant attention for its potential applications in human health treatment. This study aimed to investigate the effect of CP on the invasion and metastasis of hepatocellular carcinoma (HepG2) cells. We analyzed the protein content of CP using MS/MS techniques. The effects of CP on cell proliferation, apoptosis, metastasis, and invasion were assessed using immunofluorescence, a wound healing assay, a transwell invasion assay, and Western blot analysis, respectively. The findings indicated that CP could inhibit the proliferation of HepG2 cell lines. Additionally, CP increased caspase-3 expression, inducing apoptosis in HepG2 cells. CP treatment also reduced metastasis and invasion of HepG2 cells. Immunofluorescence and Western blot analyses revealed that CP upregulated E-cadherin expression, while downregulating MMP-2 and MMP-9 expression. Overall, this study demonstrated that CP inhibits HepG2 cell proliferation and promotes apoptosis. Furthermore, CP suppresses metastasis and invasion by increasing E-cadherin expression and downregulating MMP-2 and MMP-9. Thus, CP may serve as a promising candidate for hepatocellular carcinoma therapy.
Mycobacterium ulcerans ecovar Liflandii has recently been described as the causative agent of fatal disease in different frog species. This is the first report of this pathogen in a Cuban crocodile (Crocodylus rhombifer), which died suddenly in a Hungarian zoo. The authors' findings suggest that M. ulcerans ecovar Liflandii does not exclusively infects anurans and highlights the zoonotic risk posed by this emerging pathogen.
Tooth enamel of most mammals shows alternating light and dark bands, called Hunter-Schreger bands (HSB), in longitudinal sections caused by decussating bundles of prisms, the unit building blocks of mammalian enamel. HSB are thought to increase resistance to abrasive food and mitigate crack propagation and hence are considered a mammalian adaptation to high-efficiency mastication. Using traditional microscopy techniques as well as X-ray diffraction computed tomography (XRD-CT), here we report for the first time the presence of HSB-like features in the tooth enamel of a non-mammalian amniote, Iharkutosuchus, an extinct herbivorous crocodile with strong heterodonty and a unique chewing mechanism. XRD-CT showed that the enigmatic HSB-like pattern in Iharkutosuchus enamel, which lacks mammal-like decussating prisms, has a purely crystallographic origin. Iharkutosuchus teeth also exhibit wavy enamel, a well-known structure in herbivorous ornithopod dinosaurs with shearing-type mastication. The unexpected finding of both enamel features in this herbivorous crocodile speaks for their role in high-efficiency chewing. However, the profoundly different structural background of mammalian and crocodilian HSB demonstrated here and the phylogenetic distribution of both HSB and wavy enamel indicate nanostructure-scale convergences, highlighting the importance of mastication-related challenges in driving dental evolution of amniotes.
Assessing the species distribution and their detectability is crucial, holding wide-ranging implications for effective conservation planning and management initiatives. Although species-habitat relationships are inherently scale-dependent, few studies apply robust multivariate approaches to optimize spatial scale selection. We developed a scale-optimized habitat suitability model for the mugger crocodile (Crocodylus palustris) using binomial generalized linear models, evaluating each predictor across multiple spatial scales within the Cauvery River Basin (CRB) in southern India. Model selection based on the lowest Akaike information criterion scores identified the multiscale modeling as the best performing approach. Most predictors showed the strongest associations at finer (500 m) to moderate (1000-2000 m) scales, while a subset of topographic and hydrological variables was retained at broader scales (8000 m), showing the importance of incorporating scale heterogeneity in riverine species modeling. Key variables influencing the potential distribution of muggers across the CRB include isothermality, radius of gyration area-weighted mean of wetland, distance to roads, and terrain wetness, indicating mugger prefer stable temperature, low disturbance, and localized patches but well-distributed wetland habitats. The multiscale model estimated 2209.5 km2 of potentially suitable habitat across the CRB of which only 38.12% lies within the existing protected area network. Integrating the best performing model into a systematic conservation planning framework that maximizes species target while minimizing human impacts, the solution identified 990 km2 of priority regions, including five high-priority areas with a total area of 540 km2, outside the current protected network. The study offers a robust and resource-efficient approach to habitat delineation and conservation prioritization, improving the performance of suitability modeling across spatially varying environmental factors.
Muscle atrophy is relatively common among older adults and can markedly impair their physical function and overall quality of life. Crocodile head-derived bioactive peptides (CP), with high bioavailability and low allergenicity, show promise as a nutritional intervention. This study aimed to investigate the alleviating effects of CP on muscle atrophy and clarify their underlying action mechanisms. Results demonstrated that CP alleviated muscle atrophy-related weight loss, increasing gastrocnemius, quadriceps, and tibialis anterior muscle indices by 60%, 33%, and 28%. The lean body mass percentage increased by 3.7%, while the body fat rate decreased by 6%. Following CP administration, grip strength, motion displacement, and exhaustion time in mouse recovered to 1.71 N, 495 m, and 1130 s. Proteomic analysis revealed that CP potentially ameliorates muscle atrophy by modulating the AMPK signaling pathway to restore energy metabolism homeostasis, activating autophagy to clear dysfunctional organelles, and reprogramming lipid metabolism to suppress ectopic fat deposition in muscle tissue. Analysis of the gut microbiota further indicated that CP intervention significantly increased the abundance of beneficial bacteria, including Muribaculaceae, Allobaculum, Lactobacillus, Monoglobus, and Dubosiella. In conclusion, CP likely mitigates muscle atrophy progression via multi-target mechanisms, including modulation of energy metabolism, autophagy, lipid metabolism, and the gut microenvironment.
Pseudocarcharias kamoharai (Matsubara, 1936) (Elasmobranchii: Lamniformes) is a small, viviparous, pseudocarchariid shark distributed throughout tropical ocean waters worldwide. Although the appendicular skeleton of elasmobranchs has been explored in the literature, little is known about the clasper skeleton. Additionally, the appendicular musculature in general is not well studied in elasmobranchs, especially that of the pelvic fins and claspers. The same can be said about the siphon sac, a muscular bladder associated with the clasper and related to reproduction in male elasmobranchs. Here, we aim to expand the amount of morphological information related to the secondary sexual characteristics and to describe the pelvic musculature and skeleton, including a description of the clasper of the crocodile shark. The connected siphon sacs have a general inverted "Y" shape, with their anterior margins "M"-shaped and their posterior portions forked. The musculature of the ventral surface of the pelvic fin in both sexes is similar. However, the musculature of the dorsal surface of the male has an extra muscle in comparison to the female, the flexor internus. Moreover, the clasper has a few exclusive muscles, such as the dilatator. In relation to the clasper skeleton, its morphology follows what is seen in other lamniforms. This study marks the first report of sexual dimorphism related exclusively to the pelvic fin musculature in Lamniformes.
Population fragmentation and restricted dispersal in endangered species can sharply reduce genetic diversity and adaptive potential, making genetic monitoring vital for conservation. However, whether neutral genetic markers alone can adequately assess adaptive potential remains debated. We examined the fine-scale genetic structure and diversity of the endangered Chinese crocodile lizard (Shinisaurus crocodilurus) in three closely located streams: DaChai (DC), YuSan (YS), and ChiShui (CS). Despite their proximity, we found clear genetic differentiation, with DC especially distinct from the CS-YS group. Both subpopulations showed reduced genome-wide diversity and signs of inbreeding. Interestingly, the MHC gene region showed a different pattern. DC individuals had higher MHC diversity, while CS-YS showed signatures of recent selection. These results suggest different evolutionary pressures at immune-related loci in the two subpopulations and highlight the importance of integrating both neutral and functional genetic markers in conservation, as neutral markers alone may overlook key adaptive capacities.
暂无摘要（点击查看详情）
暂无摘要（点击查看详情）
[This corrects the article DOI: 10.3389/fmicb.2025.1697111.].
暂无摘要（点击查看详情）