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The Chinese alligator (Alligator sinensis) is currently an endangered species due to a combination of factors, including climate change, anthropogenic activities, and habitat fragmentation. Captivity plays a crucial role in mitigating the decline of the Chinese alligator population. Currently, there is a lack of clarity regarding the influence of host development and captive conditions on the gut microbiota of Chinese alligators. The aim of the study was to investigate the gut bacterial communities of Chinese alligators and their surrounding environmental bacterial communities using 16S rRNA sequencing. The primary gut flora of Chinese alligators consists of Proteobacteria, Bacteroidetes, and Firmicutes. Proteobacteria is the most abundant and efficient settler in the gut, water, and sediment. PCoA and Adonis test revealed significant differences in bacterial communities across these habitats. Venn analysis revealed overlap in OTUs among the gut, water, and sediment, varying with growth stage and density stress. Different growth stages of Chinese alligator guts harbor distinct pathogenic bacteria, requiring attention. Density stress leads to an increase in pathogenic bacteria, a decrease in gut absorption efficiency. PICRUst2 predicts more abundant metabolic pathways related to gut function during high‐density stress, possibly linked to Roseburia. SourceTracker Analysis indicated that water bacteria have a greater impact on Chinese alligator gut bacteria than sediment, and density stress significantly affects the contribution of environmental microorganisms to the gut microbes of Chinese alligator. BugBase analysis identified water body microbes as the main source of "potentially pathogenic" phenotypes in the gut microbiota. RDA analysis found dissolved oxygen (DO) in water to be the most significant factor influencing water microorganisms, positively correlated with certain pathogenic strains. These findings enhance our understanding of the significance of microbial communities in the gut and surrounding aquatic environment of the Chinese alligator. Furthermore, they provide theoretical support for environmental regulation, disease control, and healthy breeding. [ABSTRACT FROM AUTHOR]

Simple Summary: A better understanding of the hindlimb developmental process will provide data support for the comparative evolutionary and functional genomics of crocodilians. For the Chinese alligator, the hindlimb is one of the main sources of power, and its development and differentiation will directly influence the survival ability in the wild. But little is known about the molecular mechanisms regulating the development and differentiation of the hindlimb of the Chinese alligator. In this study, RNA-sequencing technology was used to analyze the gene expression of the hindlimb in the Chinese alligator embryos at different stages (on days 29, 35, 41, and 46). Differentially expressed genes (DEGs) at different stages were identified. Our results will provide data support for the embryonic development process of the Chinese alligator. Crocodilians, which are a kind of animal secondary adaptation to an aquatic environment, their hindlimb can provide the power needed to engage in various life activities, even in low-oxygen water environments. The development of limbs is an important aspect of animal growth and development, as it is closely linked to body movement, support, heat production, and other critical functions. For the Chinese alligator, the hindlimb is one of the main sources of power, and its development and differentiation will directly influence the survival ability in the wild. Furthermore, a better understanding of the hindlimb developmental process will provide data support for the comparative evolutionary and functional genomics of crocodilians. In this study, the expression levels of genes related to hindlimb development in the Chinese alligator embryos during fetal development (on days 29, 35, 41, and 46) were investigated through transcriptome analysis. A total of 1675 differentially expressed genes (DEGs) at different stages were identified by using limma software. These DEGs were then analyzed using weighted correlation network analysis (WGCNA), and 4 gene expression modules and 20 hub genes were identified that were associated with the development of hindlimbs in the Chinese alligator at different periods. The results of GO enrichment and hub gene expression showed that the hindlimb development of the Chinese alligator embryos involves the development of the embryonic structure, nervous system, and hindlimb muscle in the early stage (H29) and the development of metabolic capacity occurs in the later stage (H46). Additionally, the enrichment results showed that the AMPK signaling pathway, calcium signaling pathway, HIF-1 signaling pathway, and neuroactive ligand–receptor interaction are involved in the development of the hindlimb of the Chinese alligator. Among these, the HIF-1 signaling pathway and neuroactive ligand–receptor interaction may be related to the adaptation of Chinese alligators to low-oxygen environments. Additionally, five DEGs (CAV1, IRS2, LDHA, LDB3, and MYL3) were randomly selected for qRT-PCR to verify the transcriptome results. It is expected that further research on these genes will help us to better understand the process of embryonic hindlimb development in the Chinese alligator. [ABSTRACT FROM AUTHOR]

Although oviductal sperm storage are essential steps in reproduction for female animals with internal fertilisation, no systematic study on the identification of genes involving sperm storage has been performed in crocodilian species. In the present research, the relationship between morphological variation related to sperm storage in the oviduct and gene expression patterns derived from RNA sequencing analyses between active period (AP), breeding period (BP), and hibernation period (HP) were investigated. The corresponding results indicated that sperm were observed not only in the ciliated cells within infundibulum and mucosal layer of uterus during BP, but also been detected in the spermatosperm storage tube (SST) in the anterior uterus at HP stage. The further transmission electron microscopy analysis indicated that the differences in the number and activity of the secretory cells likely to attributed to the seasonal variation of microenvironment related to the sperm storage. Based on the RNA-sequecing, 13 147 DEGs related to the Peroxisome proliferator-activated receptors (PPARs) and FOXO signalling were identified, including these, the down-regulated ATG12 and BCL2L11 in the HP group may thus constitute an important point of convergence between autophagy and apoptosis involving the FOXO1 pathway. The genes involved in the PPARs pathway might modulate the immune response and thereby contribute to prolong the life span of stored spermatozoa in Alligator sinensis. The outcomes of this study provide fundamental insights into the mechanism of sperm storage in A. sinensis. The differences in the number and activity of the secretory cells in the oviductal epithelium is likely attributed to the seasonal variation of the microenvironment in the lumen. A list of DEGs related to the PPARs, FOXO, and steroid biosynthesis pathways were identified and comprehensively profiled. This study provides fundamental insights into the mechanism of sperm storage in A. sinensis. [ABSTRACT FROM AUTHOR]

The Chinese alligator (Alligator sinensis) is an endemic and rare species in China, and is considered to be one of the most endangered vertebrates in the world. It is known to hibernate, an energy-saving strategy against cold temperatures and food deprivation. Changes in gene expression during hibernation remain largely unknown. To understand these complex seasonal adaptive mechanisms, we performed a comprehensive survey of differential gene expression in heart, skeletal muscle, and kidney of hibernating and active Chinese alligators using RNA-Sequencing. In total, we identified 4780 genes differentially expressed between the active and hibernating periods. GO and KEGG pathway analysis indicated the likely role of these differentially expressed genes (DEGs). The upregulated DEGs in the active Chinese alligator, CSRP3 , MYG and PCKGC , may maintain heart and skeletal muscle contraction, transport and storage of oxygen, and enhance the body's metabolism, respectively. The upregulated DEGs in the dormant Chinese alligator, ADIPO , CIRBP and TMM27 , may improve insulin sensitivity and glucose/lipid metabolism, protect cells against harmful effects of cold temperature and hypoxia, regulate amino acid transport and uptake, and stimulate the proliferation of islet cells and the secretion of insulin. These results provide a foundation for understanding the molecular mechanisms of the seasonal adaptation required for hibernation in Chinese alligators, as well as effective information for other non-model organisms research. Chinese alligators (Alligator sinensis) are an endemic and rare species in China. We performed RNA-Sequencing to compare the transcriptome of the Chinese alligator during hibernation and active periods in order to better understand the mechanisms underlying seasonal differences in activity level. Results indicate that some differentially expressed genes play a crucial role in a wide range of fundamental physiological processes in Chinese alligators. These data may provide valuable and useful resources for further studies of Chinese alligators. Photo by Hongji Sun. [ABSTRACT FROM AUTHOR]

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. [ABSTRACT FROM AUTHOR]

The digestive tract is the largest endocrine organ in the body; the distribution pattern of endocrine cells varies with different pathological and physiological states. The aim of the present study was to investigate the distributed density of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS) and vasoactive intestinal peptide (VIP) immunoreactive (IR) cells in the digestive tract of Alligator sinensis during the active and hibernating period by immunohistochemical (IHC) method. The results indicated that 5-HT-IR cells were distributed throughout the entire digestive tract, which were most predominant in duodenum and jejunum. The density increased significantly in stomach and duodenum during hibernation. GAS-IR cells were limited in small stomach and small intestine. The density decreased significantly in small stomach during hibernation, while increased in duodenum. What's more, most of the endocrine cells in duodenum were generally spindle shaped with long cytoplasmic processes ending in the lumen during hibernation. SS-IR cells were limited in stomach and small stomach. The density increased in stomach while decreased in small stomach during hibernation, meanwhile, fewer IR cells occurred in small intestine. VIP-IR cells occurred in stomach and small stomach. The density decreased in small stomach, while increased in stomach during hibernation. These results indicated that the endocrine cells in different parts of digestive tract varied differently during hibernation, their changes were adaptive response to the hibernation. [ABSTRACT FROM AUTHOR]

Highlights: [•] DDTs were the most dominant PHCs in Chinese alligators. [•] Both neonates and eggs had significantly higher ∑PCBs and ∑DDTs than adult muscles. [•] DDTs in Chinese alligator eggs could cause sex reversal. [•] First report on PBDEs in crocodilia and first study on PHCs in Chinese alligators. [Copyright &y& Elsevier]

Sex steroid hormones play an important role in mediating physiological responses and developmental processes through their receptors across all vertebrates. Chinese alligator ( Alligator sinensis ) is a critically endangered reptile species unique to China. In this study, we have cloned one of the sex steroid hormone receptor genes, androgen receptor (AR) from the brain of Chinese alligator for the first time. The full-length AR cDNA is 2717 bp in length with an open reading frame (ORF) encoding 722 amino acids. Amino acid alignment analyses indicated that the ARs exhibit highly conserved functional domains. Especially, the P-box and D-box, which are essential to ensure that receptor binding to the androgen response elements, are completely conserved in selected species. Using the quantitative real-time PCR (qPCR), the spatial expression of four receptor mRNAs in all newborn brain tissues and temporal expression of them in the cerebrum during the embryonic development in Chinese alligators were investigated. The results of qPCR showed ubiquitous expression of the four receptor mRNAs in all newborn brain tissues examined and significant changes in the expression levels of these receptor mRNAs in the embryonic development. These results suggest that sex steroid hormones might play an important role in the regulation of complex neuroendocrine activities in newborn Chinese alligator. Furthermore, these data provide an important foundation for further studies on endocrinology and molecular biology of non-mammalian sex steroid hormone receptors. [ABSTRACT FROM AUTHOR]

The Chinese alligator Alligator sinensis is a critically endangered species endemic to China. Knowledge about reproductive strategies of a species contributes to their conservation. Little is, however, known about the reproductive strategies and its impact on the population. In the present study, an easy and non-invasive genetic method was used to improve the understanding of mating system of Chinese alligators and its effect on the population genetic diversity by nine polymorphic microsatellite loci. There was a high incidence of multiple paternity among 50 clutches, with a total 60% of the clutches having multiple paternity and up to three males contributing to single clutches. In addition, polyandry females choose to mate with males that are more distant in relatedness compared with monogamy females. Multiple paternity can decrease the inbreeding coefficient, while there is no significant difference between single and multiple paternity ( P > 0.05). Furthermore, there was an increased allelic diversity (though not heterozygosity) in multiple paternity sired offspring compared with the single paternity sired offspring in F2 generations ( P < 0.05), as predicted by the genetic diversity hypothesis. Multiple paternity may function as an important inbreeding avoidance compensation mechanism leading to the potential of the species to avoid extinction. These findings will not only enhance the understanding of the mating system and the biological traits of the Chinese alligator, but also improve the captive breeding program management and conservation strategies of the endangered species. [ABSTRACT FROM AUTHOR]

Estrogens play critical roles in reproductive physiology via estrogen receptors (ESRs) in vertebrates, including reptiles. Chinese alligator ( Alligator sinensis ) is an endemic and endangered reptile species in China. In the present study, we cloned ESR2 gene from the ovary using rapid amplification of cDNA ends (RACE), investigated the spatial expression of ESRs in various tissues and temporal expression of ESRs in the pituitary glands during the reproductive cycle in Chinese alligators by quantitative real-time PCR (qPCR). Bioinformatics and phylogenetic analysis of deduced ESR2 protein were also performed. The full-length cDNA of the ESR2 is 1647 bp in length, with an open-reading frame encoding 548 amino acids. The bioinformatics analysis indicated that the deduced amino acid sequence of alligator ESR2 was highly conserved with that of other vertebrate species. In addition, compared to human ESR2, the 14 amino acids in the alligator ESR2 that are essential for specific recognition of estradiol are entirely conserved. The phylogenetic analysis showed that alligators were more closely related to birds than to other reptiles. The results of qPCR showed that the tissue distribution patterns of both ESR subtype mRNAs appeared to be different. In male tissues, the highest mRNA level of both ESRs is in the liver. While in female tissues, ESR1 and ESR2 showed the highest mRNA level in the hypothalamus and pituitary gland, respectively. During the female reproductive cycle, the expression level of ESR1 mRNA increased from the initial post-hibernation period to the reproductive period, reached its peak in the reproductive period, and then decreased in the autumn active period and hibernation period. Conversely, the highest transcription level of ESR2 was observed in the hibernation period. [ABSTRACT FROM AUTHOR]

The Chinese alligator Alligator sinensis is an endangered species endemic to China, up to date, little is known about the regulation of its growth and development. Insulin-like growth factor I (IGF-I) plays a vital role in regulating vertebrate growth and development. In this study, the full-length cDNA of IGF-I in Chinese alligator (caIGF-I) was obtained for the first time, it contains 890-bp nucleotides encoding a 153-amino acid precursor, the mature caIGF-I consists of 70 amino acids by cleaving the signal peptide and C-terminal extension (E domain). The caIGF-I contains all the features of IGF-I peptide with B, C, A, and D domains and the six conservative cysteine residues involved in the stable tertiary structure. Multiple alignment analysis showed that the amino acid sequence of caIGF-I shares high identity with American alligator Alligator mississippiensis (100%) and birds (95–97%). Phylogenetic tree analysis of the IGF-I amino acid sequences indicated that alligators cluster into the bird branch. Real-time quantitative PCR technique showed that caIGF-I is widely expressed in all the examined tissues with the highest expression level in liver, higher in pancreas and oviduct while lower in heart, spleen, lung, kidney, stomach, intestines, ovary and muscles. During hibernation, the caIGF-I expression level decreased significantly in liver, pancreas, oviduct and kidney, while did not significantly change in heart, spleen, lung, stomach, small intestine, ovary and muscles. The mRNA expression changes during the two periods implicate that caIGF-I might play an important role in the regulation of feeding and growth in the Chinese alligator. [ABSTRACT FROM AUTHOR]

Chinese alligator, Alligator sinensis , is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha ( ERα / ESR1 ) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764 bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period ( P < 0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues ( P > 0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles. [ABSTRACT FROM AUTHOR]

Crocodiles are susceptible to infection with a wide array of external and internal gastrointestinal helminths, yet little is known on the histopathology following infection or the effects of these parasites. The present study was aimed at evaluating the impact of infection by Ortleppascaris sinensis (Nematoda: Ascaridoidea) on the stomach of captive Alligator sinensis. The histological examination of the stomach revealed presence of superficial ulcer in mucous layer and granulomatous inflammation in submucous layer at entire gastric walls of the Alligator sinensis. Our findings also confirm that development of Ortleppascaris sinensis is in close association with the wall of the stomach. [ABSTRACT FROM AUTHOR]

The follicle-stimulating hormone (FSH) plays a central role in vertebrate reproduction, with the actions of FSH mediated by FSH receptors (FSHRs) on the granulosa cells of the ovary. The present study reports the cloning and characterization of FSHR in Chinese alligator, Alligator sinensis (caFSHR), and its tissue distribution and mRNA expression changes during the reproductive cycle. The mature protein of caFSHR displays typical features of the glycoprotein hormone receptor family, but also contains some remarkable differences when compared with other vertebrate FSHRs. The deduced amino acid sequence of the caFSHR shares identity of 85% with Chinese softshell turtle, 84–87% with birds, 77–78% with mammals, 67–73% with amphibians and 51–58% with fishes. Phylogenetic tree analysis of the FSHR amino acid sequence indicated that alligators cluster into the bird branch. Tissue expression analysis showed that caFSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid. Expression of caFSHR in the ovary is high in May (breeding prophase) and peaks in July during the breeding period, where it is maintained at high levels through September (breeding anaphase). Expression decreases significantly in November (hibernating period) and then remains relatively low from January to March (hibernating period). These temporal changes in FSHR expression suggest that it plays an important role in promoting ovarian development during the female reproductive cycle of Chinese alligator. [ABSTRACT FROM AUTHOR]

The Chinese alligator Alligator sinensis is an endangered species endemic to China, it has a highly specialized reproductive pattern with low fecundity. Up to date, little is known about the regulation of its female reproductive cycle. Follicle-stimulating hormone (FSH), a glycoprotein hormone, plays a key role in stimulating and regulating ovarian follicular development and egg production. In this study, the complete FSHβ cDNA from the ovary of the Chinese alligator was obtained for the first time, it consists of 843-bp nucleotides, including 120-bp nucleotides of the 5′-untranslated region (UTR), 396-bp of the open reading frame, and 3′-UTR of 327-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHβ with a signal peptide of 18 amino acids followed by a mature protein of 113 amino acids. Its deduced amino acid sequence shares high identities with the American alligator (100%) and birds (89–92%). Phylogenetic tree analysis of the FSHβ amino acid sequence indicated that alligators cluster into the bird branch. Tissue distribution analyses indicated that FSHβ mRNA is expressed in ovary, intestine and liver with the highest level in the ovary, while not in stomach, pancreas, heart, thymus and thyroid. Expression of FSHβ in ovary increases in May (breeding prophase) and peaks in July (breeding period), it is maintained at high levels through September, then decreases significantly in November (post-reproductive period) and remains relatively low from January to March (hibernating period). These temporal changes of FSHβ expression implicated that it might play an important role in promoting ovarian development during the female reproductive cycle. [ABSTRACT FROM AUTHOR]