Single-nucleus transcriptome inventory of giant panda reveals cellular basis for fitness optimization under low metabolism.

BACKGROUND: Energy homeostasis is essential for the adaptation of animals to their environment and some wild animals keep low metabolism adaptive to their low-nutrient dietary supply. Giant panda is such a typical low-metabolic mammal exhibiting species specialization of extremely low daily energy expenditure. It has low levels of basal metabolic rate, thyroid hormone, and physical activities, whereas the cellular bases of its low metabolic adaptation remain rarely explored. RESULTS: In this study, we generate a single-nucleus transcriptome atlas of 21 organs/tissues from a female giant panda. We focused on the central metabolic organ (liver) and dissected cellular metabolic status by cross-species comparison. Adaptive expression mode (i.e., AMPK related) was prominently displayed in the hepatocyte of giant panda. In the highest energy-consuming organ, the heart, we found a possibly optimized utilization of fatty acid. Detailed cell subtype annotation of endothelial cells showed the uterine-specific deficiency of blood vascular subclasses, indicating a potential adaptation for a low reproductive energy expenditure. CONCLUSIONS: Our findings shed light on the possible cellular basis and transcriptomic regulatory clues for the low metabolism in giant pandas and helped to understand physiological adaptation response to nutrient stress.

Sexual Dimorphism of the Gut Microbiota in the Chinese Alligator and Its Convergence in the Wild Environment.

The gut microbiota forms a complex microecosystem in vertebrates and is affected by various factors. As a key intrinsic factor, sex has a persistent impact on the formation and development of gut microbiota. Few studies have analyzed sexual dimorphism of gut microbiota, particularly in wild animals. We used 16S rRNA gene sequencing to analyze the gut microbiota of juvenile and adult Chinese alligators, and untargeted metabolomics to study serum metabolomes of adult alligators. We observed significant sexual differences in the community diversity in juvenile, but not adult, alligators. In terms of taxonomic composition, the phylum Fusobacteriota and genus Cetobacterium were highly abundant in adult alligators, similar to those present in carnivorous fishes, whereas the gut microbiota composition in juvenile alligators resembled that in terrestrial reptiles, indicating that adults are affected by their wild aquatic environment and lack sex dimorphism in gut microbiota. The correlation analysis revealed that the gut microbiota of adults was also affected by cyanobacteria in the external environment, and this effect was sex-biased and mediated by sex hormones. Overall, this study reveals sexual differences in the gut microbiota of crocodilians and their convergence in the external environment, while also providing insights into host-microbiota interactions in wildlife.

Multi-omics analysis reveals that natural hibernation is crucial for oocyte maturation in the female Chinese alligator.

BACKGROUND: Hibernation in an appropriate environment not only is important for the survival of hibernators in winter, but also is crucial for breeding in the following season for many hibernating species. However, the genetic and epigenetic mechanism underlying this process remain unclear. In the current study, we performed an integrative multi-omics analysis of gonads collected from Chinese alligators that overwintered in wild cave and artificial warmroom to explore transcriptomic and epigenomic alternations in these organs. RESULTS: The data revealed that in the breeding season, female alligators were more strongly affected in terms of gene expression than males by non-hibernation because of overwintering in a warm room, especially for genes related to oocyte maturation, and this effect commenced in winter with the downregulation of STAR, which is the rate limiting factor of steroid biosynthesis. Further, miRNAs were found to play essential roles in this negative effect of overwintering in the warm room on hibernation. The upregulated miRNAs likely were responsible for the suppression of oocyte maturation in the breeding season. Finally, DNA methylome changes, especially hypomethylation, were found to play an important role in the alterations in ovarian function-related gene expression induced by non-hibernation. CONCLUSIONS: Our study revealed the crucial role of hibernation quality for oocyte maturation in the Chinese alligator and the underlying genetic and epigenetic mechanisms, and highlights the importance of habitat, and especially, the overwintering site, in the conservation of not only the Chinese alligator, but also other endangered hibernators.

Male density, a signal for population self-regulation in Alligator sinensis.

The regulation of population density is suggested to be indirect and occurs with a time-lag effect, as well as being female centred. Herein, we present a quantitative analysis on the precise, timely and male-dominated self-regulation of Chinese alligator ( Alligator sinensis) populations. Analysis of 31 years of data revealed gender differences in regulation patterns. Population dynamics were restricted by male density rather than population density, and population growth was halted (birth rate = 0) when male density exceeded 83.14 individuals per hectare, until some males were removed, especially adult males. This rapid and accurate response supports the notions of intrinsic mechanisms and population-wide regulation response. Furthermore, density stress affected mating success rather than parental care to juveniles, i.e. females avoided unnecessary reproduction costs, which may represent an evolutionary advantage. Our findings highlighted the importance of further studies on related physiological mechanisms that focus on four characteristics: quantity breeds quality, gender differences, male density thresholds and nonlinearity.

MHC class I diversity predicts non-random mating in Chinese alligators (Alligator sinensis).

The major histocompatibility complex (MHC) has several important roles in kin recognition, pathogen resistance and mate selection. Research in fish, birds and mammals has suggested that individuals optimise MHC diversity, and therefore offspring fitness, when choosing mates. In reptiles, however, it is unclear whether female mate choice is based on genome-wide genetic characteristics such as microsatellite DNA loci, particular functional-trait loci (e.g., MHC) or both, and MHC's effects on mate choice remain relatively understudied. Herein, we used 13 microsatellite loci and two MHC class I loci to investigate female mate choice of Chinese alligators (Alligator sinensis) in the semi-natural condition. We also determined correlations between the MHC genotype of breeding males and male reproductive success. We found that MHC-heterozygous males harbour a greater reproductive success, which probably is the reason that these males are more preferred by the females than MHC-homozygous males. Furthermore, the MHC class I amino-acid distance and functional distance of true mating pairs were higher compared with those of randomly sampled pairs. Analysis of microsatellites revealed that, despite mate choice, females did not completely avoid inbreeding. These findings are the first evidence of MHC-associated mate choice in Chinese alligators, suggesting that females may adopt different mating strategies after assessing the MHC characteristics of potential mates.

Seeing-good-gene-based mate choice: From genes to behavioural preferences.

Although vertebrates have been reported to gain higher reproductive outputs by choosing mates, few studies have been conducted on threatened species. However, species recovery should benefit if natural mate choice could improve reproductive output (i.e. pair performance related to offspring number, such as increased clutch size, numbers of fertilized egg and fledglings). We assessed the evidence for major histocompatibility complex (MHC)-based mate preference in the endangered crested ibis (Nipponia nippon) and quantified the impacts of such choice on reproductive output. We tested the hypothesis that crested ibis advertise "good genes" through external traits, by testing whether nuptial plumage characteristics and body morphology mediate mate choice for underlying genetic MHC variation. We found differences between males and females in preferred MHC genotypes, external traits used in mate choice and contributions to reproductive outputs. Females preferred MHC-heterozygous males, which had darker [i.e. lower total reflectance and ultraviolet (UV) reflectance] nuptial plumage. Males preferred females lacking the DAB*d allele at the MHC class II DAB locus, which had higher average body mass. DAB*d-free females yielded heavier eggs and more fledglings, while MHC-heterozygous males contributed to more fertilized eggs and fledglings. Fledging rate was highest when both parents had the preferred MHC genotypes (i.e. MHC-heterozygous father and DAB*d-free mother). Comparisons showed that free-mating wild and semi-natural pairs yielded more fertilized eggs and more fledglings, with a higher fledging rate, than captive pairs matched artificially based on pedigree. Conservation programmes seldom apply modern research results to population management, which could hinder recovery of threatened species. Our results show that mate choice can play an important role in improving reproductive output, with an example in which an endangered bird selects mates using UV visual capability. Despite the undoubted importance of pedigree-based matching of mates in conservation programmes, we show that free mating can be a better alternative strategy.

IDSSR: An Efficient Pipeline for Identifying Polymorphic Microsatellites from a Single Genome Sequence.

Simple sequence repeats (SSRs) are known as microsatellites, and consist of tandem 1-6-base motifs. They have become one of the most popular molecular markers, and are widely used in molecular ecology, conservation biology, molecular breeding, and many other fields. Previously reported methods identify monomorphic and polymorphic SSRs and determine the polymorphic SSRs via experimental validation, which is potentially time-consuming and costly. Herein, we present a new strategy named insertion/deletion (INDEL) SSR (IDSSR) to identify polymorphic SSRs by integrating SSRs with nucleotide insertions/deletions (INDEL) solely based on a single genome sequence and the sequenced pair-end reads. These INDEL indexes and polymorphic SSRs were identified, as well as the number of repeats, repeat motifs, chromosome location, annealing temperature, and primer sequences, enabling future experimental approaches to determine the correctness and polymorphism. Experimental validation with the giant panda demonstrated that our method has high reliability and stability. The efficient SSR pipeline would help researchers obtain high-quality genetic markers for plants and animals of interest, save labor, and reduce costly marker-screening experiments. IDSSR is freely available at https://github.com/Allsummerking/IDSSR.

Balancing selection and recombination as evolutionary forces caused population genetic variations in golden pheasant MHC class I genes.

BACKGROUND: The major histocompatibility complex (MHC) genes are vital partners in the acquired immune processes of vertebrates. MHC diversity may be directly associated with population resistance to infectious pathogens. Here, we screened for polymorphisms in exons 2 and 3 of the IA1 and IA2 genes in 12 golden pheasant populations across the Chinese mainland to characterize their genetic variation levels, to understand the effects of historical positive selection and recombination in shaping class I diversity, and to investigate the genetic structure of wild golden pheasant populations. RESULTS: Among 339 individual pheasants, we identified 14 IA1 alleles in exon 2 (IA1-E2), 11 IA1-E3 alleles, 27 IA2-E2 alleles, and 28 IA2-E3 alleles. The non-synonymous substitution rate was significantly greater than the synonymous substitution rate at sequences in the IA2 gene encoding putative peptide-binding sites but not in the IA1 gene; we also found more positively selected sites in IA2 than in IA1. Frequent recombination events resulted in at least 9 recombinant IA2 alleles, in accordance with the intermingling pattern of the phylogenetic tree. Although some IA alleles are widely shared among studied populations, large variation occurs in the number of IA alleles across these populations. Allele frequency analysis across 2 IA loci showed low levels of genetic differentiation among populations on small geographic scales; however, significant genetic differentiation was observed between pheasants from the northern and southern regions of the Yangtze River. Both STRUCTURE analysis and F-statistic (F ST ) value comparison classified those populations into 2 major groups: the northern region of the Yangtze River (NYR) and the southern region of the Yangtze River (SYR). CONCLUSIONS: More extensive polymorphisms in IA2 than IA1 indicate that IA2 has undergone much stronger positive-selection pressure during evolution. Moreover, the recombination events detected between the genes and the intermingled phylogenetic pattern indicate that interlocus recombination accounts for much of the allelic variation in IA2. Analysis of the population differentiation implied that homogenous balancing selection plays an important part in maintaining an even distribution of MHC variations. The natural barrier of the Yangtze River and heterogeneous balancing selection might help shape the NYR-SYR genetic structure in golden pheasants.

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China.

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe-DRA*02, Mobe-DQA1*01 and Mobe-DQA2*05 alleles, which may be important for pathogen resistance. A Ewens-Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.

Haplotype-resolved chromosome-scale genomes of the Asian and African Savannah Elephants.

The Proboscidea, which includes modern elephants, were once the largest terrestrial animals among extant species. They suffered mass extinction during the Ice Age. As a unique branch on the evolutionary tree, the Proboscidea are of great significance for the study of living animals. In this study, we generate chromosome-scale and haplotype-resolved genome assemblies for two extant Proboscidea species (Asian Elephant, Elephas maximus and African Savannah Elephant, Loxodonta africana) using Pacbio, Hi-C, and DNBSEQ technologies. The assembled genome sizes of the Asian and African Savannah Elephant are 3.38 Gb and 3.31 Gb, with scaffold N50 values of 130 Mb and 122 Mb, respectively. Using Hi-C technology ~97% of the scaffolds are anchored to 29 pseudochromosomes. Additionally, we identify ~9 Mb Y-linked sequences for each species. The high-quality genome assemblies in this study provide a valuable resource for future research on ecology, evolution, biology and conservation of Proboscidea species.

Patterns of genetic differentiation at MHC class I genes and microsatellites identify conservation units in the giant panda.

BACKGROUND: Evaluating patterns of genetic variation is important to identify conservation units (i.e., evolutionarily significant units [ESUs], management units [MUs], and adaptive units [AUs]) in endangered species. While neutral markers could be used to infer population history, their application in the estimation of adaptive variation is limited. The capacity to adapt to various environments is vital for the long-term survival of endangered species. Hence, analysis of adaptive loci, such as the major histocompatibility complex (MHC) genes, is critical for conservation genetics studies. Here, we investigated 4 classical MHC class I genes (Aime-C, Aime-F, Aime-I, and Aime-L) and 8 microsatellites to infer patterns of genetic variation in the giant panda (Ailuropoda melanoleuca) and to further define conservation units. RESULTS: Overall, we identified 24 haplotypes (9 for Aime-C, 1 for Aime-F, 7 for Aime-I, and 7 for Aime-L) from 218 individuals obtained from 6 populations of giant panda. We found that the Xiaoxiangling population had the highest genetic variation at microsatellites among the 6 giant panda populations and higher genetic variation at Aime-MHC class I genes than other larger populations (Qinling, Qionglai, and Minshan populations). Differentiation index (FST)-based phylogenetic and Bayesian clustering analyses for Aime-MHC-I and microsatellite loci both supported that most populations were highly differentiated. The Qinling population was the most genetically differentiated. CONCLUSIONS: The giant panda showed a relatively higher level of genetic diversity at MHC class I genes compared with endangered felids. Using all of the loci, we found that the 6 giant panda populations fell into 2 ESUs: Qinling and non-Qinling populations. We defined 3 MUs based on microsatellites: Qinling, Minshan-Qionglai, and Daxiangling-Xiaoxiangling-Liangshan. We also recommended 3 possible AUs based on MHC loci: Qinling, Minshan-Qionglai, and Daxiangling-Xiaoxiangling-Liangshan. Furthermore, we recommend that a captive breeding program be considered for the Qinling panda population.

Molecular characterization of the major histocompatibility complex class Ia gene in the black-spotted frog, Pelophylax nigromaculata.

The genes of the major histocompatibility complex (MHC) are attractive candidates for investigating the link between adaptive variation and individual fitness. We improved rapid amplification of cDNA ends to obtain the whole coding sequence of the MHC class Ia gene of the black-spotted frog (Pelophylax nigromaculata), the most common amphibian in China. We also used genome walking to characterize the partial introns adjacent to exon 3 of the MHC Ia gene. Based on the sequences obtained, we designed locus-specific primers to investigate the molecular polymorphisms of this species in southeast China. The MHC class Ia gene showed a high level of genetic diversity, indicating that this species retains a relatively high potential for survival, despite a population decline among frog species in general and many other amphibians.

Alternative telomere maintenance mechanism in Alligator sinensis provides insights into aging evolution.

Lifespan is a life-history trait that undergoes natural selection. Telomeres are hallmarks of aging, and shortening rate predicts species lifespan, making telomere maintenance mechanisms throughout different lifespans a worthy topic for study. Alligators are suitable for the exploration of anti-aging molecular mechanisms, because they exhibit low or even negligible mortality in adults and no significant telomere shortening. Telomerase reverse transcriptase (TERT) expression is absent in the adult Alligator sinensis, as in humans. Selection analyses on telomere maintenance genes indicated that ATM, FANCE, SAMHD1, HMBOX1, NAT10, and MAP3K4 experienced positive selection on A. sinensis. Repressed pleiotropic ATM kinase in A. sinensis suggests their fitness optimum shift. In ATM downstream, Alternative Lengthening of Telomeres (ALT)-related genes were clustered in a higher expression pattern in A. sinensis, which covers 10-15% of human cancers showing no telomerase activities. In summary, we demonstrated how telomere shortening, telomerase activities, and ALT contributed to anti-aging strategies.

Genomic investigation of the Chinese alligator reveals wild-extinct genetic diversity and genomic consequences of their continuous decline.

Critically endangered species are usually restricted to small and isolated populations. High inbreeding without gene flow among populations further aggravates their threatened condition and reduces the likelihood of their long-term survival. Chinese alligator (Alligator sinensis) is one of the most endangered crocodiles in the world and has experienced a continuous decline over the past c. 1 million years. In order to identify the genetic status of the remaining populations and aid conservation efforts, we assembled the first high-quality chromosome-level genome of Chinese alligator and explored the genomic characteristics of three extant breeding populations. Our analyses revealed the existence of at least three genetically distinct populations, comprising two breeding populations in China (Changxing and Xuancheng) and one breeding population in an American wildlife refuge. The American population does not belong to the last two populations of its native range (Xuancheng and Changxing), thus representing genetic diversity extinct in the wild and provides future opportunities for genetic rescue. Moreover, the effective population size of these three populations has been continuously declining over the past 20 ka. Consistent with this decline, the species shows extremely low genetic diversity, a large proportion of long runs of homozygous fragments, and mutational load across the genome. Finally, to provide genomic insights for future breeding management and conservation, we assessed the feasibility of mixing extant populations based on the likelihood of introducing new deleterious alleles and signatures of local adaptation. Overall, this study provides a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of critically endangered alligators.

Battacin (Octapeptin B5), a new cyclic lipopeptide antibiotic from Paenibacillus tianmuensis active against multidrug-resistant Gram-negative bacteria.

Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 µg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria.

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes.

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.

Sex-Biased miRNAs in the Gonads of Adult Chinese Alligator (Alligator sinensis) and Their Potential Roles in Sex Maintenance.

MicroRNA (miRNA) is a category of single-stranded non-coding small RNA (sRNA) that regulates gene expression by targeting mRNA. It plays a key role in the temperature-dependent sex determination of Chinese alligator (Alligator sinensis), a reptile whose sex is determined solely by the temperature during the incubation period and remains stable thereafter. However, the potential function of miRNAs in the gonads of adult Chinese alligators is still unclear. Here, we prepared and sequenced sRNA libraries of adult female and male alligator gonads, from breeding (in summer) and hibernating (in winter) animals. We obtained 130 conserved miRNAs and 683 novel miRNAs, which were assessed for sex bias in summer and winter; a total of 65 miRNAs that maintained sex bias in both seasons were identified. A regulatory network of sex-biased miRNAs and genes was constructed. Sex-biased miRNAs targeted multiple genes in the meiosis pathway of adult Chinese alligator oocytes and the antagonistic gonadal function maintenance pathway, such as MOS, MYT1, DMRT1, and GDF9. Our study emphasizes the function of miRNA in the epigenetic mechanisms of sex maintenance in crocodilians.

Comparative Genomics Provides Insights into Adaptive Evolution in Tactile-Foraging Birds.

Tactile-foraging birds have evolved an enlarged principal sensory nucleus (PrV) but smaller brain regions related to the visual system, which reflects the difference in sensory dependence. The "trade-off" may exist between different senses in tactile foragers, as well as between corresponding sensory-processing areas in the brain. We explored the mechanism underlying the adaptive evolution of sensory systems in three tactile foragers (kiwi, mallard, and crested ibis). The results showed that olfaction-related genes in kiwi and mallard and hearing-related genes in crested ibis were expanded, indicating they may also have sensitive olfaction or hearing, respectively. However, some genes required for visual development were positively selected or had convergent amino acid substitutions in all three tactile branches, and it seems to show the possibility of visual degradation. In addition, we may provide a new visual-degradation candidate gene PDLIM1 who suffered dense convergent amino acid substitutions within the ZM domain. At last, two genes responsible for regulating the proliferation and differentiation of neuronal progenitor cells may play roles in determining the relative sizes of sensory areas in brain. This exploration offers insight into the relationship between specialized tactile-forging behavior and the evolution of sensory abilities and brain structures.

Incubation determines favorable microbial communities in Chinese alligator nests.

Nest materials are a major heat source due to rotting promoted by microbial activity. Additionally, they are a potential microbial source given their direct contact with eggshells. Microbial dynamics during incubation have been studied in wild birds; however, similar studies in reptiles remain elusive. Here, the study characterized microbial communities in the nest materials of Chinese alligator (Alligator sinensis) using high-throughput sequencing of bacterial 16S rRNA genes and fungal internal transcribed spacer (ITS) region sequences. The results showed that significant changes in the diversity and structure of microbial communities according to different incubation periods. The diversity and richness of bacterial species increased significantly over time, but the relative abundance of the most dominant bacteria in pre-incubation period, including some pathogenic bacteria, declined after incubation. In contrast, fungal species diversity and richness decreased significantly with time. Additionally, nest material composition significantly influenced microbial community structure rather than species diversity and richness. Notably, the fungal community structure showed a stronger response than bacteria to nest material composition, which varied due to differences in plant litter composition. Our results demonstrate the significant response of microbial community diversity and structure to differences in incubation periods and nest material composition in reptiles. It is further emphasized that the importance of incubation period in the conservation of the Chinese alligator and could inform similar studies in other reptiles and birds.

Antibacterial Properties and Potential Mechanism of Serum from Chinese Alligator.

The Chinese alligator (Alligator sinensis) is an ancient reptile with strong immunity that lives in wetland environments. This study tested the antibacterial ability of Chinese alligator serum (CAS) against Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and analyzed the potential underlying mechanisms. Results showed that the CAS had a marked antibacterial effect on K. pneumoniae, E. coli, and P. aeruginosa, while S. aureus was only mildly affected. However, these effects disappeared when Protease K was added to the serum. The serum proteome analysis revealed that the antibacterial ability of CAS was produced by interactions among various proteins and that the complement proteins played a major antibacterial role. Therefore, we made relevant predictions about the structure and function of complement component 3. In addition, sequence alignment and phylogenetic analysis of complement component 3d (C3d) in four mammalian species and two alligator species showed that the amino acids that make up the acid pocket on the concave surface of alligator C3d are not identical to those in mammals. This study provided evidence that CAS elicits significant antibacterial effects against some pathogens and provides the basis for further development of novel antibacterial drugs.