Metabarcoding of fecal DNA reveals the broad and flexible diet of a globally endangered bird.

Knowing the diet of endangered wild animals is a prerequisite for species-specific conservation and habitat management. The Sichuan partridge Arborophila rufipectus is a globally endangered Galliformes species endemic to the mountains of southwest China. Existing information on the diet of this species is biased and fragmented owing to traditional observation methods. Little is known about their dietary composition or how they respond to temporal variations in food resources throughout the year. In this study, a dietary analysis was performed on 60 fecal samples using DNA Metabarcoding of invertebrates and plants to determine the primary animal and plant components of the diet across 3 critical periods of adult life history (breeding, postbreeding wandering, and overwintering). Preys from the dipteran order, followed by the lepidopteran and araneaen spp., were the predominant, animal-derived foods. Symplocos, Rubus, Celastrus, Holboellia, and Actinidia spp. supply a large abundance of fruits and seeds for this omnivorous bird. Substantial temporal dietary changes among the 3 periods and a general shift toward lower dietary diversity during the breeding season were observed, suggesting that the Sichuan partridge can adjust their diet according to the availability of food resources and their own needs. Characterizing the composition and seasonal changes in Sichuan partridge diets informs the habitat management of native flora (the plant taxa that can generate berries and seeds, such as Symplocos, Rubus, Celastrus, and Holboellia, which are likely of conservation interest) to achieve full life-cycle conservation.

Association between diet and the gut microbiome of young captive red-crowned cranes (Grus japonensis).

BACKGROUND: Exploring the association of diet and indoor and outdoor environments on the gut microbiome of red-crowned cranes. We investigated the microbiome profile of the 24 fecal samples collected from nine cranes from day 1 to 35. Differences in the gut microbiome composition were compared across diet and environments. RESULTS: A total of 2,883 operational taxonomic units (OTUs) were detected, with 438 species-specific OTUs and 106 OTUs common to the gut microbiomes of four groups. The abundance of Dietzia and Clostridium XI increased significantly when the red-crowned cranes were initially fed live mealworms. Skermanella and Deinococcus increased after the red-crowned cranes were fed fruits and vegetables and placed outdoors. Thirty-three level II pathway categories were predicted. Our study revealed the mechanism by which the gut microbiota of red-crowned cranes responds to dietary and environmental changes, laying a foundation for future breeding, nutritional and physiological studies of this species. CONCLUSIONS: The gut microbiome of red-crowned cranes could adapt to changes in diet and environment, but the proportion of live mealworms in captive red-crowned cranes can be appropriately reduced at the initial feeding stage, reducing the negative impact of high-protein and high-fat foods on the gut microbiome and growth and development.

Temporal Variations in the Gut Microbiota of the Globally Endangered Sichuan Partridge (Arborophila rufipectus): Implications for Adaptation to Seasonal Dietary Change and Conservation.

Host-associated microbiotas are known to influence host health by aiding digestion, metabolism, nutrition, physiology, immune function, and pathogen resistance. Although an increasing number of studies have investigated the avian microbiome, there is a lack of research on the gut microbiotas of wild birds, especially endangered pheasants. Owing to the difficulty of characterizing the dynamics of dietary composition, especially in omnivores, how the gut microbiotas of birds respond to seasonal dietary changes remains poorly understood. The Sichuan partridge (Arborophila rufipectus) is an endangered pheasant species with a small population endemic to the mountains of southwest China. Here, 16S rRNA sequencing and Tax4Fun were used to characterize and compare community structure and functions of the gut microbiota in the Sichuan partridges across three critical periods of their annual life cycle (breeding, postbreeding wandering, and overwintering). We found that the microbial communities were dominated by Firmicutes, Proteobacteria, Actinobacteria, and Cyanobacteria throughout the year. Diversity of the gut microbiotas was highest during postbreeding wandering and lowest during the overwintering periods. Seasonal dietary changes and reassembly of the gut microbial community occurred consistently. Composition, diversity, and functions of the gut microbiota exhibited diet-associated variations, which might facilitate host adaptation to diverse diets in response to environmental shifts. Moreover, 28 potential pathogenic genera were detected, and their composition differed significantly between the three periods. Investigation of the wild bird gut microbiota dynamics has enhanced our understanding of diet-microbiota associations over the annual life cycle of birds, aiding in the integrative conservation of this endangered bird. IMPORTANCE Characterizing the gut microbiotas of wild birds across seasons will shed light on their annual life cycle. Due to sampling difficulties and the lack of detailed dietary information, studies on how the gut microbiota adapts to seasonal dietary changes of wild birds are scarce. Based on more detailed dietary composition, we found a seasonal reshaping pattern of the gut microbiota of Sichuan partridges corresponding to their seasonal dietary changes. The variation in diet and gut microbiota potentially facilitated the diversity of dietary niches of this endangered pheasant, revealing a seasonal diet-microbiota association across the three periods of the annual cycle. In addition, identifying a variety of potentially pathogenic bacterial genera aids in managing the health and improving survival of Sichuan partridges. Incorporation of microbiome research in the conservation of endangered species contributes to our comprehensive understanding the diet-host-microbiota relationship in wild birds and refinement of conservation practices.

Molecular phylogenetics and diversity of the Himalayan shrew ( Soriculus nigrescens Gray, 1842) (Eulipotyphla, Soricidae) in Southwest China.

The Himalayan shrew, Soriculus nigrescens Gray, 1842, belongs to the monotypic genus Soriculus, which is distributed mainly in the Himalayan region. Previous authors have studied its classification based on morphological and molecular data. However, no comprehensive study of the diversity and phylogeny of this species has been performed. In this study, we investigated the molecular phylogeny, genetic diversity, and species divergence of S. nigrescens based on one mitochondrial gene and three nuclear genes. A total of 124 samples from 27 sites in Southwest China were analyzed. Our molecular phylogenetic analyses and species divergence reveal non-monophyly of Soriculus, potentially representing two genera and three clades. Populations from Yunnan (Clade YN) represent the subspecies S. n. minors and should recover the full species status. Populations from Himalayas (Clade A) represent the species S. nigrescens, while populations from southeastern Nyenchen Tanglha Mountains and southern Himalayas (Clade B) represent a new cryptic and unnamed species. Species delimitation analyses and deep genetic distance analysis clearly support the species status of these three evolving clades. The putative new genus and cryptic species should be studied and identified in the future using a more extensive sampling combined with a comprehensive morphological and phylogenetic analysis.

Revalidation and expanded description of Mustela aistoodonnivalis (Mustelidae: Carnivora) based on a multigene phylogeny and morphology.

The lacked-teeth pygmy weasel, Mustela aistoodonnivalis Wu & Kao, 1991, was originally described as being from Taibai Mountain and Zhashui county, Shaanxi, China. Subsequently, it was considered a subspecies or synonym of Mustela nivalis. In a faunal survey of northwestern Sichuan, eight specimens of M. aistoodonnivalis were collected. A molecular phylogenetic analysis of one mitochondrial and six nuclear genes clustered the specimens as a distinct clade and not with M. nivalis. Morphologically, the lack of the second lower molar differentiated them from M. nivalis, and genetic distances were typical of discrete species. These analyses confirmed that M. aistoodonnivalis is an independent species in the genus Mustela.

TGF-ß-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-ß1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-ß1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-ß-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-ß1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-ß are found in aVICs. TGF-ß transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-ß-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

Changed cecal microbiota involved in growth depression of broiler chickens induced by immune stress.

A previous study identified genes and metabolites associated with amino acid metabolism, glycerophospholipid metabolism, and inflammatory response in the liver of broilers with immune stress. The present research was designed to investigate the effect of immune stress on the cecal microbiome in broilers. In addition, the correlation between altered microbiota and liver gene expression, the correlation between altered microbiota and serum metabolites were compared using the Spearman correlation coefficients. Eighty broiler chicks were randomly assigned to 2 groups with 4 replicate pens per group and 10 birds per pen. The model broilers were intraperitoneally injected of 250 µg/kg LPS at 12, 14, 33, and 35 d of age to induce immunological stress. Cecal contents were taken after the experiment and kept at -80°C for 16S rDNA gene sequencing. Then the Pearson's correlation between gut microbiome and liver transcriptome, between gut microbiome and serum metabolites were calculated using R software. The results showed that immune stress significantly changed microbiota composition at different taxonomic levels. KEGG pathways analysis suggested that these gut microbiota were mainly involved in biosynthesis of ansamycins, glycan degradation, D-glutamine and D-glutamate metabolism, valine, leucine, and isoleucine biosynthesis and biosynthesis of vancomycin group antibiotics. Moreover, immune stress increased the activities of metabolism of cofactors and vitamins, as well as decreased the ability of energy metabolism and digestive system. Pearson's correlation analysis identified several bacteria were positively correlated with the gene expression while a few of bacteria were negatively correlated with the gene expression. The results identified potential microbiota involvement in growth depression mediated by immune stress and provided strategies such as supplement of probiotic for alleviating immune stress in broiler chickens.

Characterization and Dynamics of the Gut Microbiota in Rice Fishes at Different Developmental Stages in Rice-Fish Coculture Systems.

The rice-fish system (RFS), a traditional coculture farming model, was selected as a "globally important agricultural heritage system." Host-associated microbiota play important roles in development, metabolism, physiology, and immune function. However, studies on the gut microbiota of aquatic animals in the RFS are scarce, especially the lack of baseline knowledge of the dynamics of gut microbial communities in rice fish during different developmental stages. In this study, we characterized the microbial composition, community structure, and functions of several sympatric aquatic animals (common carp (Cyprinus carpio), crucian carp (Carassius carassius), and black-spotted frogs (Pelophylax nigromaculatus)), and the environment (water) in the RFS using 16S rRNA gene sequencing. Moreover, we investigated stage-specific signatures in the gut microbiota of common carp throughout the three developmental stages (juvenile, sub-adult, and adult). Our results indicated that the Fusobacteriota, Proteobacteria, and Firmicutes were dominant gut microbial phyla in rice fish. The differences in gut microbial compositions and community structure between the three aquatic species were observed. Although no significant differences in alpha diversity were observed across the three developmental stages, the microbial composition and community structure varied with development in common carp in the RFS, with an increase in the relative abundance of Firmicutes in sub-adults and a shift in the functional features of the community. This study sheds light on the gut microbiota of aquatic animals in the RFS. It deepens our understanding of the dynamics of gut microflora during common carp development, which may help improve aquaculture strategies in the RFS.

Molecular phylogeny and taxonomy of the genus Nectogale (Mammalia: Eulipotyphla: Soricidae).

The elegant water shrew, Nectogale elegans, is one of the small mammal species most adapted to a semi-aquatic lifestyle. The taxonomy of the genus Nectogale has received little attention due to difficulties in specimen collection. In this study, we sequenced one mitochondrial and eight nuclear genes to infer the phylogenetic relationship of Nectogale. Phylogenetic analyses revealed two large clades within Nectogale. One clade represented N. elegans, and the other was regarded as N. sikhimensis. The split between N. elegans and N. sikhimensis dated back to the early Pleistocene (2.15 million years ago [Ma]), which might be relevant to the Qinghai-Tibet Plateau (QTP) uplift. The morphological comparison showed several distinguishing characters within Nectogale: the shape of the mastoids, the first lower unicuspid (a1), and the second upper molar (M2). Overall, the molecular and the morphological evidences supported that the genus Nectogale consists of two valid species: N. elegans and N. sikhimensis.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

Multi-locus phylogeny and species delimitations of the striped-back shrew group (Eulipotyphla: Soricidae): Implications for cryptic diversity, taxonomy and multiple speciation patterns.

The striped-back shrew group demonstrates remarkable variation in skull and body size, tail length, and brightness of the dorsal stripe; and karyotypic and DNA variation has been reported in recent years. In this study, we investigated the phylogenetic structure of the group, as well as speciation patterns and demographic history in Mountains of Southwestern China and adjacent mountains, including the southern Himalayas, Mts. Bashan, Wushan, and Qinling. We sequenced a total of 462 specimens from 126 localities in the known range of the group, which were sequenced and analyzed based on 6.2 kb of sequence data from two mitochondrial, six nuclear, and two Y chromosome markers. Phylogenetic analyses of the concatenated mtDNA data revealed 14 sympatric and independently evolving lineages within the striped-back shrew group, including Sorex bedfordiae, S. cylindricauda, S. excelsus, S. sinalis and several cryptic species. All concatenated data (ten genes) showed a consistent genetic structure compared to the mtDNA lineages for the group, whereas the nuclear and the Y chromosome data showed a discordant genetic structure compared to the mtDNA lineages for the striped-back shrew group. Species delimitation analyses and deep genetic distance clearly support the species status of the 14 evolving lineages. The divergence time estimation suggested that the striped-back shrew group began to diversify from the middle Pleistocene (2.34 Ma), then flourished at approximately 2.14 Ma, followed by a series of rapid diversifications through the Pleistocene. Our results also revealed multiple mechanisms of speciation in the Mountains of Southwestern China and Adjacent Mountains with complex landscapes and climate. The uplifting of the Qinghai-Tibetan Plateau, Quaternary climate oscillations, riverine barriers, ecological elevation gradients, topographical diversity, and their own low dispersal capacity may have driven the speciation, genetic structure, and phylogeographic patterns of the striped-back shrew group.

A new species of Tamiops (Rodentia, Sciuridae) from Sichuan, China.

During a small mammal survey in Wanglang National Natural Reserve, Sichuan, China in AugustOctober 2018, we collected four specimens of an Asiatic striped squirrel (genus Tamiops) that could not be assigned morphologically to any described species. Phylogenetic analyses of CYTB and nuDNA sequences strongly supported them in the genus Tamiops. The p-distances of CYTB between these individuals and other species of Tamiops ranged from 17.4 to 19.1%. Morphologically, they differed from their congeners, including T. maritimus, T. mcclellandii, T. rodolphii, and T. swinhoei by pelage, skull, and baculum characteristics. Based on the results of morphological comparisons and molecular analyses, we describe the four specimens as the new species Tamiops minshanica sp. nov.

Establishment of Gut Microbiome During Early Life and Its Relationship With Growth in Endangered Crested Ibis (Nipponia nippon).

Gut microbiota during early life could influence host fitness in vertebrates. Studies on how gut microbiota colonize the gut in birds using frequent sampling during early developmental stages and how shifts in microbiota diversity influence host growth are lacking. Here, we examine the microbiome profiles of 151 fecal samples from 14 young crested ibis (Nipponia nippon), an endangered bird species, collected longitudinally across 13 time points during the early stages of development and investigated their correlation with host growth. Gut diversity showed a non-linear change during development, which involved multiple colonization and extinction events, mainly associated with Proteobacteria and Firmicutes. Gut microbiota in young crested ibis became more similar with increasing age. In addition, gut microbiota exhibited a strong temporal structure and two specific developmental stages; the beginning of the latter stage coincided with the introduction of fresh loach, with a considerable increase in the relative abundance of Fusobacteria and several Firmicutes, which may be involved in lipid metabolism. Crested ibis chick growth rate was negatively correlated with gut microbiota diversity and negatively associated with the abundance of Halomonadaceae, Streptococci, Corynebacteriaceae, and Dietziaceae. Our findings highlight the importance of frequent sampling when studying microbiome development during early stages of development of vertebrates. The role of microbial diversity in host growth during the early stages of development of birds warrants further investigations.

DNA metabarcoding provides insights into seasonal diet variations in Chinese mole shrew (Anourosorex squamipes) with potential implications for evaluating crop impacts.

Diet analysis of potential small mammals pest species is important for understanding feeding ecology and evaluating their impact on crops and stored foods. Chinese mole shrew (Anourosorex squamipes), distributed in Southwest China, has previously been reported as a farmland pest. Effective population management of this species requires a better understanding of its diet, which can be difficult to determine with high taxonomic resolution using conventional microhistological methods. In this study, we used two DNA metabarcoding assays to identify 38 animal species and 65 plant genera from shrew stomach contents, which suggest that A. squamipes is an omnivorous generalist. Earthworms are the most prevalent (>90%) and abundant (>80%) food items in the diverse diet of A. squamipes. Species of the Fabaceae (frequency of occurrence [FO]: 88%; such as peanuts) and Poaceae (FO: 71%; such as rice) families were the most common plant foods identified in the diet of A. squamipes. Additionally, we found a seasonal decrease in the diversity and abundance of invertebrate foods from spring and summer to winter. Chinese mole shrew has a diverse and flexible diet throughout the year to adapt to seasonal variations in food availability, contributing to its survival even when food resources are limited. This study provides a higher resolution identification of the diet of A. squamipes than has been previously described and is valuable for understanding shrew feeding ecology as well as evaluating possible species impacts on crops.

Chromosome-scale genomes provide new insights into subspecies divergence and evolutionary characteristics of the giant panda.

Extant giant pandas are divided into Sichuan and Qinling subspecies. The giant panda has many species-specific characteristics, including comparatively small organs for body size, small genitalia of male individuals, and low reproduction. Here, we report the most contiguous, high-quality chromosome-level genomes of two extant giant panda subspecies to date, with the first genome assembly of the Qinling subspecies. Compared with the previously assembled giant panda genomes based on short reads, our two assembled genomes increased contiguity over 200-fold at the contig level. Additional sequencing of 25 individuals dated the divergence of the Sichuan and Qinling subspecies into two distinct clusters from 10,000 to 12,000 years ago. Comparative genomic analyses identified the loss of regulatory elements in the dachshund family transcription factor 2 (DACH2) gene and specific changes in the synaptotagmin 6 (SYT6) gene, which may be responsible for the reduced fertility of the giant panda. Positive selection analysis between the two subspecies indicated that the reproduction-associated IQ motif containing D (IQCD) gene may at least partly explain the different reproduction rates of the two subspecies. Furthermore, several genes in the Hippo pathway exhibited signs of rapid evolution with giant panda-specific variants and divergent regulatory elements, which may contribute to the reduced inner organ sizes of the giant panda.

Automated control algorithms for silicon photonic polarization receiver.

We demonstrate greedy linear descent-based, basic gradient descent-based, two-point step size gradient descent-based, and two-stage optimization method-based automated control algorithms and examine their performance for use with a silicon photonic polarization receiver. With an active feedback loop control process, time-varying arbitrary polarization states from an optical fiber can be automatically adapted and stabilized to the transverse-electric (TE) mode of a single-mode silicon waveguide. Using the proposed control algorithms, we successfully realize automated adaptations for a 10 Gb/s on-off keying signal in the polarization receiver. Based on the large-signal measurement results, the control algorithms are examined and compared with regard to the iteration number and the output response. In addition, we implemented a long-duration experiment to track, adapt, and stabilize arbitrary input polarization states using the two-point step size gradient descent-based and two-stage optimization method-based control algorithms. The experimental results show that these control algorithms enable the polarization receiver to achieve real-time and continuous polarization management.

Multilocus phylogeny and cryptic diversity of white-toothed shrews (Mammalia, Eulipotyphla, Crocidura) in China.

BACKGROUND: Crocidura, the most speciose mammalian genus, occurs across much of Asia, Europe and Africa. The taxonomy of Chinese representatives has been studied primarily based on cursory morphological comparisons and their molecular phylogenetic analyses remain unexplored. In order to understand the phylogeny of this group in China, we estimated the first multilocus phylogeny and conducted species delimitation, including taxon sampling throughout their distribution range. RESULTS: We obtained one mitochondrial gene (cytb) (~ 1, 134 bp) and three nuclear genes (ApoB, BRCA1, RAG1) (~ 2, 170 bp) for 132 samples from 57 localities. Molecular analyses identified at least 14 putative species that occur within two major well-supported groups in China. Polyphyletic C. wuchihensis appears to be composed of two putative species. Two subspecies, C. rapax rapax and C. rapax kurodai should be elevated to full species status. A phylogenetic tree based on mitochondrial gene from Asian Crocidura species showed that the C. rapax rapax is embedded within C. attenuata, making the latter a paraphyletic group. Three strongly supported undescribed species (C. sp.1, C. sp.2 and C. sp.3) are revealed from Zada County of Tibet (Western China), Hongjiang County of Hunan Province (Central China) and Dongyang County of Zhejiang Province (Eastern China), Motuo County of Tibet, respectively. The divergence time estimation suggested that China's Crocidura species began to diversify during the late Pliocene (3.66 Ma) and the Early Pleistocene (2.29 Ma), followed by a series of diversifications through the Pleistocene. CONCLUSIONS: The cryptic diversity found in this study indicated that the number of species is strongly underestimated under the current taxonomy. We propose that the three undescribed species should be evaluated using extensive taxon sampling and comprehensive morphological and morphometric approaches. Climate change since the late Pliocene and the uplift of the Qinghai-Tibet Plateau may result in the diversification and speciation of China's Crocidura species. In short, the underestimated diversity underlines the need for a taxonomic revision of Chinese Crocidura species.

Metagenomics Reveals Seasonal Functional Adaptation of the Gut Microbiome to Host Feeding and Fasting in the Chinese Alligator.

As a natural hibernator, the Chinese alligator (Alligator sinensis) is an ideal and intriguing model to investigate changes in microbial community structure and function caused by hibernation. In this study, we used 16S rRNA profiling and metagenomic analysis to compare the composition, diversity, and functional capacity in the gut microbiome of hibernating vs. active Chinese alligators. Our results show that gut microbial communities undergo seasonal restructuring in response to seasonal cycles of feeding and fasting in the Chinese alligator, but this animal harbors a core gut microbial community primarily dominated by Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes across the gut regions. During hibernation, there is an increase in the abundance of bacterial taxa (e.g., the genus Bacteroides) that can degrade host mucin glycans, which allows adaptation to winter fasting. This is accompanied by the enrichment of mucin oligosaccharide-degrading enzyme and carbohydrate-active enzyme families. In contrast, during the active phase (feeding), active Chinese alligators exhibit a carnivore gut microbiome dominated by Fusobacteria, and there is an increase in the relative abundance of bacteria (e.g., Cetobacterium somerae) with known proteolytic and amino acids-fermentating functions that improve host protein-rich food digestion efficiency. In addition, seasonal variations in the expression of ß-defensins play a protective role in intestinal immunity. These findings provide insights into the functional adaptations of host-gut microbe symbioses to seasonal dietary shifts to maintain gut homeostasis and health, especially in extreme physiological states.

A crucial role of paralogous ß-defensin genes in the Chinese alligator innate immune system revealed by the first determination of a Crocodilia defensin cluster.

The ß-defensin, one of the antimicrobial peptides (AMPs), is a significant component of the innate immune with a broad range of antimicrobial activities. Differing from the widely-studied mammals and birds, limited information about ß-defensins has been reported in reptiles, especially in crocodilians. As a same ancient species as dinosaurs and the most endangered species of 23 crocodilians, the survival of Chinese alligator (Alligator sinensis) means a powerful immune system and possible involvement of AMPs in its immune resistance. In this study, we identified 20 novel Alligator sinensisß-defensin genes (AsBDs) from a 390 kb region using bioinformatic and experimental approaches, and successfully distinguished six orthologous AsBDs to birds and nine paralogous AsBDs undergoing gene duplication events. The amino acid alignment shows that the AsBD paralogs, like α-defensins, encode a significantly longer pro-piece comparing with the orthologs. The calculation of non-synonymous (dN) and synonymous (dS) substitutions in the mature peptide reveals that the AsBD paralogs experience a significantly higher selective pressure (dN/dS) than the orthologs, but a similar evolutionary force to α-defensins. The gene expression result indicates that the AsBD paralogs have a significantly higher expression level than the orthologos in gastrointestinal tract where the host is vulnerable to enteric pathogenic bacteria, as observed in α-defensins. These three pieces of evidence demonstrate that the AsBD paralogs do play an important role in maintaining long-term survival of this endangered reptile. Thus, this survey of AsBDs on the genomic structure, evolutionary characteristics, and expression pattern provides a genetic and immunological foundation for further investigating their antimicrobial function and alternative antibiotics potentiality.