Characteristics Of ?-Defensin Genes Cluster And Metagenomic Analysis Of Gut Microbiota On Hibernation Adaptability In The Chinese Alligator

The Chinese alligator(Alligator sinensis),the most endangered species among23 crocodilians,is an old,rare,and endemic reptile that is under first-class protection in China.As a semi-aquatic carnivore,crocodilians are always under the threat of opportunistic pathogenic bacterial infections.There are antimicrobial peptides(AMPs)with a broad range of antimicrobial activities in crocodile serum.The?-defensin,one of the AMPs,is a significant component and first immune barrier of the innate immune.Differing from the widely studied mammals and birds,limited information about?-defensins has been reported in reptiles,especially in crocodilians.Chinese alligator,the poikilotherm,naturally experiences hibernation periods of several months during which it undergoes a phase of deep dormancy,metabolic supersession and fasting due to seasonal pattern of food availability.Based on earlier studies,hibernator gut microbial communities are validated to seasonally vary with host physiology and availability of exogenous diet,but without characterizing the functional potentials of gut microbiome and interactions of host-microbiota at metagenomic level.Thus,Chinese alligator is a natural model of investigations on changes in microbial community and functional adaptations induced by hibernation.In this study,we identified 20 novel Alligator sinensis?-defensin genes(AsBDs,Alligator sinensis?-defensin genes)from a 390 kb region using bioinformatic and RACE-PCR approaches.The analysis of AsBDs on the genomic structure,amino acids sequences,evolutionary characteristics and expression pattern revealed that paralogous AsBDs were evolutionally and functionally similar to mammalian?-defensins,but differed from orthologous AsBDs,established the foundation for the subsequent research on the interactions between AsBDs and gut microbiota.Here,we used 16S rRNA profiling and metagenomic analysis to compare microbiome composition,diversity and functional capacity in hibernating vs.active Chinese alligator gut;and the relationship between the seasonal expression of AsBDs and gut microbial bacterial was also investigated.The main results are as follows:(1)We 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 mammalian?-defensins,encode a significantly longer pro-piece(36~40 amino acids)comparing with the orthologs.Homology in pro-piece peptides gives rise to more similar net charge and hydrophobicity of AsBD paralogs and?-defensins.(2)The calculation of non-synonymous(d_N)and synonymous(d_S)substitutions in the mature peptide reveals that the AsBD paralogs experience a significantly higher positive selective pressure(?=d_N/d_S)than the orthologs(paralogs:?=1.208,orthologs:?=0.499,p=0.034),but a similar evolutionary force to?-defensins.But there were no statistically significant differences in the?value in the pre-pro peptides of AsBD paralogs and orthologs(paralogs:?=0.759,orthologs:?=0.604,p=0.471).According to NJ phylogenetic tree,the nine alligator-specific paralogous AsBDs were grouped with the mammalian?-defensins instead of clustering with mammalian?-defensins,further suggesting that mammalian?-defensins might evolve from reptilian-specific paralogous?-defensins.(3)The AsBDs ubiquitously and strongly expressed in the investigated tissues,and showed the robust expressed level in the digestive tract,liver,spleen and kidney,and the least expression in the muscle,gallbladder and heart.Interestingly,the AsBD paralogs have a significantly higher expression level than the orthologs in gastrointestinal tract where the host is vulnerable to enteric pathogenic bacteria,as observed in?-defensins which interact directly with gut microbes.(4)We identified 41 bacterial phyla and 3215 operational taxonomic units(OTUs)in the gut of Chinese alligator by 16S rRNA gene sequencing.Chinese alligator harbors a core but distinct gut microbial community primarily dominated by Proteobacteria,Fusobacteria,Bacteroidetes,and Firmicutes,which made up the vast majority(>80%)of the total sequences.Based on metagenomic sequencing,we identified 51 bacterial phyla and 3907 OTUs from fecal metagenome.(5)The PCoA,UPGMA,ANOSIM and CCA methods were performed on comparative analysis of gut microbial communities in hibernating and active Chinese alligator.Our results indicate that gut microbial community compositions and structure vary significantly between hibernation and the active state.The temperature and diet are primary drivers for shaping the bacterial community and key microbial members.Additionally,colonic and faecal microbial communities in active Chinese alligator have similar microbial community.We propose that faecal samples are representative of colonic communities in such endangered species under normal physiological conditions,as a non-invasive sampling strategy.(6)The phylum Bacteroidetes and genus Bacteroides were highly represented in the microbiome of hibernating Chinese alligator,accounted for 57.95%and 55.82%respectively.These specific Bacteroides species are known to degrade and forage on host-derived mucins.Based on CAZy datasets,we identified 193 different CAZyme families comprising over 458 CAZymes.Among CAZymes,11 glycoside hydrolases families(GHs),two carbohydrate binding modules families(CBMs),and one carbohydrate esterase family(CE),which are known to bind and degrade various mucin oligosaccharides according to the CAZy database,were significantly enriched in the hibernation group.In addition,the hibernating Chinese alligator microbiome was highly enriched in 28 mucin oligosaccharide-degrading enzymes.These results demonstrate that mucin glycans are utilised as an energy source by mucin-degrading colonisers in response to low food-derived nutrient availability during hibernation.(7)In contrast,during the active phase,active Chinese alligators exhibit a carnivore gut microbiome dominated by Fusobacteria(20.37%~41.22%).Cetobacterium somerae,which accounted for nearly all of the taxa in the phylum Fusobacteria,was prevalent in hindgut of active Chinese alligator(Duodenum:17.81%vs.0.09%,p=0.05;Colon:31.40%vs.0.35%,p=0.002;Faeces:33.91%vs.0.81%,p=0.038).The enrichment of pathways and genes associated with AA metabolism was consistent with the increased abundance of bacteria capable of degrading and fermenting proteins and amino acids in the active phase,implying that the Chinese alligator's protein-based diet shapes the gut microbial community as an adaptation to a protein-rich diet in order to maximise nutrient extraction and energy production.(8)The analysis of interactions between distribution of pathogenic microbes and seasonal expression of immune-related genes showed that the opportunistic pathogens were common in the GI tracts of Chinese alligator,but the more pathogens and viral sequences(active vs.hibernation:4.03%vs.0.04%)were detected in the active group.We observed seasonal variations in the expression of?-defensins,with paralogues(AsBD105?,105?and 106?)more highly expressed during hibernation,which is regarded as immune compensation mechanisms for reduced mucosal barrier caused by bacterial mucin degradation during hibernation;and with orthologous?-defensins(AsBD5,10 and 13)predominating in the active state,in order to protected host from the more pathogenic microorganisms in the active phase.These results provide insight into functional adaptation by the gut microbiome that maintains gut homeostasis and health in the host in extreme physiological states.In summary,the insights of Chinese alligator?-defensin genes cluster provide basic information for the future study of other animals,especially for?-defensin in reptiles;provide important references for understanding the immune adaptability in alligator sinensis;provides an important theoretical and applied basis for the future development of alternatives to traditional antibiotics.Meanwhile,it is appealed that the conservators should pay more attention to the protection of gut microbial diversity in the systematic protection project of endangered animals.The metagenomic analysis of gut microbiome in Chinese alligator has important practical significance in the conservation biology of endangered animals and theoretical value in the interaction mechanism of host-microbes consortium in energy metabolism and immune response.