| The Guts of arthropods contain a large number of microorganisms,including bacteria,fungi,archaea,protozoa and viruses,which play an important role in the host's nutrition supply,digestion and absorption,growth and development,resistance to pathogen.Spiders belong to the phylum Arthropoda,class Arachnida,order Araneae,which are widely distributed and have a wide variety of species,and they are perceived as a kind of the important natural enemies of agricultural pests.However,the present studies on the microorganisms of spiders mainly focus on the detection of endosymbionts and their effects on hosts' reproduction and development,and little is known about the microbial communities of spider guts.In order to get insights into the composition and function of gut microbiotain spiders,in this study,we tested the microbial communities(bacteria and fungi)of guts and gonads from three spider species(Eriovixia cavaleriei,Larinioides cornutus and Pardosa pseudoannulata)by high-throughput sequencing.The difference of microbial composition between guts and gonads within a same spider,were analyzed,and the difference of microbial composition between female and male spiders were also analyzed.The function of intestinal and gonad microorganisms were predicted.The cultivable bacteria of two spider species(E.cavaleriei and P.pseudoannulata)which have different feeding methods were studied by using traditional microbial isolation and culture methods.This results will provide an important foundation for further research on the interaction among bacterial,fungal and spider hosts,and for geting insights into the function of intestinal microbiota of spiders.The main results are as follows:1.Morphological structures of midgut,ovary and testis of three spider species.Anatomical observation showed that the intestinal structures of different spiders species were similar: midguts were slender,and oval dung bags were expanded at the end of midguts.There were small branches around the intestinal wall.The ovaries and testicles of E.cavaleriei and L.cornutus were long and thin,while the ovaries and testicles of P.pseudoannulata were full and large.The size of the testis of P.pseudoannulata was obviously different from those of E.cavaleriei and L.cornutus.2.Diversity and differential analysis of intestinal and gonadal bacteria in three spider species.We examined the bacterial communities of guts and gonads in E.cavaleriei,L.cornutus and P.pseudoannulata by Illumina-Hiseq sequencing based on the V3-V4 high variable regions of the 16 S r RNA gene.The results showed that there were a large number of bacterial groups in the body of these three spider species.In the guts of three species of spider,Proteobacteria was the most dominant phylum in the guts of E.cavaleriei,L.cornutus and P.pseudoannulata.There was no significant difference in bacterial structure between male and female gut of the same spider at phylum level(P > 0.05).Proteobacteria in guts of E.cavaleriei had a significant higher abundance compared to the guts of L.cornutus and P.pseudoannulata(P < 0.05).At the genus level,Wolbachia had a high abundance in the guts of E.cavaleriei,and Spiroplasma had a high relative abundance in the guts of L.cornutus.Besides Lachnospiraceae?NK4A136?group,the other gut bacteria(relative abundance?1%)had no significant difference between the male and female guts in the same spider species(P >0.05).In the results of Beta diversity analysis : there was no significant difference in the bacterial communities between the male and female guts of each spider species,but the bacterial communities of guts between three spider species were different.In the ovary and testis of three spider species,the relative abundance of Proteobacteria in the ovary of E.cavaleriei(93.08%)was significantly higher than that in testis(65.19%)(P <0.05),and the relative abundance of other dominant phyla in the testis of E.cavaleriei was significantly higher than that in ovary(P < 0.05).Besides Tenericutes,other dominant phyla had no significant abundance between the ovary and testis in L.cornutus.In the gonad of P.pseudoannulata,the relative abundance of Proteobacteria,Bacteroidetes and Actinobacteria were significantly different between the ovary and testis(P < 0.05).At genus level,the relative abundance of Wolbachia in the ovary of E.cavaleriei(91.94%)was significantly higher than that in testis(57.31%)(P < 0.05).Among the top 30 bacterial genera,the relative abundance of most bacteria were significantly different between the ovaries and testicles ofeach spider species(P < 0.05).The results of Beta diversity analysis showed that there were differences in the structure of the bacterial community between the ovary and the testis in E.cavaleriei and L.cornutus,and the structure of the bacterial community between the ovary and testis in P.pseudoannulata was similar,but the structure of the bacterial community between the gonads of three spider species were different.Through the comparative analysis of the relative abundance of top 30 bacteria between gut and gonad of the same spider species,we found there were no obviously difference between female gut and ovary of the same spider species,but the relative abundance of some bacteria were different between male gut and testis.Prediction of bacterial functions in the guts and gonads showed that bacteria in spiders were associated with amino acid metabolism,carbohydrate metabolism,metabolism of cofactors and vitamins,energy metabolism,nucleotide metabolism and translation.3.Diversity and differential analysis of intestinal and gonadal fungi in three spider speciesThe data showed that a small amount of fungi were detected within the spiders.A total of252 OTUs were detected in the guts and gonads of female and male spiders.In the three spider species,Ascomycota was the main dominant fungal phylum,and the relative abundance of Ascomycota in the gut of male E.cavaleriei was as high as 92.97%,and there was significant difference in the relative abundance of Ascomycota and Basidiomycota between female and male guts of E.cavaleriei(P < 0.05).There were no significant difference in the relative abundance of Ascomycota and Basidiomycota between female and male guts of L.cornutus and the same was in P.pseudoannulata(P > 0.05).At genus level,Malassezia was the main dominant bacteria in the guts and gonads of the three spider species.Besides Candida,there were no significant difference in the relative abundance of the other bacteria between the female and male guts of E.cavaleriei and L.cornutus(P > 0.05).There was a higher fungal diversity in the guts of P.pseudoannulata,and besides Sarocladium,the relative abundance of other fungi had no significant difference between female and male guts of P.pseudoannulata.Beta diversity analysis showed that the difference of fungal composition between the male and female guts of E.cavaleriei and P.pseudoannulata were very similar,and the fungal composition between the female and male guts of L.cornutuswere different.There was some difference in the fungal structure between the guts of the three spider species.In the ovary and testis of three spider species,six phyla were annotated,Ascomycota was the dominant fungal phylum in the ovaries and testicles of three spider species.The relative abundance of Ascomycota in the testis of E.cavaleriei was significantly higher than that in ovary(P < 0.05).There were no significant difference in the relative abundance of Ascomycota and Basidiomycota between ovaries and testicles of L.cornutus and P.pseudoannulata(P > 0.05).At genus level,the relative abundance of some fungi genera were significantly different between the ovaries and testicles of E.cavaleriei,L.cornutus and P.pseudoannulata(P < 0.05).Some fungi genera had high abundance in the ovary and testis of P.pseudoannulata.The results of Beta diversity analysis showed that the structure of fungi in ovary and testis of each spider were similar,and the structure of fungi in gonads between three spider species were different.Through the comparative analysis of fungal relative abundance between gut and gonad from the same spider species,it was found that only a few fungi had difference between the guts and gonads of each spider species.4.Isolation and identification of culturable bacteria in spider gutsLB and NA medium were used to isolate and cultivate the gut bacteria of E.cavaleriei,Propionibacterium(Actinobacteria),Sphingomonas(Proteobacteria),Bacillus(Firmicutes),Paenibacillus(Firmicutes)and Fictibacillus phosphorivorans(Firmicutes)were identified in this study.The feces of P.pseudoannulata were isolated and cultured with 1/5 LB medium,the identified strains were Corticibacterium populi,Acinetobacter,Enterococcus faecalis,Pseudomonas,Staphylococcus,Sphingobacterium,Exiguobacterium and Comamonas.After the sample of P.pseudoannulata were feed with artificial food for two months,the feces were isolated and cultured,and the isolated strains were identified as Enterobacter,Acinetobacter,Pseudomonas,Staphylococcus and Chryseobacterium.Before and after artificial feed breeding,the intestinal bacteria of P.pseudoannulata belong to Proteobacteria and Firmicutes.After artificial feed breeding,the feces became lighter in color and the culturable bacteria were reduced. |
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