| The diamondback moth,Plutella xylostella(Lepidoptera:Plutellidae)is a worldwide migratory pest,attacking nearly all the cruciferous vegetable crops and causing severe economic loss.Cotesia vestalis(Hymenoptera:Braconidae)is a solitary endoparasitoid of P.xylostella larvae.Due to its wide distribution and high parasitism rate,C.vestalis has been used as a biological control agent for the management of P.xylostella.After being parasitized by C.vestalis,the P.xylostella larvae showed changes in immunity,growth and development,and nutritional metabolism.But whether the midgut microbes play an important role in the interactions bewteen parasitioid and host was largely unclear.Therefore,in this study we explored the diversity of midgut microbes of P.xylostella and C.vestalis,the effects of parasitism of C.vestalis on the midgut microbiota of P.xylostella and the possible mechanisms underlying this process,which may lay an essential foundation for further study.Three aspects were carried out:the midgut microbiota of P.xylostella larvae and their changes after parasitization;the midgut microbiota of C.vestalis larvae and their possible relationship with host gut microbiota;and the core species of midgut microbes and their possible biological significance during the interaction bewteen parasitioid and host.The main results were shown below:1)The community structure of the midgut microbes of P.xylostella and their core species of microbes.Based on prokaryotic 16S ribosomal RNA(rRNA)sequencing,we analyzed the midgut microbial communities in P.xylostellaC and P.xylostellaAd at different larval stages.Firmicutes and Proteobacteria were dominant phyla in the midgut of P.xylostella.Enterococcus and Carnobacterium were dominant microbial genera in P.xylostellac,and Enterococcus was the most abundant genus in P.xylostellaAd.Furthermore,we analyzed the midgut microbiota at the last stage of 3rd larvae(3L),middle stage of 4th larvae(4M)and last stage of 4th(4L)larvae of P.xylostellaC using metagenomic sequencing.It revealed that the microbial community of P.xylostella varies among different life stages,Enterococcus and Clostridium were abundant in the 3L larvae,while Enterococcus and Carnobacterium were dominant microbial genera in the 4th larvae.Interestingly,the most abundant genus was Enerococcus,which was dominant across the life history of P.xylostella.The qPCR results showed that the core species of P.xylostellaC was Enterococcus mundtii,while P.xylostellaAd was E.rotai.2)The effects of parasitism of C.vestalis on the microbial community diversity in the midgut of P.xylostella.To investigate whether parasitism plays a role in microbial community composition,we analyzed the midgut microbial communities in the 3L,4M and 4L larvae of parasitized and non-parasitized P.xylostellaC using metagenomic sequencing.The results demonstrated that the relative abundance of Firmicutes was significantly increased in the 4M larvae,but not altered in the 3L and 4L larvae of P.xylostella after parasitization.At the genus level,the relative abundance of Enterococcus was significantly increased in the 4M larvae of P.xylostella after parasitization.Alpha and beta diversity analyses showed that the microbial diversity decreased significantly in the 4M larvae,and tended to decrease in the 3L and 4L larvae of P.xylostella.The microbial community structure of parasitized P.xylostella underwent a drastic change.Based on traditional microbial culture methods and qPCR,we found that parasitism significantly increased the abundance of Enterococcus in the midgut of the 4th larvae of P.xylostellaC and P.xylostellaAd.Besides,the culturable bacteria and total bacteria were significantly increased in the 4M larvae of P.xylostellaC and P.xylostellaAd.3)The effects of parasitic factors of C.vestalis on the microbial community diversity in the midgut of P.xylostella.In order to explore the effect of parasitic factors on the midgut microbiota,we analyzed the midgut microbial communities in different stages of pseudoparasitized and control larvae of P.xylostellaC using metagenomic sequencing.Our results demonstrated that the variation tendency of the abundance of Firmicutes,and the dominant genus Enterococcus was similar after pseudoparasitization.The relative abundance of Firmicutes and Enterococcus were firstly increased(3L)and then decreased(4L)after pseudoparasitization.The microbial diversity has dropped significantly in the 3L larvae,unchanged in the 4M larvae,significantly increased in the 4L larvae of P.xylostella,and the microbial community structure changed dramatically in the 3L and 4L larvae of P.xylostella after pseudoparasitization according to alpha and beta diversity analyses.Comparing the pseudoparasitized larvae with the parasitized larvae,the relative abundance of the genus Enterococcus was significantly changed in the 4M and 4L larvae,but no significant difference was found in the 3L larvae of P.xylostella.The midgut microbial community structure exhibited an obvious difference between pseudoparasitized and parasitized larvae of P.xylostella,while no significant differences were found in the midgut microbial diversity of P.xylostella.Furthermore,the relative abundance of Firmicutes and Enterococcus were significantly increased in the midgut of 3L,4M and 4L larvae of P.xylostella after microinjected with CvBV or venom,respectively.Alpha and beta diversity analyses showed that the microinjection of CvBV or venom could change the microbial community structure,and reduced the microbial diversity in the midgut of 3L,4M and 4L larvae of P.xylostella,significantly.We found that CvBV and venom play an important role in the regulation of host gut microbiota.4)The microbial community composition and diversity of C.vestalis larvae.The midguts of C.vestalisc(C.vestalis larvae that grow in P.xylostellaC)and C.vestalisAd(C.vestalis larvae that grow in P.xylostellaAd)larvae were sampled and subjected to metagenomic sequencing.We observed that Proteobacteria,Firmicutes and Ascomycota,demonstrating the richness of the microbial community composition.The gut microbiota was dominated by three genera Streptomyces,Enterobacter and Enterococcus in the larvae of C.vestalisc,while the gut microbiota of C.vestalisAd larvae was dominated by Enterococcus,followed by Verticillium.No significant differences were found in the microbial diversity and microbial community structure between C.vestalisC and C.vestalisAd,according to alpha and beta diversity analyses.The Venn diagram showed that 119 microbial genera were common in the larvae of P.xylostella and C.vestalis,including the core genera Enterococcus,Enterobacter and Streptomyces.The results of microbial culture showed that C.vestalisc and C.vestalisAd shared a common bacterium with their corresponding hosts,i.e.E.mundtii presented in C.vestalisC and E.rotai presented in C.vestalisAd.Furthermore,we also demonstrated that Enterococcus species may be transmitted from the ovary to its offspring in C.vestalis by scanning electron microscope and fluorescence in situ hybridization analyses.5)The roles of Enterococcus species in the C.vestalis-P.xylostella system.Given that the abundance of Enterococcus species of P.xylostella was altered after parasitization,we explored the function of Enterococcus species in the P.xylostalla and C.vestalis system.Firstly,we successfully constructed the axenic and E.rotaicontaining P.xylostellaAd by treating the larvae with antibiotics and E.rotai,respectively.By sugar alcohol utilization experiment,we figured out E.rotai isolated from P.xylostellaAd midgut was capable of using various forms of sugars as carbon sources for the production of acids.The lactic acid concentration in the midgut of P.xylostellaAd was measured by HPLC,and the results showed that E.rotai increased the concentration of lactic acid,which was partially inhibited by parasitization.In addition,the trehalose concentration in the hemolymph of P.xylostella was lowered by E.rotai,which was negatively regulated by parasitization.Further research found that the change of trehalose concentration was achieved by regulating the expression of hexokinase PxHK,a key enzyme in the trehalose synthesis pathway.E.rotai also had a stimulating effect on the growth and development of P.xylostella and C.vestalis.In conclusion,parasitization affected the expression level of PxHK gene by changing the abundance of E.rotai in the midgut of P.xylostella,thereby regulating the sugar metabolism of P.xylostella,thus providing guarantee for the growth and development of the parasitoid wasp offspring. |
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