Diversity And Difference Of Endosymbionts In Laodelphax Striatellus From Different Developmental Stages And Host Crops

The small brown planthopper?SBPH?,Laodelphax striatellus Fallén,is one of important agricultural pests in Asia.As a euryphagous insect pest with piercing-sucking mouthpart,SBPH can feed on diverse graminaceous crops,such as rice,wheat and barley.Moreover,the different generation of SBPH can transfer between different host crops and cause a huge loss of crop yields.Searching for a safe and efficient way to control of SBPH is a key step in the current SBPH prevention and control work.Insect endosymbionts are one of important microbial resources that can be exploited and utilized in pest biological control,as they play important roles in the growth,development and reproduction of insects.In order to fully understand the diversity and ecological functions of endosymbionts in SBPH,and to also provide new ideas for developing SBPH biocontrol technologies based on the relationships of symbiosis,this study use different SBPH populations which feeding on three crop plants?rice,wheat and barley?as research objects.Firstly,the community composition and species diversity of endosymbionts are compared by metagenomic method during different developmental stages of SBPH.Secondly,the endosymbiont diversity and differential gene expression pattern among three SBPH populations feeding on under different host crops were detected based on metagenomics and metatranscriptomic techniques.The main results are as follows:1.Community composition and species diversity analysis of endosymbionts during different developmental stages of SBPH:The community compositions and species diversities of symbiotic bacteria and symbiotic fungi are detected in SBPH nymphs(1^st to 5^th instar)and adults?male and female?through bacterial 16S rDNA and fungal ITS high-throughput sequencing,respectively.The valid sequences obtained from each sample were clustered and analyzed according to the sequence similarity.As a result,79?59?,65?89?,62?80?,87?88?,68?78?,55?98?and 57?73?OTUs of symbiotic bacteria?symbiotic fungi?were obtained from 1^st to 5^th instar nymphs?B1 to B5?,female?BF?and male?BM?adults of barley-feeding SBPH,respectively.The total OTUs of the symbiotic bacteria?symbiotic fungi?in all samples were annotated into 12?3?phyla,24?17?classes,42?31?orders,74?40?families,139?53?genera and 156?71?species,respectively.At the levels of phylum,class,and order,the most dominant bacteria in each sample were from Proteobacteria?95.27%-99.94%?,?-Proteobacteria?89.19%-99.56%?and Rhodospirillales?23.54%-89.73%?,while the predominant symbiotic fungi all belonged to Ascomycota?94.28%-98.99%?,Sordariomycetes?56.41%-97.35%?and Hypocreales?54.97%-95.64%?,respectively.At the family level,the dominant symbiotic bacteria with the highest abundance belonged to Acetobacteraceae?23.54%-89.76%?and Anaplasmataceae?7.18%-74.88%?and the dominant symbiotic fungi were all from an undetermined family in Hypocreales?49.85%-95.38%?and Trichocomaceae?Trichocomaceae,0.66%-38.96%?.At both the genus and species levels,the dominant symbiotic bacteria and symbiotic fungi were all annotated as unidentified genus or species.The abundance of the species with full name,such as Acinetobacter baylyi,Stenotrophomonas maltophilia,Penicillium variabile and Sterigmatomyces halophilus were all generally low?less than 2%?.Diversity index analysis indicated that the most diverse bacterial communities of SBPH in order were detected in B2,BM,B3,B5,B4,BF and B1,while the most diverse fungal communities was observed in BM,successively followed by B4,B2,BF,B1,B5 and B3,respectively.2.Community composition and species diversity analysis of endosymbionts in SBPH from different host crops:Through bacterial 16S rDNA and fungal ITS high-throughput sequencing,the community compositions and species diversities of symbiotic bacteria and symbiotic fungi are determined in the newly emerged female adults?24 h after emergence?of three SBPH populations,which were feeding on barley?BF?,wheat?WF?and rice?RF?.Consequencely,48?109?,69?59?and 96?52?OTUs of the symbiotic bacteria?symbiotic fungi?were detected in BF,WF and RF,respectively.Among them,16 OTUs of symbiotic bacteria and 35 OTUs of symbiotic fungi were shared between the three samples.The community compositions of endosymbionts were basically similar among BF,WF and RF,although the relative abundances of those were different.The total OTUs of symbiotic bacteria?symbiotic fungi?in BF were annotated into 5?5?phyla,8?16?classses,14?29?orders,19?36?families,27?46?genera and 27?56?species,respectively.The total OTUs of symbiotic bacteria?symbiotic fungi?in WF covered 5?4?phyla,8?9?classes,16?11?orders,23?13?families,30?17?genera and 33?20?species,while a total of 5?3?phyla,12?9?classes,23?12?orders,36?14?families,59?20?genera and 63?22?species were annotated in RF,respectively.The most dominant symbiotic bacteria in the three samples both belong to Proteobacteria?97.49-99.95%?,Alphaproteobacteria?83.84-97.84%?and Rhodospirillales?6.88-83.94%?.At the level of family and genus,the dominant bacteria in both BF and WF belonged to Acetobacteraceae?83.95%for BF/67.92%for WF?and an unclassified genus in Acetobacteraceae?83.94/67.75%?,while the dominant bacteria were from Anaplasmataceae?76.83%?and Wolbachia?76.77%?in RF,respectively.In general,the abundance of bacteria annotated into specific species was extremely low,as all of which were less than 5%.The most dominant symbiotic fungi in the three samples were all belonged to Ascomycota?94.10-99.88%?,Sordariomycetes?87.78-99.18%?,Hypocreales?73.08-94.55%?,and an unclassified family?72.62-90.29%?,genus?72.62-90.29%?and species?72.63-90.29%?in Hypocreales.the values of Alpha diversity indices,such as Chao1,Shannon and Simpson,indicated that both the symbiotic bacteria and symbiotic fungi in the BF,WF,and RF were highly rich and diverse,and the symobtic community structure in SBPH was significantly affected by its host crops.The highest diversity of symbiotic bacteria was observed in RF,while the lowest shown in BF.The rank for the diversity of symbiotic fungi in SBPH was BF>WF>RF from high to low.3.Quantitative analysis and verification of three dominant endosymbionts in SBPH:Quantitative dynamic of three dominant endosymbionts?Noda,Wolbachia and Acetobacteraceae?in SBPH during different developmental stages and under different host crop rearing conditions was analyzed and verified by qPCR?quantitative PCR?.The results showed that the number of three dominant bacteria was significantly changed during the entire developmental stages of SBPH.The number of Noda?Hypomyces chrysospermus?gradually increased in nymph of SBPH from 1^st to 3^thh instar,decreased at the 4^th instar,rebounded at the 5^th instar and then peaked in female adults.A significant difference in the number of Noda was also detected among 1^st,2^nd,5^th and male adult of SBPH under different host plant rearing conditions.The number of Wolbachia increased steadily with the increase of nymphal instar of SBPH and rapidly increased at the adult stage.The number of Wolbachia in rice-feeding SBPH was significantly higher than that of barley-or wheat-feeding poupulations.The number of Acetobacteraceae bacteria in barley-and wheat-feeding SBPH was significantly different between nymphal and adult stages,as the number of which was reached the highest level at female adults.However,the number of Acetobacteraceae bacteria in rice-feeding SBPH did not change significantly during nymphal stages,but significantly different in adult stage.The number of Acetobacteraceae bacteria in male adults was significantly higher than that in female adults.Overall,The results of the quantitative analysis of three dominant endosymbinots are basically consistent with those of obtained above two chapers,and hence could verify,which the accuracy of the high-throughput sequencing data in a certain extent.4.Metatranscriptome construction and gene difference expression analysis of endosymbionts in SBPH from different host crops:The Illumina HiSeq^TM4000sequencing platform was used for metatranscriptomic sequencing in SBPH,which were feeding on barley?SBPH-B?,wheat?SBPH-W?and rice?SBPH-R?,respectively.The differentially expressed genes?DEGs?were obtained by the function annotation and the related bioinformatics analysis of the metatranscriptomic sequencing data.As a result,the number of DEGs between SBPH-B and SBPH-W was significantly lower than that between SBPH-B and SBPH-R or between SBPH-W and SBPH-R.As presented,only3,450 DEGs?1,747 upregulated and 1,703 downregulated?were found betwwen SBPH-B and SBPH-W.However,32,228 DEGs?10,195 upregulated and 22,033downregulated?and 30,659 DEGs?8,646 upregulated and 22,013 downregulated?were observed between SBPH-B and SBPH-R,and between SBPH-W and SBPH-R,respectively.GO enrichment and KEGG pathway enrichment analysis indicated that the function of DEGs was highly correlated to the type of host crops which was SBPH fed on.For example,GO function enrichment analysis showed that mycelium development is the main biological process,meanwhile structural constituent of ribosome related gene expression was active in SBPH-B and SBPH-W.However,the main biological process determined in SBPH-R is transport and the expression rna-directed dna polymerase activity genes were active.KEGG pathway enrichment analysis showed that endosymbionts in barley-and wheat-feeding SBPH were mainly involved in metabolic pathways such as ribosome and biosynthesis of amino acids,while the metabolic pathways in rice-feeding SBPH are mainly enriched in peroxisome,amino acid metabolism?Lysine/Glyine/Serine/Threonine?and others.