| The brown planthopper(Nilaparvata lugens St?l,BPH)is one of serious insect pests of rice in China.BPH not only directly sucks sap from rice phloem that can lead to serious wilt of the whole plant,but also spreads rice virus disease which can further cause a huge loss of rice yield.At prsent,an economically efficient method for BPH control is the utilization of resistant rice cultivars.However,the life-span of resistant rice varieties is prone to extremely shorten because of the rapid variation in the virulence of BPH.In view of key roles that insect gut microbes performed in the food degradation,detoxification metabolism and immune regulation,the gut microbes of BPH are highly prossible to participate in the process of virulence variation of the host.However,the current understanding of the gut microbial diversity and its relationships between different BPH virulence populations is stillquite limited.Therefore,this study uses two BPH populations with different virulence levels which are seperatedly reared on the susceptible rice variety TN1(TN1 population)and the resistant rice variety ASD7(ASD7 population)as research objects.Based on the amplicon high-throughput sequencing and macrotranscriptome technology,the complete community structure,species diversity,biological function of the gut microbes in BPH and its differentiation during different developmental stages and between two different BPH virulence populations were detected and analyzed.The main results are as follows:1.Gut microbial diversity analysis in BPH across different developmental stages: The gut bacterial 16 S rRNA and the fungal ITS gene fragments were sequenced by Illumina MiSeq(PE300)for BPH nymphs(first to fifth instar)and 24 h-emergence adults(male and female)which were reared on TN1 rice.Based on clustering analysis of the valid sequences from each sample at 97% similarity level,132(424),238(510),195(573),169(574),88(724),179(309)and 250(388)OTUs(Operational taxonomic unit)of gut bacteria(gut fungi)were obtained from the gut microflora of first to fifth instar nymphs,female and male adults of BPH,respectively.According to the species classification analysis of OTUs,the gut bacteria were annotated into 19 phyla,50 classes,85 orders,165 families and 306 genera,while the gut fungi were annotated into 5 phylum,15 classes,28 orders,38 families and 47 genera 59 species,respectively.At the level of phylum,class and order,the dominant bacteria in each sample were from Proteobacteria(68.68%-96.22%),Gammaproteobacteria Proteobacteria(58.64%-92.94%)and Enterobacteria(50.29%-83.43%),while the dominant gut fungi were Ascomycota(98.24%-99.89%),Sordariomycetes(92.22%-98.71%)and an unclassified order in Sordariomycetes(89.17%-96.76%).At the family level,the abundance of Enterobacteriaceae was the highest(50.29%-83.41%),followed by Moracaceae(5.34%-35.84%).Remarkably,the abundance of Moraxaceae in nymphs was significantly higher than that in adults.The dominant fungi were from an unclassified family in Sordariomycetes,with the relative abundance ranging from 93.06% to 96.76%.At the genus level,the dominant genera of gut bacteria in BPH across all developmental stages were Acinetobacter,with the relative abundance varing from 41.36% to 75.85%.The dominant fungal genus with the highest abundance(93.06%-96.76%.)was an unidentified genusin Sordariomycetes.The diversity index analysis showed that the highest diversity level of gut bacteria and fungi was observed in male adult,followed by female adult,and the lowest was fifth instar nymph.2.Gut microbial diversity and difference analysis in two BPH populations with different virulence level: Gut microbial microbial diversity and its difference in TN1 and ASD7 populations were analyzed based on 16 S rRNA and ITS amplicon high-throughput sequencing.16 S rRNA sequencing generated 80156 and 80336 valid sequences for TN1 and ASD7 samples,respectively.519 and 352 bacterial OTUs were finally obtained by sequences clustering at 97% similarity level.Of those,271 OTUs were shared by two samples.After quality control,a total of 36 367 valid sequences of ITS were obtained from both TN1 and ASD7 samples,and 268 fungal OTUs were clustered.Among them,251 OTUs were from TN1 sample and 249 OTUs were from ASD7 sample.The number of shared fungal OTUs was 232.Based on the OTU classification,the gut bacteria(fungi)of TN1 sample were annotated to 20(4)phylum,29(7)class,64(8)orders,107(10)families and 174(11)genera,while the gut bacteria(fungi)of ASD7 sample covered 13(3)phyla,19(6)classes,43(7)orders,81(9)families and 131(14)genera.The most dominant bacteria in the both samples affiliated to Proteobacteria(90.00%-95.44%),Gammaproteobacteria(84.95%-85.59%),Pseudomonas(52.57%-83.26%)and Moramoraceae(52.57%-83.20%),while the most dominant fungi all belonged to Ascomycetes(96.44%-99.77%),Sordariomycetes(74.66%-76.64%),Hypocreales(61.37%-63.84%)and an unidentified family(63.60%-66.06%)in Hypocreales.At the genus level,the dominant gut bacteria and fungi with the highest abundance belonged to Acinetobacter(52.41%-83.18%)and an unclassified genus in Hypocreales(63.60%-66.06%),respectively.The results based on statistical analysis of the Alpha diversity indices,such as Shannon,Simpson and Chao1,showed that the community abundance and species diversity of TN1 samples were significantly higher than those of ASD7 samples.Our results preliminarily indicated that gut microbes might play an important role in the process of virulence variation in BPH.3.qPCR verification of the high-throughput sequencing results: qPCR technology was used to verify the effectiveness of the high-throughput sequencing results.Four gut microbes,which were annotated by high-throughput sequencing,were randomly selected from the TN1 population at different development stages.These four gut microbes included two bacterial species(Acinetobacter sp.and Lactobacillus sp.)and two fungal species(Wallemia mellicola and Cryptococcus sp.)The copy number of gut microbes was determinated by qPCR technology(absolute quantitative).The results showed that the copy numbers of Acinetobacter sp.in BPH nymphs(first to fifth instar)and 24 h-emergence adults(male and female)were 21 245,28 253,33 265,35 240,46 363,43 208 and 38530,respectively.The copy numbers of Lactobacillus sp.in BPH nymphs(first to fifth instar)and 24 h-emergence adults(male and female)were 332,760,585,423,267,489 and 4026,respectively.The ratios of the two were similar to those obtained by high-throughput sequencing.In addition,in nymphs(first to fifth instar)and 24 h-emergence adults(male and female),the number of copies of Wallemia mellicola in the gut of BPH was 12635,22366,35645,45231,48220,50273 and 68656,respectively.The copy number of Cryptococcus sp.was 4263,4013,1253,2536,4921,8996 and 1423,respectively.The ratio of the two was also consistent with the results in high-throughput sequencing.The results of the quantitative analysis of four gut microbes were basically consistent with those of the high-throughput sequencing,and thus verifyed that the high-throughput sequencing data had a high reliability.4.Metatranscriptome construction and gene difference expression analysis of gut microbes in two BPH populations with different virulence levels: Metatranscriptomic sequencing for gut microbes associated with BPH was conducted and then gene difference expression analysis were performed between TN1 and ASD7 populations.The results showed that the gene expression of gut microbes between TN1 and ASD7 populations was significantly different.Compared with TN1 population,there were 2,151 differentially expressed genes(DEGs)in gut microbes of ASD7 population.Of those,71.27%(1533)were up-regulated and 28.73%(618)were down-regulated significantly.The GO enrichment analysis showed that the number of DEGs which distributed in biological processes,cell components and molecular functions were 495,403 and 212,respectively.Among them,peptidoglycan biosynthetic process,small/large ribosomal subunits and NADH dehydrogenase activity related gene expressions were active.The KEGG enrichment analysis showed that DEGs were mainly enriched in 149 metabolic processes in ASD7 sample,such as environmental adaptation,amino acid metabolism and carbohydrate metabolism.These results lay a foundation for revealing the mechanism of BPH virulence variation from the perspective of symbiotic microbes. |
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