Genetic Diversity Of Diaphorina Citri In China And The Influences Of Its Host Plants

Asian citrus psyllid,Diaphorina citri Kuwayama(Hemiptera:Liviidae),is the main pest of citrus in the new shoot stage.This insect pest sucks sap from citrus phloem and transmits Candidatus Liberibacter asiaticus(CLas)as a vector.Huanglongbing(HLB)caused by CLas is a devastating citrus quarantine disease and results in a great threat to the citrus industry all over the world.In this study,we analyzed the genetic diversity,genetic structure and demographic history of citrus psyllid population in China by using mitochondrial DNA(mtDNA)and microsatellite loci(SSR).On those results,the major force to drive genetic differentiation of D.citri—host plant‘influences were further studied,including:host plant influence on feeding selection,growth,development,reproduction and life table parameters of D.citri;host plant influence on the composition and structure of gut microbiota of D.citri;and the influence of host plant-borne CLas on the transcriptome of D.citri.This study provides background information for revealing the genetic diversity and the internal mechanism of D.citri host adaptive differentiation and has important theoretical and practical significance for promoting the ecological regulation and integrated control of D.citri.The main results are as follows:1.SSR markers and mitochondrial genes were used to investigate the genetic diversity,genetic structure and demographic history of 225 samples of D.citri from 7provinces in China.1)Based on the whole genome sequence,nine SSR loci with high polymorphism were selected.Then,we found that the genetic diversity of D.citri in China was high with a mean observed heterozygosity of 0.50 by using those nine SSR loci as molecular markers.The genetic structure detected by analysis of molecular variance(AMOVA),cluster analysis and principal coordinate analysis(PCoA),suggested that there was a significant genetic differentiation in D.citri of China.D.citri that feeds on Shatangju,NF mandarin and Ponkan were the most closely related and clustered one group;D.citri feeding on Common jasmin orange and Lemon were clustered one group;and D.citri feeding on Citrus unshiu,Bingtangcheng,Summer orange and Navel were clustered the other group.In summary,the results showed that genetic differentiation driven by host plants existed in D.citri of China.2)A total of 26 haplotypes of D.citri were identified from the concatenated mtDNA(COI,Cytb and ND5),with which all populations showed low diversity at both the nucleotide(?:0.00082)and haplotype(Hd:0.4957)levels.Cluster analysis showed that there was no obvious genetic differentiation,which indicated that the intraspecific variation of D.citri was limited at mtDNA,and that the evolution history of D.citri in China was short.We suggested the host-adapted divergence is likely to have occurred very recently.Further Mantel tests of isolation by distance revealed no significant correlation between genetic differentiation and geographical distance(r~2<0.01,p?0.32).The results of mismatch analysis(unimodal distribution)and neutral test(Tajima‘s D=-2.61,p<0.01;Fu‘s Fs=-5.23,p<0.01)suggested a recent demographic expansion.2.Based on the above research that the host plant is the main driver of the genetic differentiation of D.citri in China,we further studied the influences of the host plant to D.citri.The influence of host plants on the growth,development,reproduction and life table of D.citri were studied under laboratory conditions.The results showed that the selectivity of D.citri to five host plants was significantly different(p<0.01),and the preference was:Navel>Shatangju,Common jasmin orange,Lemon>Ponkan.The total nymph duration of D.citri on different host plants was significantly different(p<0.01),with the shortest duration on Shatangju(12.78 d)and the longest on Lemon(14.18 d).Host plants significantly affected the pre-oviposition duration of D.citri(p<0.01),and the pre-oviposition duration of adult female psyllid was the shortest on Shatangju(7.56 d)and the longest on Ponkan(8.38d).Studies on the life table showed that the net reproductive rate of D.citri that feeds on Shatangju(162.12)and Common jasmin orange(155.05)was significantly higher than that on other host plants.The age-specific survival curve indicated that D.citri that feeds on Shatangju and Common jasmin orange had a significant high survival rate(p<0.01).To sum up,host plants can significantly affect the nymph duration,pre-oviposition duration and population growth rate of D.citri.Among five host plants tested,D.citri had the highest suitability on Shatangju and Common jasmin orange.3.The results of the study on gut microbial diversity of D.citri based on 16S rDNA showed that:1)Host plants significantly affected the richness and diversity of gut microbiota.The gut microbial richness and diversity of D.citri that feeds on Ponkan and Common jasmin orange was significantly higher than that on Navel,Lemon and Shatangju(p<0.01).The average number of observed OUT of D.citri that feeds on Common jasmin orange and Ponkan were 266 and 259,respectively,which was significantly higher than that on Navel(97),Lemon(149)and Shatangju(119)(p<0.01).Chao1 index in D.citri that feeds on Ponkan(290)was higher than that on Navel(145),Lemon(186)and Shatangju(195)(p<0.01).Shannon index was the highest in gut sample of D.citri feeding on Ponkan(1.812),followed by Common jasmin orange diet(1.498),and the lowest was in Shatangju diet(0.554)(p<0.01).Simpson index was higher in D.citri gut samples that feeds on Shatangju(0.796)than that on Ponkan(0.288),Lemon(0.408),Common jasmin orange(0.466)and Navel(0.490)(p<0.01).2)Host plants significantly influence the composition and structure of gut microbiota of D.citri.Proteobacteria,Actinobacteria,Firmicutes and Bacteroidetes were present in all D.citri gut samples.Wolbachia,Escherichia-shigell,Candidatus Profftella,Pantoea,Lactobacillus and Stenotrophomona were the core gut microbiota of D.citri.However,host plants can significantly affect the composition of psyllid gut microbiota.Acidobacteria was only found in D.citri gut samples feeding on Common jasmin orange,Ponkan and Lemon;Spirochaetes was only found in Common jasmin orange,Ponkan,Navel and Lemon feeding D.citri samples;and Deinococuus-Termus and Cracilibacteria were only found in Ponkan feeding D.citri samples.In addition,host plants also significantly affected the predominant gut microbiota of D.citri.The predominant gut microbiota of D.citri that feeds on Common jasmin orange was Wolbachia(68.13%);the predominant gut microbiota of D.citri feeding on Ponkan were Escherichia-shigell(31.31%)and Wolbachia(48.45%);the predominant gut microbiota of D.citri feeding on Navel was Escherichia-shigell(82.91%);and the predominant gut microbiota of D.citri feeding on Lemon were Wolbachia(45.38%)and Escherichia-shigell(21.91%).4.RNA-Seq technology was used to study the influence of host plant-borne CLas on transcriptome of D.citri.Using more stringent criteria(FDR?0.01 and Log2 fold change?1),200 differentially expressed genes related to D.citri's response to CLas were obtained,and 543 differentially expressed genes related to D.citri's successful resistance to CLas were obtained.These genes are mainly concentrated in two pathways of energy metabolism and immune defense.1)CLas infection mainly affects the metabolism of D.citri.There were 41 unigenes in total,and 34 of them were up-regulated,indicating that CLas infection would enhance the metabolic activity of D.citri.All 9 unigenes related to vitellogenin were up-regulated,indicating that CLas can promote the reproductive ability of D.citri.Against the infection of CLas,D.citri initiated lysozyme pathway for immune defense,and there are 4 differentially expressed unigenes,among which 3 were up-regulated.No other genes were involved in immune pathway.2)Two immune pathways were selected from the differentially expressed genes related to CLas resistance.Among them,there are 15 unigenes related to cellular immunity,including lysozyme pathway,endocytosis and phagosome pathway and 10 unigenes were up-regulated,indicating that cellular immunity played an important role in D.citri resistance to CLas.The peritrophic is the first natural barrier against invading pathogens.In this study,a total of 11 peritoneal protein-related unigenes were detected,and the expression of these unigenes was down-regulated,suggesting that D.citri might initiate the peritoneal membrane excretion mechanism to eliminate CLas.In the energy metabolism pathway,the expression of unigenes,which is related to carbohydrate transport and metabolism,and lipid transport and metabolism,were significantly up-regulated,indicating that D.citri needs to consume a lot of energy in the process of resisting to CLas.However,62%of unigenes related to amino acid transport and metabolism were down-regulated,suggesting that D.citri might prevent CLas from colonization and reproduction by limiting the supply of essential amino acids.