| RNA viruses,carrying molecules of ribonucleic acid(RNA)as their genetic material,are the most widely distributed viruses.Due to the error-prone function of the RNA polymerase RNA-dependent RNA polymerase(Rd Rp),the evolution of RNA viruses is extremely fast.Insects are often infected with a variety of RNA viruses,including lethal,asymptomatic,and latent infection types.The traditional virus identification methods are not effective enough to discover the asymptomatic and latent viruses.With the development of high-throughput sequencing technology,more and more insect viruses have been discovered.Due to their strong environmental adaptability and wide host range,Tephritidae flies could cause serious damage to the fruit and vegetable industry.Recently,the damaged areas of Bactrocera dorsalis are rapidly expanded,and this pest is gradually spreading to high latitude and low humidity areas in China.There have been many reports about the effect of symbiotic bacteria on improving the environmental adaptability of B.dorsalis,while the symbiotic viruses have not been reported yet.Besides,it is known that symbiotic viruses in insects could improve the environmental adaptability of the host.Therefore,exploring the symbiotic viruses in Tephritidae flies and their interaction with the host could increase the understanding of the environmental adaptability of those flies,and provide a new perspective for control of those pests.For this purpose,this thesis used high-throughput sequencing technology to discover RNA viruses of Tephritidae flies,and carried out subsequent studies in B.dorsalis.The main results are as follows:1.The discovery and identification of RNA viruses in Tephritidae flies based on transcriptome and small RNA sequencingThe transcriptome libraries of B.dorsalis,Bactrocera correcta,Bactrocera minax,Zeugodacus tau and Zeugodacus cucurbitae were constructed and sequenced,and the raw data of transcriptome of seven species of flies including Bactrocera frauenfeldi,Bactrocera kraussi,Bactrocera latifrons,Bactrocera musae,Bactrocera oleae,Bactrocera tryoni,and Bactrocera zonata were downloaded from NCBI.A total of 47full-length or nearly full-length sequences of RNA viral sequences were obtained.These viruses were comprised of 24 positive-stranded RNA viruses,8 negative-stranded RNA viruses,and 15 double-stranded RNA viruses.Sequence analysis and function annotation showed that they all had the conserved structure of RNA viruses,Rd RP.Phylogenetic analysis showed that there were abundant with Dicistrovirus,Negev virus,Orthomyxovirus,Totivirus,and Paritivirus.In terms of the distribution of RNA virus among the fly species,the B.dorsalis contained the most abundant viral species(12 RNA viruses).Further analysis of virus-derived small RNA by small RNA sequencing showed that most viruses could replicate in the host and trigger the host’s RNAi antiviral immune response,while a small number of viruses had weak signals in small RNA and no obvious si RNA characteristics.Notably,the phylogenetic tree showed that Bc TLV is similar to a virus in Trypanosoma,and the characteristics of its small RNA also consistent with the characteristics of the Trypanosoma’s RNAi pathway.Besides,a large number of Trypanosoma sequences were found in the library,implying that Bc TLV is a Trypanosoma virus in B.correcta.2.The infection patterns of RNA viruses at the population and individual levels in B.dorsalisThe infection patterns of 12 viruses in different developmental stages and tissues were analyzed by RT-q PCR.All of the 12 viruses were detected at each developmental stage.Except for Bd TLV2,the others were mainly infected in the pupae and adults.These viruses mainly infected the gut,fat body,and central nervous system.These viruses could also be detected in eggs and excretion,indicating that they could be transmitted horizontally and vertically.These viruses were also detected in all four field-collected populations with multiple infection patterns.Besides,the viral particles were observed by transmission electron microscopy.The patterns of viral infection at the individual level were also detected by RT-q PCR,and the results showed that the infection patterns of the above 12 viruses were different at the individual level from the population level.Bd CV exhibited intestinal tissue tropism at the individual level,and Bd NLV exhibited systemic infection at the individual level.Correlation analysis of viral loads showed that there was a strong correlation between some viruses in the gut and fat body,which indicated that they might be a synergistic effect.Groupe analyses were performed based on the load level of Bd CV in the gut.Intriguingly,the load of Bd TLV1 and Bd PLV in the high Bd CV load group was significantly higher than the low Bd CV load group.In addition,the correlation between viral loads was enhanced by grouping strategy,suggesting that Bd CV may play an important role in virus-virus interaction.3.Gene annotation of innate immunity pathways in B.dorsalis and its correlation with the viral infectionIn total,56 immune-related genes were annotated from the genome data of B.dorsalis by taking the available genes from the innate immunity pathways of D.melanogaster.Compared with the gene numbers in D.melanogaster,additional genes were found in Cactus(1),Domeless(2),while the IKK2 gene was missed,and reduced gene number in Toll and Autophagy were found in B.dorsalis.The expression patterns of the key genes of the five immune pathways in different tissues at the individual level were analyzed by RT-q PCR.Three key genes of the RNAi pathway(Dicer2,R2D2 and AGO2)showed the highest expression levels in the gut.The expression of three key genes(Myd88,Tube and pelle)of the Toll pathway were the highest in the ovary.Two key genes of the IMD pathway,IMD and Tak1,were also expressed at the highest level in the ovary.Two key genes in the JAK-STAT pathway,Hop and Stat92 E,were also showed the highest expression in the ovary.In the autophagy pathway,Akt was highly expressed in the gut,Toll7 was highly expressed in the fat body.The correlation analysis between viral loads and expression of immune genes showed that the infection of Bd CV,Bd TLV1,Bd TLV2,and Bd PLV2 in some tissues were highly correlated with the expression of some immune genes,suggesting that these viruses might be against by these immune pathways.Using the grouping strategy analysis mentioned in section 3,it was found that Bd TLV2 had a strong correlation with immune genes in multiple tissues.4.Bd CV could affect the feeding and digestion behavior of female adult hostsThe adults of low Bd CV load were obtained through isolation.Compared with this low Bd CV load files,the amount of food ingestion of female adults significantly increased in the adults with high Bd CV load,but there was no significant difference in excreting frequency.It was found that there was no significant difference in body weight between files with low and high Bd CV loads,but the contents of energy storage including triglyceride,trehalose,and glucose contents could be significantly increased with higher Bd CV load.In summary,RNA viruses were discovered and identified in 12 Tephritidae flies,and then the infection patterns of RNA viruses were analyzed at the population and individual levels in B.dorsalis.Moreover,the innate antiviral immune genes of B.dorsalis were identified and the relationships between its expression and viral infection were analyzed.Finally,it was revealed that the infection of Bd CV could promote the feeding and digestion behavior of the female adult.This work provides a new perspective for the pest control based on RNAi and RNA viruses. |
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