| Diaphorina citri(D. citri) is a vector of Huanglongbing, a destructive disease for citrus. One of the principal methods to control the spread of HLB is to repress the field population of Diaphorina citri(D. citri) by applying insecticides. However, the inappropriately applications of conventional insecticides to the field population of D. citri have led to high level of resistance. The resistance in D. citri to chemicals has been reported in China, USA and other citrus growing regions in the world. So the resistance study can point the way out for the effective control of D. citri. Pyrethroids are a class of neurotoxic pesticides used worldwide to control arthropod pests of medical, veterinary and agricultural importance. The toxicological target of pyrethroids is the voltage-gated sodium channel, which is a transmembrane protein that initiates and propagates the rising phase of action potentials of most excitatory cells. Pyrethroids preferentially bind to the open state of the channel, resulting in prolonged opening of the channel, which causes paralysis and subsequent death of the insect. Non-synonymous mutations in target-site would reduce the susceptibility of insects to insecticides. Simultaneously, the variants of sodium ion channel, consisting of different alternative exon combinations, have diverse properties in the interaction between proteins of the variants and pyrethroids. Transcriptomics, a method studying gene function, has been applied in multiple fields of science and technology. Transcriptomics has advantages including the ability of high throughput and the advanced algorithm, and is able to find the interested genes, for example the resistance related genes. According to above reasons, this thesis investigated the levels of susceptibility to conventional insecticides for field population of D. citri, tested the effects of host plants on the activities of detoxification enzymes in D. citri, characterized the sodium ion channel gene of D. citri and predicted the potential mutations, comprehensively analyzed the alternative exon combinations in single molecules of sodium ion channel gene and the frequencies of alternative exons in 7 developmental stages of D. citri. Finally, this thesis identified many resistance related genes. The main results are as following:1 Detection of susceptibility for D. citri to insecticides in FloridaThe susceptibility levels of D. citri to 7 insecticides were determined by topical assay using field populations from 5 counties in Florida. The results indicated that the susceptibility of D. citri to insecticides varied among counties in Florida. The susceptibility of psyllids from Lake Alfred to fenpropathrin was significantly lower than psyllids from Winter Garden. The susceptibility of psyllids from La Belle to carbaryl, dimethoate and thiamethoxam was significantly lower than psyllids from Fort Pierce. The susceptibility of psyllids from Fort Pierce to imidacloprid was significantly lower than psyllids from La Belle. The susceptibility of psyllids from Winter Garden tothiamethoxam was significantly lower than psyllids from Fort Pierce. Therefore, the data indicated that the D. citri in Florida might potentially develop resistance due to the reduced susceptibility of D. citri to insecticides.2 The effects of host plants on activities of detoxification enzymes in D. citriThe activities of detoxification enzymes P450, general esterase and glutathione S-transferase were determined using adults D. citri reared on the 3 host plants for 12 generations by established methods. The P450 activities were significantly higher for psyllids reared on Murraya paniculata than on Citrus sinensis and Murraya koenigii, but the latter two had no statistical difference. There were no differences for general esterase activities among the three samples. The activities of glutathione S-transferase for psyllids reared on M. paniculata and M. koenigii were higher than C. sinensis. The topical assays were also conducted to test the susceptibility of the D. citri populations from the three plants to fenpropathrin. The result shows that the susceptibility between the three D. citri samples was no significant differences, but was consistent with the activities of detoxification enzymes. Therefore different host plants can affect insects? detoxification enzyme system.3 Identification, analyses and mutation prediction of sodium ion channel gene in D. citriWe cloned and sequenced 18 fragments of sodium ion channel in D. citri, which cover the full CDS of the gene. 36 exons of sodium ion channel for D. citri were successfully defined by both bioinformatic analysis and more sequencing. The potential hotspot mutations were V410, M918 and F1534 in sodium ion channel in D. citri according to the usage of codon. We also identified the alternative exon j, i, a, b, k and l by using blastx search and fragments sequencing.4 Alternative exon combinations and their frequencies in developmental stages of D. citriA PCR system that was developed in this thesis called Variant-detecting PCR. It is a very important technology because it can be used in other organisms to detect alternative exons. 13 alternative exon, including j, i, a, b, f, k and l, combinations were identified by Variant-detecting PCR in 66 clones made from 7 developmental stages. The frequency of optional exon j was very low in eggs, whereas the frequency for optional exon b was relatively higher in 1st instar than other stages. For optional exon f, it was gradually down-regulated from 1st to 5th instar.5 Transcriptome assembly, gene-function annotation and identification of resistance related genes in D. citriTranscriptome assembly and analyses were conducted by using Trinity software for 4 samples of D. citri. 58,059 Unigenes were obtained from the assembly for all the samples and 20,191 Unigenes were annotated with function through the bioinformatic analyses. By gene function annotation, 84, 49, 46, 16, 16, 14, 9, 9, 2 and 2 sequences of ABC transportor, P450, NADH dehydrogenase, glutathione S-transferase, carboxylesterase, calcium channel, Catalase, chloride channel, GABA receptor and ryanodine receptor were identified respectively.In summary, the D. citri in Florida might potentially develop resistance due to the reduced susceptibility of D. citri to insecticides. It indicates that the real-time monitoring the level of insecticides resistance for field populations is needed to provide information in scientific use of insecticides. Host plants would reduce the susceptibility of D. citri to pyrethroids and other insecticides; therefore the management of D. citri should consider the effects of host plants on D. citri resistance. Mutiple mutations in sodium ion channel often confer the pyrethroids resistance in insect and have cumulative effect. Therefore if the resistance level is not monitored by bioassay, then the mutation in sodium ion channel could be checked to estimate the resistant risk for pyrethroids. The characterization and mutation prediction of sodium ion channel in D. citri paved a way for quickly detecting of insecticides resistance at molecular level. The investigation of alternative splicing in developmental stages provides new information to study the mechanism of resistance to pyrethroid. The insecticides resistance-related genes were identified using transcriptome data. Consequently, our study lays the foundation for the research of insecticides resistance at molecular level in D. citri. |
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