| Bactrocera dorsalis originated from South Asia.In more than 100 years since its first discovery,it has become a major fruit and vegetable pest in more than 70 countries,including China,Southeast Asia,the United States,Australia,India and the Pacific island countries,causing major economic losses.Short-term extreme temperature is an important factor affecting the seasonal dynamics of insect population in the field.In recent years,due to the more complex climate,the B.dorsalis has mostly suffered from abnormally high and low temperature environment.It is necessary to carry out the study on the population fitness comparison and adaptation mechanism of B.dorsalis under high and low temperature stress,in order to better understand the tolerance strategy of B.dorsalis,predict the population occurrence rule of the pest,and explore its feasible control measures.Therefore,by selecting 12 hours of low temperature(0,5,10℃)stress and 12 hours of high temperature(31,36℃)stress,taking 26℃ as the control group,this paper systematically studied the effects of high and low temperature stress on the growth,development and reproduction of B.dorsalis population under laboratory conditions,and used high-throughput sequencing technology and gene expression characteristics to study and record the molecular regulatory mechanism of B.dorsalis under high and low temperature stress,Finally,the physiological changes of B.dorsalis in the resistance to high and low temperature stress were determined by measuring the content of sugar metabolism substances in the body of the oriental fruit fly.The main results were as follows:1.Comparison of short-time temperature stress on the growth,development,and reproduction of B.dorsalisBy analyzing the biological characteristics and differences of the survival rate,reproduction,growth and development of B.dorsalis under different temperature stresses,it was found that the survival rate,life span and reproduction of the pest were significantly affected under single temperature stress.After high or low temperature treatment,the survival rate of all stages of B.dorsalis decreased gradually with the increase or decrease of different temperature.The development duration and preoviposition period were significantly prolonged by temperature stress,With the temperature change of high and low temperature treatment,the life span of male and female adults shortened,and the number of single female eggs increased first and then decreased gradually in low temperature treatment.,However,certain low temperature(10℃)treatment can promote the fecundity of female adults.To sum up,a single high temperature of 36℃ and a low temperature of 0℃ and 5℃ have adverse effects on the survival rate,life span,reproduction and development duration of B.dorsalis,and have greater response to low temperature for B.dorsalis.2.Transcriptome characteristics of B.dorsalis at different temperature stress were describedIn order to explore the changes of gene expression level of adult B.dorsalis under high temperature and low temperature stress,the transcriptome sequencing of adult was carried out using Illumina sequencing platform.After assembling the sequencing data of control group(26℃),the high temperature stress group(36℃)and the low temperature treatment group(0℃),a total of 60.01 Gb of clean data was obtained.The clean data of all samples reached 5.85 Gb,and the quality was qualified.A total of 1757 differentially expressed genes were obtained in the high temperature and low temperature stress groups.A total of 2021 differentially expressed genes in the low temperature stress and control groups.Among them,the heat stress group and the control group had the least differential genes.A total of 405 differential genes were obtained.The differentially expressed genes were enriched in GO and KEGG databases and found that high/low temperature stress caused significant changes in the expression of genes related to antioxidant pathway(HSP70,HSP60,UTP et al.)in sea horses.In addition,in the 0℃ vs 36℃ comparison group,the top 30 GO terms were related to biological processes.According to the proportion of the differentially expressed genes,the sequence was 30 genes involved in the intercellular translation process,27 genes enriched in stress response,18 genes enriched in protein folding,and 16 genes enriched in defense response;In the analysis of KEGG under high and low temperature stress,it was found that most of the enriched KEGG pathways were related to substance metabolism.Nine differential genes such as HSP70 and HSP30 were selected for transcriptome reliability verification,and the results showed that the results of RT-qPCR were basically consistent with those of sequencing.3.Cloning and identification of TPS genes from B.dorsalisBased on the information of trehalose synthesis-related genes screened by transcriptome,the gene of trehalose synthetase protein TPS was cloned by PCR and analyzed by bioinformatics,and the spatiotemporal expression characteristics and temperature stress response of BdTPS in B.dorsalis were analyzed by RT-qPCR.The results showed that the full-length cDNA sequence of B.dorsalis’ s TPS was 2693 bp,the length of open reading frame(ORF)was 2502 bp,encoding 833 amino acids,without signal peptide and transmembrane structure.The type of tertiary structure oligomer of TPS protein is homodimer,and they all have α-Helix and β-Folding structure.The consistency of TPS amino acid sequence with that of other insects is88.94%,which is highly conservative.The expression of TPS gene is the highest in larval and adult stage,and the lowest in pupal stage.The tissue distribution results show that the expression of TPS gene is higher in abdomen,fat body and hemolymph.After low temperature(5℃)and high temperature(36℃)stress,the relative expression of TPS in B.dorsalis adults was significantly higher than that in the control,and reached the peak at 5℃ stress for 6 hours.4.Changes in sugar metabolism substances of B.dorsalis under temperature stressThrough the analysis of carbohydrate content and important enzyme activity in the adult of B.dorsalis.Our results showed that after high and low temperature stress,the levels of glucose,trehalose and TPS increased,but the level of glycogen decreased.The glycogen content in the adult was lower than that of the control under high temperature or low temperature.Moreover,the glycogen content was the lowest at 0℃.The contents of trehalose and glucose in adults gradually increased with the increasing or decreasing temperature.The trehalose-6-phosphate synthase activity level of B.dorsalis was significantly higher than that of the control treatment at 0℃,5℃ and 10℃,while the synthetase activity showed an upward trend under high temperature.However,there was no significant difference with the control treatment.In short,B.dorsalis can improve its cold resistance by regulating its physiological metabolism level and accelerating the conversion of glycogen,trehalose or other carbohydrate substances into small molecular cold resistant substances under temperature stress.In conclusion,there were significant differences in the response of the population adaptability of B.dorsalis to high and low temperature stresses.To explore this difference,we systematically explored the response mechanism of B.dorsalis at the levels of ecological adaptation,physiology,biochemistry,and molecular biology in this study.By analyzing the differences in transcriptome information of the fly under different temperature modes,explored the molecular adaptation mechanism of trehalose-6-phosphate synthase under various temperature treatments.In addition,we further verified the changes in the content of four important trehalose metabolites in B.dorsalis.Our results provide clues for exploring the significant fluctuations in the population of B.dorsalis during high temperature situation in summer and low temperature in spring or autumn,facilitating the understanding of its population dynamics,prediction,and scientific management,which can provide a preliminary exploration for revealing the role of TPS as a potential molecular regulatory target in response to temperature stress in B.dorsalis. |
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