Functional Studies Of MicroRNAs In The Regulation Of Energy Metabolism In Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel)is one of the most dangerous horticultural pests in China.Currently,baits and chemical insecticides are mainly used for pest control,and the misuse of chemical pesticides has caused a series of problems,such as pollution of the ecological environment,pesticide residues and resistance,so there is an urgent need to develop new efficient green control technologies.Energy metabolism is the most basic life activity of living organisms,and the regulation of metabolic homeostasis is maintained by an interwoven regulatory network,which is crucial to the physiological processes of insects,such as growth and development,molting,metamorphosis and reproduction.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs of 21 nt in length that mainly regulate gene expression at the post-transcriptional level and can participate in various biological processes in insects,such as development,differentiation,immunity and host–microorganism interactions.However,the functions of miRNAs in insect energy metabolism and their molecular regulatory mechanisms are still poorly understood.However,the functions of miRNAs in insect energy metabolism and their molecular regulatory mechanisms are still poorly understood.In this study,we investigated the biological functions and molecular mechanisms of miR-275/305,miR-8-3p and miR-989-3p in the regulation of energy metabolism in B.dorsalis by using transcriptomics,RNAi and CRISPR/Cas9 techniques.The main findings are as follows:1.Identification and primary screening of miRNAs related to energy metabolism in B.dorsalisIn this chapter,we first verified the effect of dietary yeast treatment on adult energy metabolism and the expression of miRNA pathway genes,and further performed Illumina sequencing of miRNA and m RNA on abdominal tissues of B.dorsalis treated with different yeasts.The results showed that:(1)Feeding yeast can significantly increase the content of TAG,glycogen,and total sugar in whole bodies,and promote the transcription of miRNA biosynthesis pathway genes.The RNAi interference with AGO1 and DCR1 resulted in a significant decrease in TAG content and an increase in glycogen and total sugar content,suggesting that miRNAs may be involved in the regulation of energy metabolism in B.dorsalis.(2)small RNA sequencing analysis identified 44 differentially expressed miRNAs,and target gene prediction of 19 highly expressed miRNAs yielded a total of 4136 target genes.Enrichment analysis showed that these targets were mainly enriched in oogenesis,regulation of glucose metabolic process and lipid particles,indicating that miRNAs can participate in the regulatory process of energy metabolism by acting on these target genes.(3)A total of 5742 differentially expressed genes were obtained from transcriptome sequencing,and GO/KEGG enrichment analysis showed that the differentially expressed genes were mainly concentrated in proteolysis,oogenesis,oxidation-reduction process Glycolysis / Gluconeogenesis and other pathways.(4)A total of 317 miRNA-m RNA relationship pairs were identified through target prediction and two-omics association analysis and construction of relationship regulatory networks.The Refed method was then employed to screen miRNAs potentially involved in energy metabolic processes.These results laid the foundation for a deeper investigation of miRNAs involved in the regulation of energy metabolism.2.Research on the function of miR-275 /305 to maintain the energy metabolism homeostasis of adult B.dorsalis through the insulin pathway.In this chapter,we employed CRISPR /Cas9 technology to mutate miR-275 and miR-305,respectively,studied the effect of mutation on the energy metabolism of adult flies,and further analyzed the role of the insulin signal in the metabolic regulation mediated by miR-275 /305.The main results show that:(1)The implementation of loss-of-function mutation studies using CRISPR/Cas9 and Co-CRISPR strategies,resulted in a high mutation rate of over 95% in the G0 generation and a 100% probability of miRNA mutation in eye-color mutant individuals.The homozygous mutation rate of G1 heterozygous crosses was 4.46%-14.75%,and there was no fertile offspring generated from homozygous crossing,indicating that the miR-275 and miR-305 mutations were semi-lethal mutations.(2)Knockout of miR-275 and miR-305 resulted in a significant decrease in the TAG and glycogen content of the flies,while the total sugar content increased significantly(TAG:miR-275,P = 0.0046,miR-305,P < 0.001;glycogen: miR-275,P = 0.0089,miR-305,P =0.0351;glucose and trehalose: miR-275,P = 0.0011,miR-305,P = 0.0097).In addition,the mutation affects the flight ability and significantly reduces the survival rate of adults and hatching rate.(3)Based on association analysis of omic data and prediction analysis of bioinformatics targets,22 and 46 candidate targets were predicted for miR-275 and miR-305,respectively.Through Refed assay,Dual luciferase reporter assay and other experiments,it was confirmed that miR-275 and miR-305 combined with 3 ’UTR of SLC2A1 and GLIS2 respectively,to inhibit their expression.Further,the interaction between miR-275 /SLC2A1 and miR-305 /GLIS2 was confirmed in vivo by RNA immunoprecipitation and in situ hybridization.(4)Knockdown of SLC2A1 and GLIS2 showed metabolic phenotypes opposite the miRNA mutants.Mixed injections of ds RNA and antagomiR can partially rescue the metabolic defect phenotype.(5)Further research found that injection of insulin could significantly promote the transcription of miR-275/305 and inhibit the expression of SLC2A1 /GLIS2,while interference with the insulin receptor substrate IRS showed the opposite trend.Knock down of TOR can also affect the transcription of target genes.These results indicate that the metabolic network mediated by miR-275 /305 cluster is regulated by the insulin signaling pathway.3.miR-8-3p targeted Fuc TB to regulate the accumulation of sugar and lipid in B.dorsalis.In this chapter,we employ q RT-PCR,CRISPR /Cas9 and other techniques to explore the function of miR-8-3p in the energy metabolism process of B.dorsali larvae.The results indicated that:(1)The depletion of miR-8-3p using CRISPR /Cas9 technology significantly decreased the survival,eclosion rate,fecundity and embryotic hatching rate,while the low hatching rate is caused by females rather than males.The female longevity of the mutant strain was significantly prolonged,while the male longevity was significantly shortened,and the mating competitiveness was decreased;(2)It was found that the contents of TAG and glycogen in the larvae of the mutant strain increased significantly,while the total sugar content decreased significantly(TAG: P < 0.001;glycogen: P < 0.002;glucose and trehalose: P = 0.0088).Mutations led to decreased activity of ecdysone and juvenile hormone pathways,while insulin signaling pathway activity was significantly enhanced.q RT-PCR showed that the expression of fatty acid synthase FASN1 was increased and the expression of lipase Brummer was decreased,indicating that the ability of lipid synthesis was increased and the ability of lipolysis was decreased.These results showed that miR-8-3p maintained metabolic homeostasis by regulating the accumulation of glycogen and lipid in larvae.(3)The RNAseq sequencing analysis of the fat body of the mutant larvae identified a total of 1391 differentially expressed(611 up-regulated and 780down-regulated),in which key enzyme genes in the glycolysis,TCA cycle,fatty acid synthesis pathway,and fatty acid β-oxidation pathways showed different degrees of differential expression.These results suggest that mutant miR-8-3p leads to high metabolic activity in 3rd instar larvae with enhanced anabolism and diminished catabolism,resulting in excessive TAG and glycogen accumulation.(4)Target prediction analysis using bioinformatics software showed that there were 38 and 77 targets bound to the 3’UTR and CDS regions of the gene,respectively,and 15 and 11 GO annotations involved in the metabolic pathway,respectively.The dual-luciferase,RIP assay showed that miR-8-3p could bind to the CDS region of α-1,3-fucosyltransferase Fuc TB to repress its transcription.The above results suggest that miR-8-3p avoids excessive accumulation of sugars and lipids by negatively regulating the α-1,3-fucosyltransferase Fuc TB,thereby maintaining the homeostasis of larval energy metabolism.4.miR-989-3p participates in the function of energy metabolism and ovarian development of B.dorsalis female.In this study,we constructed deletion mutant strain using CRISPR/Cas9 to investigate the function of miR-989-3p in the reproduction and metabolism of B.dorsalis females.The results showed that:(1)miR-989-3p is a highly expressed miRNA specific to female ovarian tissue,and its expression increased with the increase of ovarian development.By optimizing the secondary structure of sg RNA,sg RNA with ex vivo cleavage activity was produced.miR-989-3p expression in mutant heterozygotes was comparable to that of wild type,while that of pure heterozygotes was only 1/230 of that of wild type.(2)Anatomical observations revealed that ovarian development was arrested in homozygotes,with no mature oocytes in the ovary and maintained in previtellogenesis.Metabolic assays showed that TAG,glycogen and total sugar contents were significantly lower than those of the wild type(TAG: P < 0.0001;glycogen: P < 0.0001;glucose and algose: P = 0.0005).q RT-PCR showed that the expression of vitellogenin Vg1 and Vg2,vitellogenin receptor vg R and adaptin were significantly lower in mutant ovaries,while expression of the calathrin was significantly higher(Vg1: P = 0.0098;Vg2: P = 0.0346;vg R: P = 0.0037;adaptin: P =0.0025;calathrin: P = 0.0167).WB test showed that the content of VG protein in the hemolymph of homozygotes was significantly higher than that of the wild type(P = 0.0002).These results suggest that knockdown of miR-989-3p affects the ovarian cytokinesis process,resulting in its inability to capture free vitellogenin or lipid in the hemolymph,and therefore exhibits a stalled ovarian development phenotype that is maintained in the previtellogenesis.In summary,we identified and screened miRNA molecules potentially involved in the regulation of energy metabolism in B.dorsalis by performing small RNA sequencing of yeast-treated abdominal tissues.Using CRISPR/Cas9 gene editing technology to mutate miRNA,systematically study the key role of miR-275/305 cluster in the energy metabolism of B.dorsalis adults,and clarify insulin signaling as the upstream regulator of miR-275/305 regulatory cascade important role.In addition,this study also studied the important functions of miR-8-3p and miR-989-3p in the glucose and lipid metabolism of B.dorsalis larvae and the ovarian development of females,respectively.These results provide new insights into the molecular mechanisms regulating metabolic homeostasis in insects and lay the theoretical foundation for the development of new molecular targets for application in agricultural pest control,as well as providing a reference for the study of glycogen and lipid metabolism in humans and other mammals.