Analysis Of Transcriptome Differences In Aedes Albopictus Induced By Haedoxan A

Haedoxan A（HA）is a dioxodicyclooctane lignan compound isolated from the perennial herb Pharyma leptostachya L.,HA is effective against a variety of pests.Especially for housefly and mosquito larvae have higher insecticidal activity.Early symptomological observations showed that the tested insects had muscle relaxation after HA treatment,involuntary tremor of the worm,anesthesia and eventually death.According to the poisoning symptoms of the insects,HA may be applied to insect neurons or muscle receptors.However,its specific mechanism of action and target are still unclear.Based on high-throughput transcriptome sequencing technology,it is possible to screen drugs and their related mechanisms of action from the gene and transcriptional levels.Therefore,this study used Illumina high-throughput sequencing technology to sequence the transcriptome of Aedes albopictus third instar larvae and untreated group after 24 h of HA sublethal dose treatment,and analyzed the HA induced Aedes albopictus from bioinformatics.Differentially expressed genes were analyzed and validated in terms of the biosynthesis pathway of ganglion glycosphingolipids,which provided a certain reference for further exploration of HA mechanism.The main results are as follows:1.The virulence test of haedoxan A（HA）on the 3rd instar larvae of Aedes albopictus showed that HA had high insecticidal activity against Aedes albopictus,LC₅₀ was 0.31μg/mL,so the sublethal dose induced concentration LC₃₀ was 0.2μg/mL.2.The transcriptome sequencing was carried out.The sequencing results produced a total of 139,734,078 raw data（Raw Reads）.After the rigorous data filtering and quality evaluation,the raw data generated 137,375,042 clean data（Clean Reads）,and the raw data obtained by sequencing was submitted to NCBI’s SRA database,accession number:SRP190133.3.The Clean Reads generated were compared with the reference genome sequences of Aedes albopictus on NCBI.We identified 416 differential genes with|Log2（FoldChange）|>1 and qvalue<0.005 as screening criteria.Among them,328 genes were up-regulated and88 genes were down-regulated.Significant enrichment analysis of GO function indicated that differential genes were mainly enriched in five biological functions of“proteolysis”,“carbohydrate metabolism process”,“transport activity”,“substrate-specific transporter activity”and“trypsin activity”.in.The KEGG metabolic pathway analysis pooled 136differential genes into 47 KEGG metabolic pathways.The differential genes are mainly enriched in four metabolic pathways:"Glycosphingolipid biosynthesis-ganglio series","Glycosaminoglycan and other glycan degradation","One carbon pool by folate","Pantothenic acid and CoA biosynthesis".4.The relative expression levels of 10 differential genes selected from the transcriptome differential genes were detected by qRT-PCR.The results were consistent with the sequencing results,indicating that the transcriptome sequencing results were reliable.5.The differential gene is mainly enriched in the biosynthesis pathway of ganglioside glycolipid.It is speculated that after HA treatment of Aedes albopictus,the biosynthesis of gangliosides in glycosphingolipids is significantly affected,thereby blocking intercellular communication.It is neurotoxic to insects;this speculation is preliminarily verified by measuring the enzymatic activity of the key enzymeβ-galactosidase（GLB1）in the ganglioside biosynthesis pathway.These results provide a reference for further study on how HA acts and regulates the biosynthesis pathway of ganglioside glycolipid,which leads to the mechanism of insect neurotoxicity.