Identification And Characterization Of Genes Involved In The Terminal Pathway Of Sex Pheromone Biosynthesis And Metabolism In Bombyx Mori

Insect sex pheromones play an important role in the process of courtship and mating.The general pathway of sex pheromone biosynthesis has received widespread attention,while the process of terminal synthesis and metabolism is still unclear.Bombyx mori is domesticated from the wild silkworm(Bombyx mandarina),but their sex pheromone components have certain differences.The domestic silkworm employs a pheromone blend containing(E,Z)-10,12-hexadecadienol(bombykol)and analogous(E,Z)-10,12-hexadecadienal(bombykal).While its wild ancestor B.mandarina uses only bombykol.The two closely related moths might be a good model for exploring the genes involved in aldehyde pheromone synthesis and metabolism.In this study,by deep sequencing and analyzing the sex pheromone gland(PG)transcriptomes,we identified116 candidate genes related to pheromone biosynthesis,metabolism,and chemoreception,including 40 genes that putatively related to the terminal pathway,namely alcohol oxidase(AO),aldehyde reductase(AR),and aldehyde oxidase(AOX)family,respectively.Spatiotemporal expression profiles,differentially expressed analysis,and phylogenetic analysis revealed that those potential candidate genes might be involved in the terminal pathway.Finally,we performed functional exploration for Bmor AO1 and Bmor AO2.The main results obtained in this study are as follows:(1)Comparative analysis of PG transcriptomes between the domestic and wild silkworms.The sex pheromone glands of B.mori(PG＿D)and B.mandarina(PG＿W)were performed RNA-sequencing(RNA-Seq).Approximately 47.4 and 44.9 million raw reads obtained.After removing the adaptors and low-quality sequences,the clean reads were assembled,resulting in 19,828 unique transcripts,of which 13,800 unigenes showed expression signals(RPKM≥1 at least one of the two samples).In total,we identified 1382 DEGs in the PGs,including 838 up-and 544 down-regulated genes in the domestic silkworm.GO enrichment analysis showed that those DEGs mainly enriched in the oxidation-reduction process,the single-organism metabolic process,in oxidoreductase activity,and in the extracellular region.KEGG analysis revealed that the most enriched pathways were glyoxylate and dicarboxylate metabolism,carbon metabolism,lysosome and peroxisome.(2)Identification of genes involved in terminal pathway of pheromone biosynthesis.We performed homologous BLAST search and protein domain annotation for the candidate genes involved in pheromone biosynthetic and metabolic pathway.Finally,we identified 116 putative candidate genes,which were associated with pheromone biosynthesis,metabolism and transportation.Among of them,40 genes related to the terminal pathway,including 25 AOs,11 ARs and four AOXs.Spatiotemporal expression profiles and differentially expressed analysis revealed that four alcohol oxidases(Bmor AO1,Bmor AO2,Bmor AO3,and Bmor AO4),one aldehyde reductase(Bmor AR1),and one aldehyde oxidase(Bmor AOX5)might be involved in the terminal pathway.Mating experiments showed that all these candidate genes in the terminal pathway displayed significantly down-regulated expression after mating,suggesting that mating may affect the expression levels of pheromone biosynthesis-related genes.In addition,the phylogenetic analysis indicated that AOs did not show a conversed orthologous relationship among moths,whereas,ARs and AOXs phylogenetically conserved.(3)Functional exploration of the terminal pathway-related genesWe pperformed RNAi-mediated gene silencing for two candidate genes(Bmor AO1and Bmor AO2).Then,GC-MS system used for checking pheromone blend ratios of those RNAi-processed PGs.Results indicated that the m RNA expression levels of Bmor AO1 and Bmor AO2 significantly decreased after RNA interference.GC-MS analysis revealed that the pheromone production ratios(bombykol/bombykal)were larger in RNAi-processed individuals compared with the controls.Moreover,there was a significant negative correlation between the pheromone production ratios and gene expression levels(R~2>0.6).These results indicate that the silkworm Bmor AO1 and Bmor AO2 may be important candidate genes for transforming bombykol into bombykal,and their expression level changes can regulate the bombykol/bombykal ratio.In summary,we identified six potential candidate genes for the terminal pathway of bombykal synthesis.RNAi and GC-MS proved that both Bmor AO1 and Bmor AO2function in the process of bombykal synthesis and have a regulatory effect on the bombykol/bombykal ratio in the domestic silkworm.This study not only provides useful information for the study of the terminal pathways of sex pheromone synthesis in moths,but also provides potential targets for the biological control of lepidopteran pests.