Studies On Lipid Biosynthesis Of The Major Pest Of Mulberry Trees,Glyphodes Pyloalis,Spodoptera Litura And Their Parasitoid Wasp

Effective control of mulberry pests is the key issue to limit the development of sericulture industry.Chemical pesticides are widely used in the control of mulberry pests because of its fast effect and low cost.However,the problems of silkworm poisoning and serious pollution caused by chemical control are becoming increasingly stern,therefore,it is urgent to develop alternative control strategy.Unsaturated fatty acids in insects play an important role in various physiological activities and behaviors.To clarify their composition,content and synthesis mechanism will provide an important reference for the development of new targets for pest management based on individual physiological factors.Moreover,parasitoids located in the upper trophic level of mulberry pests play an important role in the population growth of mulberry pests.However,the regular pattern of lipid synthesis and its influencing factors have not been revealed,which limits the potential of biocontrol based on lipid synergism.Therefore,based on the"mulberry pest parasitoid"system,this study also aims to reveal the lipid dynamics of parasitoids and pests,clarify the mechanism of unsaturated fatty acid synthesis related genes in their lipid synthesis,form a new understanding of the lipid metabolism of the host pests and their parasitoids in mulberry ecosystem,and provide new strategies or targets for mulberry insect pests management.It can provide basic data for stimulating the natural control potential of natural enemies,and new ideas for exploring the sources of unsaturated fatty acids of mulberry insects and effectively carrying out the comprehensive utilization of silkworm resources.The main results are as follows:（1）To determine the composition and content of the important unsaturated fatty acids in Glyphodes pyloalis,the content of unsaturated fatty acids in different developmental stages of G.pyloalis was determined by GC.The results showed that the content of linoleic acid andα-linolenic acid on one-day-old pupae was the highest,which was 3.54±1.98μg and 21.90±8.71μg,repectively.To explore the molecular mechanism of unsaturated fatty acid synthesis in the pupae of G.pyloalis,two fatty acid synthase genes（Gpyl FASs）and five desaturase genes（Gpyl Desats）were identified from the transcriptome data of the pupae of G.pyloalis.q RT-PCR results showed that the expression levels of Gpyl FAS1,Gpyl Desat1,Gpyl Desat2,Gpyl Desat3and Gpyl Desat5 were highest in pupal stages.After silencing Gpyl FAS1 and Gpyl Desat5 by RNAi,the contents of palmitic acid,oleic acid,linoleic acid andα-linolenic acid in the pupae of G.pyloalis were significantly increased,which indicated that Gpyl FAS1 and Gpyl Desat5 played key roles in fatty acid synthesis;（2）To investigate the effects of diet on the composition and content of unsaturated fatty acids in mulberry insects,the effects of food sources on the content of unsaturated fatty acids of Spodoptera litura were compared by feeding on different food（mulberry leaves,soybean leaves and artificial diet）.The results showed that the body weight of S.litura fed on mulberry leaves increased significantly faster than that fed on soybean leaves and artificial diet.The results of GC showed that the pupae of S.litura fed on mulberry leaves had the highestα-linolenic acid content on one-day-old pupae（36.83±4.97μg）,which was significantly higher than that of that fed on soybean leaves（2 28.92±10.15μg）and artificial diet（7.79±1.96μg）.To further explore the molecular mechanism of unsaturated fatty acid synthesis in polyphagous insects,we constructed the differential transcriptome database（SRR14297442～SRR14297450）of one-day-old pupae of S.litura fed on different foods,and screened the genes related toα-linolenic acid synthesis.The results of q RT-PCR showed that the expression of Slit PLA₂was the highest in the one-day-old pupae of S.litura fed on mulberry leaves,and Slit Desat1 was also had a higher expression in pupal stage.Then the effects of Slit PLA₂and Slit Desat1 on the synthesis of unsaturated fatty acids in S.litura pupae were studied by RNAi.After knockdown of Slit PLA₂,the contents ofα-linolenic acid in the pupae of S.litura fed on artificial diet significantly decreased,after knockdown of Slit Desat1,the contents of palmitic acid,oleic acid,linoleic acid andα-linolenic acid in the pupae of S.litura fed on artificial diet significantly decreased.In the pupae of S.litura fed on mulberry leaves,α-linolenic acid content significantly decreased after Slit PLA₂was knocked down.In S.litura fed on soybean leaves,the contents ofα-linolenic acid remained stable after knocking down Slit PLA₂and Slit Desat1.The results indicating that Slit PLA₂and Slit Desat1 had different functions in fatty acid synthesis of S.litura fed on different foods;（3）To explore the fatty acid synthesis of parasitic natural enemies in mulberry multi-layer trophic level,the dynamics and molecular mechanism of fatty acid synthesis in parasitoid were studied by using S.litura and M.pulchricornis as the research system.The results of isotope labeling showed that fatty acids,such as palmitic acid and stearic acid could be synthesized during the adult stage.The lipid content of parasitoid was the highest from larval stage and to five-day-old pupae,and began to decline at the six-day-old pupae,and continue to decline after eclosion.Four fatty acid synthase genes（Mpul FASs）were screened out from the transcriptome data of M.pulchricornis.Mpul FAS1 and Mpul FAS2 had the highest expression levels in adult stage,and Mpul FAS3 and Mpul FAS4 had the highest expression levels in larva stage.After silencing four Mpul FASs,respectively,the lipid content decreased significantly.The results showed that parasitoids can still synthesize lipids in the adult stage,and Mpul FASs plays an important role in this process;（4）To further explore the molecular mechanism of fatty acid synthesis in adult stage of M.pulchricornis,we set three food treatments,including feeding,starvation and feeding after starvation,and 16 differentially expressed genes related to fatty acid metabolism were identified from the differential transcriptome database including 8Mpul LELOVLs,one Mpul FAS,two Mpul FARs and five Mpul Car Es.q RT-PCR results showed that feeding after starvation could significantly induce the expression of genes related to fatty acid synthesis,while honey solution feeding could reduce the expression of genes related to lipid consumption,which proved that the genes related to lipid synthesis could still play a role in the adult stage of the parasitic wasps,suggesting that the parasitic wasps may still have the ability of lipid synthesis;（5）The results of indoor experiment showed that the longevity,parasitism rate and lipid content of parasitoids were significantly affected by supplementary nutrition.The longevity of M.pulchricornis fed on sucrose,glucose,trehalose and fructose was the longest and had nosignificant difference.The number of cocoons produced by M.pulchricornis fed on honey and sucrose solution was the highest,which were 15.1±6.3 and 14.3±5.2,respectively.The end-point lipid content of parasitoids fed honey,trehalose,glucose and fructose was higher than water control.The results of field experiments showed that the field parasitism rate of M.pulchricornis could be significantly increased by setting sucrose and honey solution feeding device during the first and second field release.When the wasps were released in the field for the third time,the parasitism rate of the treatment group with sucrose solution supplement device was the highest,while the parasitism rate of the treatment group with honey sollution and water supplement device had no significant difference.The results indicated that the parasitism rate of parasitoids in both indoor and outdoor conditions could be significantly increased by supplementing sugar nutrition,which might be related to the fact that supplementing sugar nutrition could counteract the decrease of fitness index caused by the decrease of lipid content.Therefore,the hypethsis of“Nutrition supplentment delays the lipid decline in parasitoids,thereby increases their parasitization rates in the fields”was predicted.The results showed that the pupae of G.pyloalis and S.litura were rich in unsaturated fatty acids.The fatty acid synthase genes（Gpyl FASs）,desaturase genes（Gpyl Desats）and phospholipase A₂genes（Slit PLA₂）played an important role in unsaturated fatty acid biosynthesis.In the future,we can develop new control targets for G.pyloalis and S.litura.The results showed that the lipid synthesis ability of parasitoids,the parasitic wasp of G.pyloalis and S.litura,was not lost in adult stage,and the classical hypothesis of“loss of lipid synthesis in parasitoid wasps”was questionable.The field experiment also proved that the loss of lipid synthesis could be compromised and the control effect could be improved by supplementing sugar nutrition.It provides a sufficient basis for the practice of biological control of mulberry pests based on the synergistic effect of parasitoid lipids.