Functional Analysis Of PxTret1-Like And PxHacd2 In Temperature Adaptation Of Plutella Xylostella

Global warming frequently leads to extreme temperatures,which pose a new challenge to insects,which must adapt to these extreme temperatures.Insects,and ectotherms,are highly sensitive to climate change.Plutella xylostella(L.)(Lepidoptera: Plutellidae)is a serious agricultural pest of cruciferous vegetables,with annual damage and treatment costs of US $4-5 billion worldwide.P.xylostella has high levels of genetic diversity and remarkable plasticity,and it was distributed across all continents except Antarctica.The laboratory team has performed whole-genome and transcriptome sequencing of high-temperature strain,low-temperature strain,and normal temperature strain of P.xylostella.The candidate genes were associated with significant SNPs and significant differences in expression.PxTret1-like and PxHacd2 genes were selected for functional verification.The knockout mutations of PxTret1-like and PxHacd2 were generated using a CRISPR/Cas9 system by targeted knockout.This study proved the temperature adaptation mechanisms of the PxTret1-like and PxHacd2 genes in P.xylostella,according to the combined metabolite–transcript analysis in different strains,age-stage-specific sex life table and extreme temperature stress.The main research contents and results are as follows:1.Both PxTret1-like and PxHacd2 were expressed in different states of P.xylostella,and the expression levels were affected by ambient temperature.PxTret1-like was significantly expressed in the midgut and malpighian tubule of fourth instar larvae in P.xylostella.PxHacd2 was significantly expressed in the fat body of fourth instar larvae in P.xylostella.2.The three mutant lines were generated using a CRISPR/Cas9 system by targeted knockout.The trehalose content and trehalase activity of mutant P.xylostella increased at different developmental stages(4th instar larvae,pupa,female adult and male adult).The trehalose content increased in the fat body of the 4th-instar P.xylostella,and decreased in the hemolymph,and there was no significant change in glucose in the fat body and hemolymph.Mutant strains of P.xylostella showed a significantly reduced survival rate,fecundity and ability to withstand extreme temperatures.The results showed that PxTret1-like could affect the development,reproduction and temperature adaptability of P.xylostella by regulating the trehalose content in the fat body and hemolymph.3.The knockout mutation of PxHacd2 was generated using a CRISPR/Cas9 system by targeted knockout.The VLCFAs content was decreased in different states(4th instar larvae,pupa,female adult and male adult)of the PxHacd2 mutant strain.The PxHacd2 mutant strain was found to have increased epidermal permeability when dyed with the water-soluble Eosin Y dye for the 4th instar larvae.The survivability of the PxHacd2 mutant strain was significantly lower than that of the wild strain under different humidity conditions.Survival and fecundity of the PxHacd2 mutant strain of the moth were significantly reduced at different temperatures,with a reduced ability to withstand temperature extremes.The results indicated that PxHacd2 can affect the development,reproduction,and temperature adaptability of P.xylostella by altering the epidermal permeability.This study demonstrates for the first time that PxTret1-like and PxHacd2 affect temperature adaptation in P.xylostella through distinct pathways,and evaluates the potential of PxTret1-like and PxHacd2 as potential targets for novel insecticides.