Relationship Between Thermosensitive TRP Channel Genes And Heat Tolerance Of Agasicles Hygrophila(Coleoptera: Chrysomelidae)

Temperature is a critical environmental factor affecting insects life activities such as growth,development,reproduction and survival.Agasicles hygrophila is a monophagous enemy insect that is used to control an exotic invasive weed,Alternanthera philoxeroides.The temperature adaptability of A.hygrophila is poor.Temperature has a significant impact on its geographical distribution and seasonal changes in populations.During hot summers,while A.philoxeroides proliferate rapidly,the population of A.hygrophila is significantly inhibited by high temperatures.The previous ecological experiments proved that low heat tolerance of the eggs was a major factor responsible for the reduction of A.hygrophila populations.Transient receptor potential(TRP)is a kind of important cation channel located on the cell membrane.It can participate in and regulate the perception of insects towards ambient temperature and participate in the formation of many other sensations,such as vision,smell,hearing,mechanical sense,etc.,which is essential for the survival and behavioral response of insects.To reveal the relationship between the TRP genes and heat tolerance of A.hygrophila,we studied the effects of high temperatures on the population dynamics of A.hygrophila through the two-sex life table.Based on the transcriptome data using Pac Bio SMRT technology,three TRP genes of A.hygrophila were screened,cloned and identified.The expression patterns of TRP genes under different high temperatures and different developmental stages were analyzed by q RT-PCR.The main results are as follows:1)The effect of high temperature on the population dynamics of A.hygrophila.Age-stage two-sex life table results indicated short-term high temperature treatment of the A.hygrophila eggs at 37.5°C,which had significant effects on population parameters such as hatch rate,developmental duration,longevity and net reproductive rate.2)Cloning and identification of TRP genes.Based on the transcriptome data using Pac Bio SMRT technology combined with bioinformatics methods,we identified three TRP genes,i.e.TRPA1,Painless and Pyrexia.The phylogenetic tree indicated that all three TRP genes belonged to the TRPA subfamily.Bioinformatics analyses further indicated that all three TRP channels were localized on the cell membrane and predicted the key functional binding sites of the ligand.3)Screening of reference genes.The stability of 9 candidate reference genes of A.hygrophila was analyzed by reference gene screening softwares ge Norm,Norm Finder and Best Keeper.And RPS18 and ?-actin were selected as the reference genes under heat stress of A.hygrophila,then RPL13 a and RPS18 were used as reference genes at different developmental stages of A.hygrophila.4)The relationship between TRP genes and high temperature tolerance of A.hygrophila.The expression patterns of three TRP genes under different high temperatures of A.hygrophila were analyzed by q RT-PCR.The results showed that expressions of Painless and Pyrexia had no obvious regularity on temperature changes.However,there was no significant difference of the expression level of TRPA1 among 25-35°C,and it had the highest expression level at 37.5°C,then the expression level decreased gradually after 37.5°C.Combined with ecological data,it is speculated that the TRPA1 may not only participate in the perception to temperature,but more importantly,it can be used as a direct sensor for noxious heat to indicate the heat tolerance of A.hygrophila.5)The expression of TRP genes at different developmental stages of A.hygrophila was investigated.The results showed that the relative expression level of TRP genes were significantly different at different developmental stages of A.hygrophila.Generally,the expression level of the three TRP genes increased gradually and then decreased with the development of A.hygrophila.The expression level of TRPA1,Painless and Pyrexia reached the highest in pupal stage,3rd and 2nd instar larval stages,respectively.However,the expression levels of the three genes were lower at the adult stage.The results showed a close relationship between the TRP genes and the growth and development of A.hygrophila.It helps to explain the mechanism of limited control effect of A.hygrophila to A.philoxeroides in summer.