A Novel Regulatory Mechanism Underlying The Regulation Of Ambient Temperature On Larval Growth And Development In Silkworm

Insect growth and development are regulated by both environmental and genetic factors.The external environmental factors mainly include temperature and others.As a key internal genetic factor,20-hydroxyecdysone(20E),an active form of the steroid hormone ecdysone,is produced by the prothoracic gland(PG)and directly triggers larval molting and larval-pupal-adult transition through a periodic fluctuation of its titer.In the silkworm(Bombyx mori),although massive progresses on endocrine regulation of the growth and development have been achieved,how external environmental factors is involved in the growth and development remains poorly understood.During silkworm rearing,ambient temperature is the most important external environmental factor and affects its development;the suitable temperature is 20 ～30 ℃.However,whether ambient temperature can mediate the action of 20 E and other endocrine hormones to regulate silkworm growth and development remains unclear.Therefore,exploring the regulatory roles of ambient temperature on the growth and development of silkworm and other insects can provide novel insight into the interaction between environmental factors and genetic factors.In this study,using silkworm and fruit fly(Drosophila melanogaster)as research models,we performed multiple experiments,including genetic manipulation,biochemistry,and molecular biology,to explore the influence of ambient temperature on larval growth and development and to investigate the regulation of ambient temperature on 20 E biosynthesis.The main findings were as follows:1.Low temperature retards the growth and development of silkworm larvaeWe reared silkworm larvae during fifth larval instar at 25 ℃ and 18 ℃,and 25 ℃ is a suitable temperature for silkworm growth and development.We observed that when silkworm larvae at 25 ℃ stopped feeding and began to wander(before pupariation),silkworm larvae at 18 ℃ were still feeding.Statistical analysis showed that compared with 25 ℃,the treatment with 18 ℃ delayed larval-pupal transition for about 4 days.At the same time,we also recorded the change of growth rate of silkworm larvae under different temperatures.After the treatments,the body weight of silkworm larvae was calculated every eight hours until wandering.The results showed that silkworm larvae at 25 ℃ first reached critical weight for the initiation of larval-pupal transition,and the critical weight was also lower.This suggested that low temperature impaired growth rate of silkworm larvae.2.Low temperature disturbs 20 E biosynthesis in silkwormGiven that low temperature retards the growth and development of silkworm larvae,and 20 E is involved in regulating silkworm development,we therefore used the enzyme immunoassay(EIA)method to analyze the concentration of 20 E in the hemolymph of silkworm individuals at 25 ℃ and 18 ℃ when silkworm individuals began wandering at25 ℃.The results showed that compared with the control at 25 ℃,with while the peak value of ecdysone appeared in the larvae treated at 25 ℃,the treatment with 18 ℃ impaired 20 E concentration.In addition,because the change of 20 E concentration will affect the expression of downstream response genes,we further performed RT-q PCR experiment to analyze the expression difference of E75 B as a 20 E signal transducer in the fat body,a 20 E target tissue,in silkworm individuals with different temperature treatments.The results showed that low temperature of 18 ℃ delayed the appearance of high peak of E75 B expression.These data indicated that low temperature inhibited 20 E biosynthesis in silkworm.We further investigated the effects of low temperature on the expressions of steroidogenic enzyme genes in the PG.RT-q PCR experiments revealed that during last(the fifth)larval instar,steroidogenic enzyme genes have a high expression in the PG of silkworm individuals before wandering,under different temperature treatments.However,low temperature of 18 ℃ delayed the appearance of high expressions of steroidogenic enzyme genes,indicating low temperature impaired the expressions of steroidogenic enzyme genes in the PG of silkworm larvae.Collectively,suitable temperature promotes the expressions of steroidogenic enzyme genes to promote 20 E biosynthesis,thereby modulating the growth and development of silkworm larvae.3.Downregulating thermosensitive channel TRPA1 disturbs insect development(1)TrpA1 knockout affected the development and 20 E biosynthesis in silkwormTo further investigate the role of ambient temperature on silkworm development,we performed site-specific knockout of the TrpA1 gene,which encodes a thermosensitive channel that plays an important role in thermal sensation,using the CRISPR/Cas9 system.The wild type as the control and silkworm TrpA1 mutant were reared at 29 ℃.Further analysis showed that TrpA1 knockout delayed larval-pupal transition,namely TrpA1 mutant still kept feeding while the control larvae had stopped feeding and entered the wandering stage.Statistical analysis showed that TrpA1 knockout delayed wandering for about 2.5 days.These data revealed that TrpA1 knockout retarded developmental progression of silkworm larvae,phenotyping the effects of low temperature treatment.We also examined the effects of TrpA1 knockout on 20 E biosynthesis in silkworm.EIA analysis showed that TrpA1 knockout impaired 20 E biosynthesis.RT-q PCR analysis revealed that TrpA1 knockout inhibited the expressions of both steroidogenic enzyme genes in the PG and E75 B in the fat body as a target tissue of 20 E action.Collectively,TrpA1 knockout impaired the expressions of steroidogenic enzyme genes and 20 E biosynthesis,thereby disrupting the development of silkworm larvae.(2)TrpA1 knockdown in the PG of the fruit fly disturbs larval development and20 E biosynthesisBased on the Gal4/UAS system-mediated RNAi,we used Phm-Gal4 to drive the knockdown of TrpA1 in the PG of the fruit fly.The results showed that under the normal temperature of 25 ℃,PG-specific TrpA1 knockdown delayed larval-pupal transition compared with the control.Moreover,RT-q PCR and immunostaining assays revealed that TrpA1 knockdowns in the PG of the fruit fly inhibited the expressions of steroidogenic enzyme genes in the PG.Taken together,our data in silkworm and fruit fly suggested thermosensitive channel TRPA1 promoted 20 E biosynthesis to modulate larval development.