| Liriomyza trifolii(Burgess)and Liriomyza sativae Blanchard(Diptera:Agromyzidae)are two closely-related species that feed on a variety of vegetables and horticultural crops.These two species have distributed globally,accompanied by rapidly interspecific competition and substitution.The capacity of insects to enhance their cold tolerance after short-term exposure to mild coldness is known as rapid cold hardening(RCH).It is a rapid and critical adaptation of insects to sudden temperature changes.However,it is often overlooked or underestimated as a component of survival.Many studies have investigated various aspects of the long-term acclimation of both species.However,no study was carried out on short-term acclimation of L.trifolii and L.sativae.The present study was undertaken to identify the effects of RCH on the cold tolerance of L.sativae and L.trifolii and to compare the differences in RCH capacity between them.Furthermore,the mechanisms of RCH in these two species were discussed on the physiological,biochemical and molecular levels.The main findings of this study are as follows:(1)To investigate the difference in basal cold tolerance and RCH capacity of L.trifolii and L.sativae,the lethal low temperature for 80%of the population(critical temperature)for each species was identified.Our result showed that in both species,5-days old pupae showed the lowest critical temperature(LT80)which is-11.6 and-8.9 for L.trifolii and L.sativae respectively.In a separate set of experiments,insects were cooled to one of six non-lethal temperatures(0-5℃)for 4-h and then cooled to the LT80 for 2-h to check RCH response.The cold tolerance of pupae in both species were significantly enhanced after acclimation at nonlethal temperature for a short period,indicating RCH response in pupae of both L.trifolii and L.sativae.The maximum survival was recorded by acclimating 5-days old pupae of L.sativae and 7-days old pupae of L.trifolii for 4 h at 1℃ before it is exposed to critical temperatures,resulting in a survival of 91%and 84%respectively.The effect of RCH lasted for 4 hours in L.sativae but only 2 hours in L.trifolii.Furthermore,RCH also significantly increased pupae survival at temperatures lower than its critical temperature,indicating that pupae exposed to RCH are more cold-tolerant than pupae directly exposed to cold shock.Interestingly,adults of L.trifolii also expressed RCH response,but acclimation failed to elicit RCH response in L.sativae adults at all six non-lethal temperatures.(2)Supercooling points and contents of fatty acid and several cryoprotectants including glycerol,trehalose and glucose were evaluated in order to uncover the physiological mechanism of RCH in leafminers.Results showed that short-term acclimation lowers the supercooling points significantly in pupae of both the species,but the change of supercooling point is greater in L.sativae than in L.trifolii.After acclimation at 1℃ for 4-h,the supercooling point of L.sativae pupae significantly decreased from-10.65 to-12.48℃,while in L.trifolii only slight changes was found,which from-19.1 to-19.6℃.It was observed that the overall degree of unsaturation showed no significant difference after four-hour hardening under 1℃ in L.trifolii and L.sativae,which indicates that fatty acid composition remodeling is not necessary nor sufficient for the RCH response in leafminer.Furthermore,trehalose and glucose were not induced by RCH in leafminer,which indicates that trehalose and glucose are not involved in the RCH phenomenon of both species.However,glycerol plays a specific role in biochemical adjustments in pupae of L.trifolii and L.sativae during cold hardening process.(3)Transcriptomic analysis was used to analyze transcriptional changes in L.trifolii,and L.sativae due to RCH.RNA-seq revealed 93,166 assembled unigenes,and 34,303 were annotated in the L.trifolii and L.sativae transcriptome libraries.Results showed that after 4hour treatment under 1℃(RCH),268 and 606 unigenes were regulated in L.trifolii and L.sativae,respectively.Comparing pupae acclimated before cold shock with pupae directly exposed to cold shock,60 and 399 unigenes were differentially expressed in L.trifolii and L sativae,respectively.Among them,some genes were commonly expressed in both L.trifolii and L.sativae,such as cytochrome P450,cuticle protein.Additionally,serval pathways including galactose metabolism and peroxisome were significantly enriched during RCH.Our results indicate that RCH activated significant transcriptional regulation in leafminers,but more transcriptional changes were identified in L.sativae than in L.trifolii.This study measured and compared the RCH capacity of L.trifolii and L.sativae.Liquid chromatography,gas chromatography-mass spectrometry and transcriptome sequencing were used to reveal the physiological,biochemical and molecular mechanisms of RCH in leafminers.In the context of global warming,especially thermal unpredictability in early spring and late autumn has increased in recent decades,which makes the ability of RCH to play a more important role in the overwintering survivorship of Liriomyza populations and may contribute to their intraspecific competition.Our study of the RCH phenomenon will provide a perspective that should be considered when predicting the intraspecific competition of L.sativae and L.trifolii in the future.Studies of the RCH mechanism also have important scientific value for understanding the thermal adaptability of insects. |
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