Responses Of Sitobion Avenae To Water-deficit Stress And The Underlying Mechanisms

In the context of global climate change,the global ecosystem has become more fragile as both frequency and intensity of drought increase.The impact of drought on insects is also becoming increasingly evident,but the physiological mechanisms of aphids’responses to dry environments are still not well understood.Aphids,feeding on plant phloem sap,can be more seriously affected by environmental change.The English grain aphid,Sitobion avenae（Fabricius）,is a global grain pest,mainly affecting wheat crops.It has been shown that S.avenae presents some characters of local adaptation in various wheat regions,and some aphid clones from arid areas have stronger adaptability under water-deficit stress.Therefore,in this study,we collected S.avenae clones from arid,semi-arid and moist areas in northwestern China,and screened them for desiccation resistance.Finally,we have identified some clones with desiccation resistance and desiccation non-resistance,providing direct evidence for the differentiation of S.avenae populations in different arid areas.Facing changing environments,aphids can show a series of responses.This study compared physiological characteristics of S.avenae clones with different levels of desiccation resistance under water stress,and the underlying adaptive mechanisms are explored.Our results provide new insights into how aphids adapt to drought conditions,and have implications for predicting pest outbreaks in future warming scenarios.The main results of this study are as follows:1.S.avenae clones from arid,semi-arid and moist areas were identified using six microsatellite loci.The results showed that"origin","clones"and their interaction significantly affected the survival time and water balance traits of S.avenae under desiccation treatment.In this study,we analyzed the correlation between fresh mass,dry mass,water loss rate,water content and LT₅₀(i.e.,LT₅₀,Lethal time to kill 50%of aphids)of test clones,and found that the fresh mass of aphid clones was positively correlated with LT₅₀ for all three areas.Negative correlations between water loss rate and LT₅₀ were identified for all three areas.Both desiccation-resistant and-nonresistant clones were identified.Resistant clones of wingless S.avenae showed longer survival time and LT₅₀ under the desiccation stress than nonresistant clones,and wingless individuals tended to have higher desiccation resistance than winged ones.Fresh mass,dry mass,absolute and relative water contents did not differ between the two kinds of clones.Resistant clones had lower water loss rates than nonresistant clones for both wingless and winged individuals,suggesting that modulation of water loss rates could be the primary strategy in resistance of this aphid against desiccation stress.2.We compared life-history traits in two groups of S.avenae clones with different levels of desiccation resistance.The results showed that drought treatment had no significant effects on the total developmental time or pre-reproduction time,but significantly affected adult weight and 10-d fecundity of aphids.For desiccation non-resistant clones,adult weight and 10-d fecundity were significantly decreased under water-deficit conditions,while for resistant clones,only10-d fecundity decreased,suggesting that the effects of water-deficit treatments on resistant clones were less severe.Secondly,life history traits of S.avenae were examined after desiccation training.Regardless of clones,the developmental time of first instar nymphs significantly increased after desiccation training,while the total developmental time and adult weight were not affected.When adult aphid individuals were treated,the daily fecundity of nonresistant aphid clones decreased significantly.The daily fecundity for resistant clones decreased only on day 7,and the total 7-d fecundity also decreased,suggesting that resistant clones were more resistant to desiccation stress.In addition,for resistant clones,7-d fecundity was negatively correlated with the water loss rate.These results indicated that less water loss rate could improve desiccation resistance of S.avenae.3.We compared the contents of nutrient substances in two groups of S.avenae clones with different levels of desiccation resistance,mainly including trehalose,glucose,protein and free amino acid.After 12 h of desiccation stress（5%RH）,the trehalose content of desiccation nonresistant clones was decreased.While,for desiccation-resistant clones,the trehalose content was higher than that of the control group from 12 h to 36h of desiccation stress.For winged aphids,the trehalose content of resistant clones showed increase only after 12-24 h treatments,while it decreased for non-resistant clones,indicating that resistant clones tended to accumulate trehalose under desiccation stress in both wing morphs.Protein concentrations showed similar trends in both groups of clones.Groups and desiccation treatments also significantly affected the concentration of free amino acids.Compared with the non-resistant group,lysine and arginine concentrations were increased in the desiccation-resistant group.After desiccation treatments,proline levels in the desiccation resistant group were significantly higher than those in the desiccation non-resistant group,suggesting that these amino acids might play a role in desiccation resistance of S.avenae.4.We compared the cuticle hydrocarbons in two groups of S.avenae clones with different levels of desiccation resistance.A total of 27 kinds of cuticle hydrocarbons（CHCs）were detected in S.avenae,including 14 n-alkanes and 13 methyl-branched alkanes.The CHC contents of two groups of aphids were different.Wing morphs,groups and desiccation treatments all significantly affected the content of CHC.For wingless aphids,total CHC content of desiccation resistant aphid was significantly higher than that of nonresistant aphids under the control treatment.Total CHC contents of aphid clones in both groups were significantly increased under drying treatments,indicating that both groups of aphid clones could quickly respond to desiccation stress.The response trends for winged aphids were similar to those for wingless aphids.We performed multivariate statistical analyses using contents of 27 CHCs.Among these,n-C25 was found to provide a CHC signature to distinguish between desiccation-resistant and nonresistant individuals.Compared with linear alkanes,methyl-branched CHCs appeared to display higher plasticity in rapid responses to desiccation,especially for 2-Me C26.This CHC increased significantly in both groups after desiccation,implying that 2-Me C26could be signature compound to response desiccation stress.Thus,both constitutive and induced CHCs can contribute to adaptive responses of S.avenae populations under desiccation environments.5.Transcriptome differences were examined for S.avenae clones with different levels of desiccation resistance.A total of 12140 genes were detected in S.avenae under the treatments.Under the control treatment,compared with desiccation-nonresistant aphids,expression of 370genes was significantly up-regulated in desiccation resistant aphids.These differentially expressed genes（DEGs）were enriched in oxidoreductase activity,peptidase activity,transmembrane transport and carbohydrate metabolism.Under the desiccation treatment,1237DEGs were detected in desiccation resistant clones and 1051 DEGs were detected in desiccation nonresistant clones.In both groups of aphids,these DEGs were enriched in lysosome,metabolism of xenobiotics by cytochrome P450,ascorbate and aldarate metabolism and fatty acid metabolism pathways.The expression levels of these genes were significantly increased under desiccation stress in desiccation resistant aphids,but significantly reduced in desiccation-nonresistant aphids.Therefore,it is speculated that these related genes might play a role in desiccation resistance of S.avenae.6.The function of the fatty acid elongase（ELO）gene was explored by the RNAi method.Eleven ELO genes were identified in the transcriptome of S.avenae.The expression pattern analyses showed that Sa ELO3,4,7 and 10 were highly expressed in the cuticle,suggesting that these genes might be involved in cuticle synthesis.The expression of Sa ELO2,7,8 and 9 were induced by desiccation stress,suggesting that these genes might be related to desiccation resistance of S.avenae.RNA interference was performed with Sa ELO7,10 and 11.One day after injection with ds RNA,relative expression levels of ELO decreased by 71%,21%and 57%,respectively.Relative expression levels of ds Sa ELO7 remained low during the four days after injection,indicating that expression of this gene was successfully inhibited.After the injection of ds Sa ELO7,the survival rate of nymphs and adults under the desiccation treatment was significantly reduced,suggesting that this gene plays important role in desiccation resistance of S.avenae.And a large number of aphids showed abnormal molting,suggesting that the silencing of ds Sa ELO7 would affect the molting process of S.avenae.Moreover,the content of CHCs decreased,mainly in n-alkanes,implying that Sa ELO7 might affect drought resistance of S.avenae by regulating the content of normal CHCs.In conclusion,S.avenae from different drought regions showed differentiation in desiccation resistance.In drought environment,the survival time of desiccation resistant clones were longer than desiccation nonresistant clones,and the difference of water loss rate between two groups of S.avenae was particularly important.In addition,10-day fecundity,daily fecundity,proline concentration,cuticle hydrocarbons contents and the expression levels of genes between two groups of S.avenae were significantly different.Methyl branched-alkanes were more sensitive to desiccation.While some n-alkanes may be used to distinguish between individuals with different desiccation resistance.The expression of ELOs may affect the drought resistance of S.avenae,which provides a new target for the integrated control of this aphid.