Cloning And Differential Expression Of Hsp70 And Hsp90 In Anaphothrips Obscurus (Müller)

The grass thrip,Anaphothrips obscurus（Müller）is a cosmopolitan insect pest that is distributed throughout the temperate region.The feeding damage from A.obscurus results in linear rust-like markings on the leaves of crops such as timothy,corn and wheat.A.obscurus adults have three wing morphs:brachypters,macropters and an intermediate phenotype（unusual）.Changes in wing phenotypes can be induced by environmental conditions such as photoperiod,host plant quality and population density.Brachypters and macropters differ greatly in morphology,reproductive biology,behavior,and life history traits.Brachypterous A.obscurus populations can live in wider temperature ranges than macropters.However the molecular aspects of A.obscurus thermotolerance have been unclear until now.In this study,we exposed adults of A.obscurus to five temperatures（40,41,42,43,and44°C）to compare the difference in thermal resistance between brachypters and macropters.The cDNA transcripts of A.obscurus Hsp70 and Hsp90genes were cloned.Subsequently,their expression patterns in brachypters and macropters were studied using qRT-PCR.We also compared the different expression levels of Hsps between macropters and brachypters and discussed the molecular mechanisms of thermotolerance in A.obscurus adults.1.The cDNA sequence ofAohsp70 had 2382bp containing a 138 bp 5’untranslated region（UTR）,a 327 bp 3’UTR and a 1917 bp open reading frame（ORF）encoding a protein with638 amino acid residues.The cDNA sequence of Aohsp90 was 2504 bp with a 94 bp 5’UTR,a247 bp 3’UTR and a 2163 bp ORF encoding 720 amino acid residues.Aohsp70 and Aohsp90contained the conserved sequences and characteristic motifs of the Hsp70 and Hsp90 families,respectively.Homology alignments showed that the predicted amino acid sequences of Aohsp70 and Aohsp90 share close relationships with Hsps in Frankliniella occidentalis,were87%and 94%respectively.Phylogenetic analysis showed that Hsp70 and Hsp90 are highly diverse among insect orders.2.A RT-qPCR was conducted to determine the expression patterns of Aohsp70 and Aohsp90 during different developmental stages.The expression levels of Aohsp70in macropters reached a peak level in pupae and then decreased in adults,while the expression levels in brachypters were relatively constant.Also,the expression levels of Aohsp70 in second instar nymphs（3.5 times）and pupae（12 times）were significantly higher in macropters than brachypters Aohsp90 had similar expression patterns in macropters and brachypters and was high in both brachypterous and macropterous adults The different expression patterns of Aohsp70 and Aohsp90 in the tested development stages simplied that Aohsp70 and Aohsp90 may have different biological functions in A.obscuresand there is difference between winged morphs.3.After heat shock treatment with an ascending series of temperature,brachypters presented a higher Ltem₅₀（43.2°C）than macropters（42.5°C）.It indicated that brachypters are more heat tolerant than macropters.The expression levels of Aohsp70 in macropters and brachypters increased with the increase of heat shock temperature and reached its peak at44°C in both macropters（approximately 90-fold）and brachypters（approximately 280-fold）.Also,the expression levels were significantly higher in brachypters than in macropters at42°C,43°C and 44°C,respectively.The expression of Aohsp90 did not show a continued increase with the temperature increase and the peak expression level was found at 43°C in macropters（approximately 2.4-fold）and at 41°C（approximately 1.8-fold）in brachypters.These results indicated that the expression patterns of Aohsp70 are highly associated with the thermal resistance of A.obscurus.