| Aphis gossypii and Acyrthosiphon gossypii are the two main aphid species in cotton field in Xinjiang Uygur Autonomous Region,China.The previous studies preliminarily suggested that Ap.gossypii and Ac.gossypii may response differently to the different temperatures,and the seasonal population dynamics of these two aphid species were also different during cotton growth stage.In addition,the continuous high temperature often occurs during cotton growth stage in Xinjiang.Therefore,the population fitness and mechanism of adaption for Ap.gossypii and Ac.gossypii under heat stress need to be further study,which can provide scientific basis to understand the population dynamic of these two aphid species.Therefore,the effects of the three different temperatures(29?,32?,and 35?)on the survival rate and reproduction of adult for Ap.gossypii and Ac.gossypii were tested,and the feeding behavior of these two aphids at three temperatures were analyzed using electrical penetration graph(EPG)technique,which can help us to detect the impacts of high temperature on fitness of Ap.gossypii and Ac.gossypii.Then,we analyzed the molecular mechanism of Ap.gossypii and Ac.gossypii to heat stress based on transcriptome sequencing and gene expression pattern analysis.Finally,the RNA interference technology was used to verified the functions of four genes of under heat stress for Ap.gossypii.The main results were as follows:1.The effects of three different temperatures(29?,32?,and 35?)on survival rate,reproduction,and feeding behavior of Ap.gossypii and Ac.gossypii adult were assessed.The results shown that the survival rate,longevity,and fecundity of Ac.gossypii were significantly lower than that of Ap.gossypii under all temperature treatments.With the increase of temperature,the survival rate and fecundity of Ac.gossypii decreased faster than that of Ap.gossypii.The feeding behavior data indicated that the feeding time spent on xylem for Ac.gossypii was longer than that on phloem at all temperatures,which was contrary to Ap.gossypii.The time of phloem feeding(E2)of Ap.gossypii at 32? was significantly higher than that at29? and 35?,but there was no significant difference between 35? and 29 ?.However,the time of phloem feeding of Ac.gossypii at 35? and 29? was shortened with the increase of temperature,which indicated that the feeding behavior of Ac.gossypii was more sensitive to temperature increase than that of Ap.gossypii.These results indicated that high temperature(35?)could affect the survival rate,reproduction,and feeding behavior of the Ap.gossypii and Ac.gossypii,especially to Ac.gossypii.2.Transcriptome sequencing based on Illumina His Seq4000 platform was used to analyzed the transcriptional level of adults of Ap.gossypii and Ac.gossypii that were exposed to three different temperature treatments(29?,32?,and 35?)for 24 h.For Ap.gossypii and Ac.gossypii,the 77807 Unigene and 65233 Unigene were obtained after assembling and de-redundancy,respectively;and the N50 of Unigenes was 1644 and 2062,respectively.Different expressed genes(DEGs)analysis showed that a total of 538 genes(186 up-regulated and 352 down-regulated),559 genes(366 up-regulated and193 down-regulated),and 1044 genes(436 up-regulated and 608 down-regulated)shown significant different expression level in the comparison of 32? vs 29?,35? vs 32?,and 35? vs 29? of Ap.gossypii,respectively.The total number of DEGs of Ac.gossypii in the comparison of 32? vs 29? was three times of Ap.gossypii,and the number of upregulated genes was five-fold of Ap.gossypii,which indicated that Ac.gossypii was more sensitive to 32? than Ap.gossypii.For Ac.gossypii in the comparison of 35? vs 32?,there were fewer up-regulated genes(443)than those at 32? vs 29?,and only 364 up-regulated genes in 35? vs 29? comparison groups,which contrary to Ap.gossypii.Therefore,we suspected that the expression of some genes of Ac.gossypii was inhibited under 35? or gene expression levels have been downregulated due to the time of heat stress too long.There are few genes in common among the three temperature treatments for both aphid species,indicating that the different genes or metabolic pathways were induced in response to 32? and 35?.Further analysis was obtained from GO enrichment analysis showed that the large number of DGEs of Ap.gossypii in the comparison of 35? vs 32? and 32? vs 29? were significantly enriched in macromolecule biosynthetic and metabolic process,and transmembrane transport,respectively.However,the oxidation-reduction activity and process were significantly enriched in the above two comparison groups for Ac.gossypii,and many of genes involved in this pathway were downregulated at 35? vs 32?.The results from KEGG enrichment analysis shown that the genes were mainly related to longevity regulating pathway,endocytosis,lysosome,oxidoreductive activity,and amino acid metabolic pathways for both Ap.gossypii and Ac.gossypii.However,the significance of metabolic pathway enrichment and the number of gene were different between the Ap.gossypii and Ac.gossypii.In addition,many genes related to Buchnera genome were found in the DEGs of Ap.gossypii and Ac.gossypii,but the expression pattern and gene number were different between species.These differences may be responsible for the differences in heat resistance among these two cotton aphids.3.The expression profiles of three heat shock protein genes(Hsps)and two cathepsin B genes(Cath B)of Ap.gossypii were tested.The results suggested that the all of genes were expressed in six tissues of Ap.gossypii including antennae,head(excluding salivary gland),salivary gland,gut,leg,thorax and abdomen.The Hsps were expressed highly in salivary gland,and Cath B were expressed highly in gut of Ap.gossypii.These genes were expressed in whole nymph stage of Ap.gossypii,and the expression levels of genes were induced under high temperature.In addition,all of genes were significantly expressed higher in the first instar than those of other instars after heat stress.It was found that the expression levels of these five genes were the highest after adults exposed to 32? and 35? for 24 h,and expression levels were decreased with the increase of processing time increased.No significant differences in the expression level of cathepsin B genes were observed between 35? and 29? after 48 h.While the expression levels of three Hsp genes of adult exposed to 35? was drop slowly compared with cathepsin B genes,which still significantly higher than the control treatment at 96 h.4.In order to detect the function of two Hsp genes(Ap Hsp70A1-1 and Ap Hsp68)and two Cath B genes(Ap Cath B01 and Ap Cath B02)of Ap.gossypii under heat stress,the nanocarrier-based transdermal ds RNA delivery system was used to silenced the expression level of genes.The results of RNA interference efficiency indicated that the expression level of four genes were decreased significantly compared with the control with non-filtered ds RNA between 24 h and 96 h,and the expression levels were the lowest at48 h.When the expression levels of Ap Hsp70A1-1,Ap Hsp68,and Ap Cath B01 was decreased,the survival rate of Ap.gossypii fed on 29? also decreased at late age,indicating that these genes may play important roles in growth of Ap.gossypii.When aphid exposed to heat stress,the survival rate and fecundity of aphid were declined sharply,which suggested that the suppression of these genes expression improved the aphid sensitivity to high temperature treatments.However,there was no significant differences in aphid survival after Ap Cath B02 was interfered.The down-regulated expression level in Ap Hsp70A1-1and Ap Hsp68 had a greater impact on survival rate and fecundity than those in the Ap Cath B01 and Ap Cath B02.These results indicated that the Ap Hsp70A1-1,Ap Hsp68,and Ap Cath B01 play a crucial role in the thermo-tolerance of Ap gossypii.In conclusion,the sensitivities of population fitness of Ap.gossypii and Ac.gossypii to heat stress were different.In order to explain to these differences,the transcriptome sequencing was used to analyze the features and differences of transcriptional level among these two cotton aphids,and a large number candidate genes involved in heat stress were selected and analyzed,and the molecular mechanism of aphids was explored.In addition,the function of four genes of Ap.gossypii under heat stress were determined.These results provided clues to understand better the Ac.gossypii under high temperature condition in summer was collapsed early than Ap.gossypii on cotton field,and provided theoretical basis for revealing the molecular regulation of Ap.gossypii to heat stress.Meanwhile,these results will serve as the basis for predicting the aphid population dynamic and its pest management in summer. |
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