Mechanisms Of Cotton Biotype Of Aphis Gossypii Glover Adapting To Cucumber Through The Feeding Experience On Pre-infested Cucumber By Cucumber Biotype

Cotton-melon aphid,Aphis gossypii Glover,is a pest with worldwide distribution that damages many economic crops.Cotton-melon aphid has several host-specialized types,such as cotton biotype and cucumber biotype that exhibit significantly reduced fitness or cannot survive on non-native host plants.The underlying mechanisms remains unknown.In previous researches,we found that although cotton biotype cannot feed on healthy cucumber leaves,they can feed on cucumber leaves pre-infested by cucumber biotype,and the cotton biotype,as well as their offspring,can use healthy cucumber as host after two weeks of feeding experience on the pre-infested cucumber.Based on those observations,we put forward two questions:why cotton biotype can use cucumber that was pre-infested by cucumber biotype,but cannot use healthy cucumber,and why cotton biotype can adapt to healthy cucumber through the feeding experiences on pre-infested cucumber.To address the first question,we think it may be due to changes in the composition of phloem sap of pre-infested cucumbers,so we extracted the phloem sap from healthy and pre-infested cucumbers and performed component analysis and feeding experience.Regarding the second question,it may be due to the feeding experience on pre-infested cucumber leaves that alters the gene expression of Buchnera,an obligate endosymbiont of aphids,and then affects the aphid-plant interaction.So,we determined the role of Buchnera in suppressing plant defenses for cucumber biotype,and compared the differential proteins expressed by Buchnera between the two host biotypes.This study aims to reveal the mechanism underlying host specialization of A.gossypii Glover.The results are as follows:1.Metabolomic profiling and feeding assays of phloem sap of cucumber leaves pre-infested by cucumber biotypePre-infested cucumber leaves were obtained by infesting cucumber leaves with cucumber biotype.Total sugar determination showed that the total sugar content of phloem sap of healthy cucumber leaves,and cucumber leaves pre-infested for 7 d and 14 d were1210μg/m L,1903μg/m L,and 3674μg/m L,respectively.Primary metabolomic profiling was conducted on phloem sap of healthy cucumber leaves and 14 d pre-infested cucumber leaves.A total of 279 metabolites were detected,including 111 differential metabolites.Pre-infestation significantly upregulated 79 metabolites and downregulated 32 metabolites.Among the differential sugar compounds,82.13%were upregulated,including sucrose,inositol galactoside,and gluconic acid phosphate.Feeding assays showed that cotton biotype grew and reproduced more successfully on phloem sap of 14 d pre-infested cucumber leaves than on that of healthy cucumber leaves,with intrinsic rate of increase（r_m）increase of 0.31 and net reproductive rate（R₀）increase of 34.97.These results indicate that infestation by cucumber biotype significantly increases sugar content in phloem sap in cucumber leaves,thereby improving the fitness of cotton biotype on these leaves.2.The role of Buchnera in the interaction between cucumber biotype of A.gossypii and cucumberThe performance of cucumber biotype on cucumber was tested after the aphids were removed of Buchnera using a mixed antibiotic.The results showed that the body length and weight of cucumber biotype significantly decreased in the absence of Buchnera,with a decrease of 40.56%and 24.19%,respectively.Life table data showed that the reproductive rate（R₀=3.19）of Buchnera-free cucumber biotype was significantly lower than that of healthy cucumber biotype（R₀=72.58）.Electrical penetration graph（EPG）analysis showed that Buchnera-free cucumber biotype produced almost no E1 and E2waves,indicating that they could not obtain nutrients from the phloem normally.The content of H₂O₂ was measured in cucumber leaves at 0 d,2 d,7 d,and 14 d after infection,and the results showed that Buchnera-free cucumber had higher levels of H₂O₂accumulation than healthy cucumber biotype at all time points.At 2 d after infection,the level of H₂O₂ in Buchnera-free cucumber biotype was 1.52 times higher than that in healthy cucumber biotype,indicating that Buchnera significantly reduced the accumulation of H₂O₂ in cucumber leaves.The content of jasmonic acid（JA）and salicylic acid（SA）in cucumber leaves was determined 2 d,7 d,and 14 d after infection,and the results showed that there was no significant difference in JA and SA content between Buchnera-free and healthy cucumber biotype at 2 d after infection,while JA content was significantly higher in the Buchnera-free treatment at 7 d and 14 d,and SA content was significantly lower in the Buchnera-free treatment at 7 d and 14 d.These results suggest that Buchnera helps cucumber biotype inhibit JA-related plant defense,and stimulate SA instead.Finally,the fitness of cucumber biotype on cucumber leaves pre-infested by Buchnera-free and healthy cucumber biotype for 14 d was tested,and the reproductive rate of cucumber biotype on healthy cucumber biotype pre-infested leaves was significantly higher than that on Buchnera-free cucumber biotype pre-infested leaves.These results indicate that Buchnera plays an important role in suppressing JA signal-related plant defenses,thus facilitating normal infection by cucumber biotype on cucumber.3.Proteomic analysis of Buchnera in cucumber and cotton biotypeCotton biotype were acclimated to cucumber by being reared on pre-infested cucumber leaves for 2 generation.By this method,we got cotton biotype and cucumber biotype with the same genetic background.Proteomic analysis of Buchnera in cucumber biotype and cotton biotype was conducted,and a total of 396 proteins were identified,with101 differentially expressed proteins,of which 23 were upregulated and 78 were downregulated in the cucumber biotype.Functional annotation analysis of the proteins using GO,KEGG,and COG revealed that the top-ranking enriched proteins were related to amino acid metabolism,nutrient metabolism,and chaperone protein function,with over98%of proteins functioning in the cytoplasm.Based on previous research and combined with the results of this study,it is suggested that five chaperone proteins,ten proteins related to nutrient metabolism,and five proteins with significantly different expression levels may determine the host specificity of A.gossypii and their functions warrant further investigation.In summary,the reason why cotton biotype can adapt to pre-infested cucumber is that the pre-infestation of cucumber biotype increases the sugar content of cucumber phloem sap,which facilitates the feeding and colonization of cotton biotype on cucumber.Buchnera was found to aid cucumber biotype by suppressing plant defenses.Significant differences in Buchnera protein expression were observed between cucumber and cotton biotype,and 20 key differential Buchnera proteins that may determine the host specificity of A.gossypii were identified.The present study shed light on the molecular mechanisms underlying host specialization of A.gossypii.