Omics Study Of Conogethes Punctiferalis (Lepidoptera:Crambidae) Immune Response Against Beauveria Bassiana (Hypocreales:Cordycipitaceae) Infection

The yellow peach moth,Conogethes punctiferalis,as an agricultural insect pest,its damage to maize ears has become more and more serious in Huang-Huai-Hai maize-producing areas of China in recent years,threatening the safe production of maize and the food safety.At present,the control of C.punctiferalis is mainly based on chemical insecticides,and the long-term use of large amounts of chemical insecticides will develop resistance.Compared with chemical insecticides,fungal insecticides,as environmentally friendly biological insecticides,have been widely used in insect pest control.Because insects themselves live in a complex environment and face complex and diverse exogenous factors,they have formed a strong innate immune system in their long-term evolution,which to a certain extent weakens the insecticidal ability by using fungal insecticides,and limiting their widespread promotion.Therefore,studying the innate immune system of insects is the basis for effective control,and understanding the molecular mechanism of its action can improve the insecticidal efficiency of fungal insecticides.Based on transcriptomics and proteomics techniques,the immune response mechanisms of C.punctiferalis larvae infected with Beauveria bassiana were studied.The results can provide a theoretical basis for the biological control of C.punctiferalis using the entomopathogenic fungi.The main results are as follows:1.The survival rate and phenotypic changes of C.punctiferalis larvae infected with B.bassiana were clarified.The survival rates of larvae were 40%,26.67%and 10%by injecting with 2×10²,2×10³ and 2×10⁴ conidia at 144 h post infection,respectively.All larvae injected with 2×10⁵ and 2×10⁶ conidia died at 120 h and 72 h post infection,respectively.The toxicity regression equation between mortality（y）and the dose of conidia（x）was got as y=0.819 x+1.732（r=0.9053）,LC₅₀=9817 conidia/μL,95%confidence limit=4760-20192 at 72 h post- infection.The phenotypic changes of C.punctiferalis larvae after infected with B.bassiana were obvious at different time points.Firstly,larvae appeared black spots on the cuticle,dead larvae rigidized and turned dark red,and finally the hyphal of B.bassiana grew through the cuticle,and larvae were surrounded by white hyphal and conidia.2.Five types of circulating hemocytes were identified from C.punctiferalis larvae,including prohemocytes,plasmatocytes,granulocytes,spherulocytes and oenocytoids.The number of granulocytes and plasmatocytes was the largest,accounting for 86.17%.The total hemocyte counts decreased significantly at 0.5 h,2 h,12 h,24 h and 48 h after infection with B.bassiana;when prohemocytes at 12 h and 48 h,plasmatocytes at 0.5 h,2 h,12 h,24 h and48 h,granulocytes at 0.5 h,12 h,24 h and 48 h,spherulocytes at 0.5 h,12 h and 48 h,and oenocytoids at 24 h and 48 h,the number of hemocytes significantly decreased.The hemocyte-mediated phagocytosis and nodulation were initiated in the hemolymph of C.punctiferalis larvae by B.bassiana conidia challenge.And DEAE-Sepharose Fast Flow beads elicited a strong encapsulation response in the hemolymph of C.punctiferalis larvae.The hemolymph phenoloxidase activity of C.punctiferalis larvae increased overall with increasing concentration of B.bassiana.3.Using the Illumina Nova Seq 6000 sequencing platform for transcriptome sequencing,the total of 33648 and 57396 genes were identified,respectively,and 114（76 up-regulated and38 down-regulated genes）and 7354（5331 up-regulated and 2023 down-regulated genes）differentially expressed genes were identified,respectively,after C.punctiferalis larvae infected with B.bassiana at 12 h and 36 h.The total of 289 immune-related genes were further identified,including 75 recognition molecules,39 signal modulation molecules,48 signal transduction molecules and 127 effectors.Eight immune-related genes randomly selected were validated for q RT-PCR,and the results showed that the results of transcriptome sequencing were consistent with those of q RT-PCR,indicating that the accuracy of the transcriptome sequencing was high.4.Using i TRAQ quantitative proteome sequencing technique,the total of 3431and 4195proteins were identified,respectively,and 197（109 up-regulated and 88 down-regulated expressions）and 198（126 up-regulated and 72 down-regulated expressions）differential protein at 12 h and 36 h,respectively,after C.punctiferalis larvae infected with B.bassiana at 12 h and36 h.Six immune-related proteins associated target genes randomly selected were validated for q RT-PCR,and the results showed that the results of proteome sequencing were consistent with those of q RT-PCR,indicating that the accuracy of the proteome sequencing was high.5.By combined transcriptome and proteome analysis,there were 3377 and 4122transcriptome and proteome correlations,respectively,and 3 and 51 differentially expressed gene-differentially expressed proteins,respectively,after C.punctiferalis larvae infected with B.bassiana at 12 h and 36 h.Five immune-related signaling pathways were obtained by KEGG pathway enrichment analysis,namely phagosome signaling pathway,lysosome signaling pathway,melanogenesis signaling pathway,Toll and Imd signaling pathway and JAK/STAT signaling pathway.6.The full-length c DNA sequence of C-type lectin IML4 of C.punctiferalis larvae was987 bp and the open reading frame was 963 bp,encoding 320 amino acids,with a predicted molecular weight of 35315.97 and an isoelectric point of 5.84.Analysis of the conserved domain revealed a signal peptide consisting of 24 amino acids,and two carbohydrate recognition domains containing 123 and 241 amino acids,respectively.Spatiotemporal expression profiling showed that IML4 was expressed in different developmental stages of C.punctiferalis（eggs,1st-5th instar larvae,pupae and adults）,with the lowest expression in eggs and the relative expression was rose in the larval stage;IML4 expression was also detected in different tissues of larvae（head,midgut,fat body,hemolymph and cuticle）,with the highest relative expression in hemolymph,followed by fat body,and the lowest expression in midgut and head.The immune function of IML4 was studied by using RNAi,and its interference efficiency reached 68%at 24 h after ds IML4 injection.The results showed that IML4interference accelerated the larval death with B.bassiana infection,and the phenotype of C.punctiferalis larvae changed significantly,which proved that IML4 was indeed involved in antifungal immune response.The gene expressions of PGRP-1,Toll-1,SP-7 and Alo-3 were detected after interfering with IML4 expression,and the results show that the expression levels of PGRP-1 and Toll-1 increased significantly,and the expression levels of SP-7 and Alo-3decreased significantly,implying that interfering with IML4 expression induced the expression of PGRP-1 and Toll-1 and inhibited the expression of SP-7 and Alo-3 expression.