| The insects are important for both economic and health reasons as they can cause crop damage,food losses,and food shortages.Ostrinia furnacalis is a main agricultural pest and causes severe damage and up to 20%yield losses.O.furnacalis can be infected by a variety of pathogens,including bacteria.It is severally treated with different concentrations of Bacillus thuringiensis(Bt).Bt is a gram-positive,soil-dwelling bacterium capable of producing δ-endotoxins(Cry),cytolytic proteins(Cyt),and vegetative insecticidal proteins(VIP).On the other hand,they are more successful in controlling O.furnacalis and other insects,and have been used to manage insects and pests for several decades.It is unknown well that if B.thuringiensis(sub-lethal concentration LC30)effect on O.furnacalis development,and immunity.To address these questions,this study carried out the following experiments and analyses:1.To test the effect of Bt on development,nutritional,and innate immunity parameters of O.furnacalis,the nutritional signs(lipid,protein,and trehalose),humoral immune response(PO and lysozyme activity),and cellular immune response(total haemocyte count(THC)and different haemocyte count(DHC))of the O.furnacalis,and development(weight,growth,mortality,longevity,and egg laying)parameters were determined after an oral infection with B.thuringiensis at a sub-lethal concentration(LC30).In this study,the results showed that the development and immunity of O.furnacalis decreased significantly(p<0.05)in the body weight of O.furnacalus larvae(by 27%on day15),pupae(by 14%on day 5),and adults(male and female)after Bt infection.The number of eggs significantly decreased(by 21%)in the treated group.Furthermore,lipid at 36 h(p<0.01),48 h(p=0.05),60 h(p<0.037),72 h(p<0.04),and 84 h(p=0.05);protein at 36 h(p=0.0437),48 h(p=0.01),60 h(p<0.02),and 72 h(p<0.01);and trehalose levels at all time point except at 24 h were found to be significantly lower compared with control.On the contrary,Bt infection increased the life span of O.furnacalus larvae(by 28%)and pupae(by 51%)(delayed development)and the larvae mortality(p<0.01)compare with control.On the other hand,the longevity of O.furnacalis female adult significantly reduced by 34%compared to control group(p<0.05).For insects have immunity against infections,hemocytes in the hemolymph must be present.The effects of Bt on O.furnacalis larvae were investigated.The results showed higher increase in lysozyme(by 32%at 36 h)and PO(by 57%at 48 h)activities.On the other hand,the results showed decrease in the content of THC(at 36,48,60,72,and 84 h)(p<0.05),and DHC(PR,GR,PL,OE,and SC)also highly decreased by 57%,48%,67%,62%,and 69%at 48,24,60,48,and 60 h post-infection with Bt.Bt can make clear adverse effects on the growth and development of O.furnacalis,indicating that sublethal concentration Bt as a bio-insecticide has a great effect on O.furnacalis.2.There are some gaps of the knowledge about how Bt toxins affect the regulation of immune-related genes and signaling pathways in O.furnacalis.The roles of PRRs(βGBPs,PGRPs,C-type lectin),Toll pathway components(MyD88),and antimicrobial peptides(AMPs),such as Gloverin,Moricin,Cecropin,and Lysozyme in O.furnacalis for defense against Bt bacteria is clear.We used RNA-seq and the transcriptomic analysis to analyze the expression profiling of immune-gene in O.furnacalis larvae exposure to Bt infection in the current investigation.At different time points(24,36,48,and 60 h),transcriptomic analysis was performed for the control group and O.furnacalis larvae treated with a sub-lethal concentration LC30 of Bt.In the present study,the results showed that a total of 225 million clean reads were obtained from five RNA-seq libraries,including 44 million and "45,44,45,and 45" million from the uninfected and treated group at different times points(24,36,48,and 60 h),respectively.The Q20%and GC%for control and treated samples were 97.82%and(97.74%,97.86%,97.89%,and 97.8%),respectively.GO and KEGG enrichment were used to further investigate distinct expression genes(DEGs).A large change of 10,021 differentially expressed genes(DEGs)in response to Bt treatment was detected at all time periods,with more up-regulated genes(5,505)than down-regulated genes(4,516).Several immune-related genes(341)that are important in defending against invading Bt,such as pattern recognition(βGRPs,PGRPs,C-type lectin),Toll components(MyD88),and AMPs(Gloverin,Moricin,Cecropin,and Lysozyme)were detected in these DEGs,and showed more down-regulated genes(237)than up-regulated genes(104).The qPCR analysis for MyD88,Gloverin,Moricin,Cecropin,βGRP3,and Lysozyme was utilized to confirm the expression levels of these genes.The qPCR results showed that they were up-regulated.We noticed that some expression levels of these genes(MyD88,Cecropin,Moricin,βGRP3,and Lysozyme)at specific time points(24,36,60,60,and 36 h)matched the RNA-seq data,respectively.3.To further investigate the effect of Bt on O.furnacalis immune response and the molecular function of O.furnacalis,myeloid differentiation factor 88(MyD88)gene was cloned.MyD88 is a pivotal adapter protein involved in activating nuclear factor NF-κB of the Toll pathway in insect innate immunity.MyD88 has been extensively studied in vertebrates and Drosophila,but the information ascribed to MyD88 in Lepidoptera is still very scarce,especially since its function remains obscure.B.thuringiensis was used to test the immune function of MyD88 in larvae of O.furnacalis.Our results showed that the transcription factor MyD88 was cloned using Rapid Application of cDNA End(RACE)in O.furnacalis larvae.The cDNA(GenBank accession number:MN906311)designated OfMyD88 was 804 bp.It consists of a 630 bp ORF(open reading frame),which involves 209 amino acids,and one RNA instability motif.Structure analysis showed OfMyD88 had the death domain(122135 aa),an intermediate domain(172-185 aa),and the TIR domain(188-208).There was no evidence of a putative polyadenylation signal or a transmembrane domain.The results showed that OfMyD88 was widely expressed in immune-related tissues such as hemocytes,fat body,midgut,and integument,with the highest expression level in hemocytes,and the lowest expression level in integument.The expression levels of Attacin and Gloverin,Cecropin,Moricin,βGRP3,and Lysozyme were regulated by OfMyD88.The result showed that Bt had significant up-regulated effects on O.furnacalis immune genes(AMPs,Lysozyme,and PPO2 expression)and PO and lysozyme activity,and then induced its immune response against gram-positive bacteria.Thus,the results confirmed that OfMyD88 is a key component of the Toll pathway but not of the PPO pathway in O.furnacalis.Through the activation of AMPs mentioned above,it can defend against Bt.In general,our research sheds insight on O.furnacalis’ immune response to Bt infection,which will aid in understanding how Bt affects O.furnacalis innate immune system and provides a good model for controlling O.furnacalis with Bt. |
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