| Silkworm(Bombyx mori) is the representative species of Lepidoptera and one of the important economic insects, which took a long time to be domesticated. I has been well studied and becomes an important model organism for the research on mechanisms of insect immune defense and insect-pathogen interaction. Beauveria bassiana is one of the fungal pathogens that are mostly investigated and widely applied as the best biological control strains for controlling agricultural and forestry insect pests due to its wide host range, high pathogenicity, high adaptability, high compatibility and easy operation in culture and harvest. At the same time, it always causes the white muscardine of economic insects such as silkworm, resulting in huge economic losses to sericulture industry and other economic insect industry. In this research, B. bassiana was used as the pathogen and silkworm as the host insect to study the mechanisms of host-pathogen interaction, including infection and pathogenicity of B. bassiana and the silkworm immune defense. Major achievement was summarized as follows.1. Mechanisms of B. bassiana infection to silkwormContact infection is the main pathways for B. bassiana to invade hosts insects, during which extracellular hydrolases play important roles. In this study, the activities of extracellular proteases and extracellular chitinases and the expression levels of related genes of 11 B. bassiana strains were measured, based on which their relationship to invasion virulence were analyzed using statistical approaches. Moreover, the effects of extracellular protease and extracellular chitinase inhibitors on the invasion virulence of B. bassiana were investigated. Besides, the capability of yeast expressed B. bassiana cuticle-degrading enzyme(Bb CDEP1) to decompose silkworm integument was also analyzed. The results showed that the virulence of 11 B. bassiana strains were positively correlated to their extracellular proteases activities and expression level of Bbcdep1, and the fitted equation was accorded with Logistic equation. Inhibition of the protease activity resulted in a lowered virulence of B. bassiana. In culture condition, the expression levels of Bbcdep1 in different B. bassiana strains could be up-regulated by silkworm components in the medium, but the level of up-regulation was different significantly among the strains. During the infection process to the silkworm larvae, the expression of Bbcdep1 was up-regulated at the earlier infection stage, which was corresponding to the process of conidia germination and cuticular penetration into the silkworm body. The Bb CDEP1 expressed in yeast could effectively decompose the silkworm cuticle, with the activities equivalent to 50% Protease K activities.The activities of extracellular chitinases were not significantly different among the 11 B. bassiana strains and not statistically correlated to their virulence to the silkworm. However, the inhibition of extracellular chitinase caused the lower invasion virulence. The two B.bassiana chitinases, chitinase 1(Bbchit1) and chitinase 2(Bbchit2), exhibited significantly different expression patterns under induction with silkworm components in the medium, i.e. the expression levels of Bbchit1 were up-regulated in all the B. bassiana strains, but the expression levels of Bbchit 2 up-regulated in only some of the B.bassiana strains. While, the expression of both the Bbchit1 and Bbchit2 were up-regulated during the spore germination and cuticular penetration process to the silkworm, but the expression of Bbchit2 was still significantly up-regulated even in the later stage of infection, suggesting that Bbchit2 might play a specific role also in the pathogenesis process in the silkworm.All these results indicated that extracellular proteases and extracellular chitinases were the key virulence factors for B. bassiana to invade silkworm, but their gene expression patterns varied significantly. Bbcdep1 might be used as the key gene for genetically engineering to improve the infectivity and Bb CDEP 1 could also be used as the biological protease additives. Besides, chitinases might play specific roles not only in the invading process to but also in the pathogenesis process in the silkworm infected by B.bassiana.2. Mechanisms of B.bassiana pathogenesis in silkwormPathogenic fungi could absorb nutritional components, secrete mycotoxins and break down the redox balance to kill the host insects. In this study, the B. bassiana toxins were extracted and their effects on survival rate of silkworm and on Bm N cells were determined. The changes of expression levels of genes related to glucose metabolism and antioxidant enzyme system were detected using q RT-PCR. In addition, changes of total antioxidant capacity, superoxide dismutase(SOD) and Catalase(CAT) activities in silkworm infected by B.bassiana and the effects of exogenous antioxidants on B. bassiana virulence were also analysed. The results showed that, Beauveria toxins could significantly reduce the survival time of silkworm larvae and cause the agglutination and disintegration of Bm N cells. The expression levels of genes related to glucose metabolism were significantly down-regulated in silkworm infected by B. bassiana, suggesting that B. bassiana could affect the sugar XI metabolism and destroy the energy metabolism in silkworm. The total antioxidant capacity, SOD and CAT activities were greatly reduced and the expression levels of genes related to antioxidant enzyme system were significantly down-regulated in silkworm infected by B.bassiana. However, the survival time of silkworm larvae could be extended by injection of SOD and CAT, indicating that the the B. bassiana infection to the silkworm could damage the redox balance of the host. These results implied that B, bassiana could kill the silkworm through secreting toxins into, damaging energy metabolism and destroying redox balance of the host.3. Silkworm immune defense against B.bassianaInsect innate immune defense system is an important barrier to resist invasion of pathogenic microorganisms. Different pathogens could activate different signaling pathways and immune effectors, causing different immune responses. In this study, the expression patterns of genes encoding different components of Toll signaling pathway, Imd signaling pathway, Jak/STAT signaling pathway, melanization and antimicrobial peptides(AMPs) were analyzed using q RT-PCR in the silkworm larvae challenged with B. bassiana. The signal transduction of Toll signaling pathway and Jak/STAT signaling pathway were analyzed using RNAi knock-down method. Moreover, the effects of signaling pathway inhibitors on antifungal activity in larvae hemolymph were also tested. In addition, the silkworm AMPs were expressed using prokaryotic expression system and the antifungal activities were determined too. The results showed that Toll signaling pathway, Imd signaling pathway, Jak/STAT signaling pathway and melanization in silkworm were all activated by B. bassiana, then the expression levels of related genes were obviously altered, but their temporal regulation mode was significantly different. Based on the results of Bombyx mori beta-1,3-glucan recognition protein(BmβGRP) RNAi assay, two probable sub-paths of Toll signaling transduction, i.e. BmβGRP 1 – Bmtoll 3 – Bmtoll 6 – Bmtoll 9-Bmspaetzle – Bmgloverin 2 and BmβGRP 2 or BmβGRP 3 – Bmtoll 7 – Bmlysozyme,might be proposed for the silkworm response to B.bassiana infection.The expression levels of Bm PGRP-Lc, Bmdredd, Bmfadd, Bmtak 1 and Bmrelish 1 were obviously up-regulated in different stages of infection, while the Bmrelish 2 was obviously down-regulated, indicating that although Imd signaling pathway in silkworm could be activated by B. bassiana, but the transcription and expression of target genes might mainly be regulated by Bmrelish 1.The expression levels of Bombyx mori C-type lectin 5(Bmctl5), Bmhop, Bmstat were all significantly up-regulated in the silkworm larvae infected with B. bassiana, and the known negative regulatory factors such as Bmsocs2, Bmsocs6 and Bmken were down-regulated significantly, while Bmdrk(another regulator of Jak/STAT signaling pathway) was significantly up-regulated at the last stage of infection, suggesting that Jak/STAT signaling pathway in silkworm could also be activated by B. bassiana infection. However, the JAK/STAT signaling pathway could not be induced by LPS and Bombyx mori cytoplasmic polyhedrosis virus(Bm CPV). Besides, the results of Bmctl5 RNAi assay indicated that Bm CTL5 was the important immune pattern recognition receptor for Jak/STAT signaling pathway.The expression patterns of 10 Bombyx mori serine protease inhibitors(Bmserpins) were significantly different. The expression levels of Bmserpin 1, Bmserpin 5, Bmserpin 7, Bmserpin 8 and Bmserpin 10 were down-regulated in different infection stages, suggesting that they might be involved in serine protease cascades and melanization. However, the expression levels of Bmserpin 2, Bmserpin 3, Bmserpin 4, Bmserpin 6 and Bmserpin 9 were up-regulated during the whole infection process. It could be speculated that they might inhibit the activities of B. bassiana serine proteases(Bb CDEP) to improve the antifungal vitality of silkworm. The expression levels of genes related to phenol oxidase activation cascade were up-regulated during the whole infection process, suggesting that a strong melanization could be activated by B. bassiana infection, which further confirmed the antifungal effects of melanization in silkworm.Antimicrobial peptides are direct immune effectors in insect innate immune response. The expression levels of Bmmoricin 1, Bmdefencin, Bmgloverin 2, Bmlysozyme, Bmcecropin A, Bmcecropin B, Bmattacin and Bmlebocin1 could all be significantly up-regulated in the silkworm infected by B. bassiana. However, the antibacterial activity assay showed that only Bm Cecropin A, Bm Cecropin B and Bm Attacin had antifungal activity to B. bassiana. It could be speculated that the up-regulated expression of the other antimicrobial peptides might be the results from the activation by B. bassiana to prevent the larvae from being infected by other competitive pathogenic microorganisms and to benefit the multiplication of B. bassiana itself.At last, the roles of detoxification and antioxidation system in antifungal process in the silkworm larvae were determined in this study. The variations in GST(glutathione-S-transferases), GSH-Px(glutathione peroxidase) and GR(Glutathione Reductase) activities, GSH(glutathione) content and in expression levels of GST and GSH-Px genes in hemolymph, midgut and fat body of silkworm larvae infected with B. bassiana or injected with Beauveria toxins were examined. Moreover, the effects of GSH and Beauveria toxins on the survival rates of silkworm larvae infected with B. bassiana were also validated. The results showed that the enzyme activities of GSTs, GSH-Px and GR and the expression levels of Bmgstd1, Bmgsts1, Bmgsto1 and Bmgsh-px genes in fat body, midgut and hemolymph were all significantly increased against B. bassiana infection. Meanwhile, GSH(glutathione) content was significantly decreased, indicating that silkworm larvae could up-regulate the enzyme activities and expression levels of the detoxification and antioxidation factors to enhance the anti fungal abilities. Toxins injection assay showed that the detoxification in silkworm larvae against Beauveria toxins was mainly depended on GSTs, GR and GSH in fat body and haemolymph. Besides, the results of quantitative real time PCR showed that the expression levels of Bmgsts1 and Bmgsto1 genes were significantly up-regulated at 24 hours after injection with toxins, suggesting that they were the key genes for detoxification in silkworm larvae. Furthermore, the bioassay results showed that GSH could relieve the damage generated by Beauveria toxins and extend the LT50(about 6 hours). Based on these results, it was confirmed that the detoxification and antioxidation system consisting of GSTs and GSH-Px plays an important role in the antifungal resistance of the silkworm.The results obtained in this study would provide a new perspective and new basis for the breeding of resistant silkworm varieties, the development of new antifungal chemicals and new biological control agents. |
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