Defense Response Of Aedes Aegypti Against Bacillus Thuringiensis And Optimization Of Cost-effective Medium For Bt Production

It has been estimated that over one million people worldwide die from mosquito transmitted diseases every year. So far, there are no effective vaccines or treatments against dengue fever yet. Consequently, it relys on the control of mosquitoes. However, control of mosquitos is largely depends on harmful chemical insecticides. Therefore, environmental friendly alternative strategies for mosquito control are needed. Bacillus thuringiensis subsp. israelensis (Bti) is one of the few Bt strains that are highly pathogenic to mosquito larvae and is widely used for control of mosquito populations. But application of this insecticide is minor,accounting for only 2% of the insecticidal market, because of the high cost and relative lower mosquitocidal activity, as compare with conventional pesticides.Therefore, how to enhance the mosquitocide efficacy and reduce the cost of Bti insecticides has been a difficult issue which we are confronted. The molecular mechanism of how mosquitoes defense against Bti infections is largely unknown.Understanding of Bti-mosquito interactions will help to develop new Bti toxins for mosquitoes and mosquito-borne disease management.Bti strain LLP29, which has been isolated in previous study, showed high activity against Aedes aegypti, one of the major vectors of yellow fever and dengue virus. The present study was intended to understand the dynamic transcription and physiological changes in Aedes aegypti during LLP29 infection, building the knowledge of pathogenesis on the pathways and regulatory networks, and clarifying the key genes that play major roles in Bti infection response; explore the possibility of utilizing spent mushroom substrate extract as the sole carbon source for production of Bacillus thuringiensis-based biopesticide; develop a cost-effective method for the mass production of Bacillus thuringiensis by solid-state fermentation. Results from this work will pave a scientific way for the future development of novel cost-effective anti-mosquito biological agents for mosquito pest management.To find some biotechnological applications for enhancing the activity of Bti toxins against mosquitoes, this study investigate mosquito-Bti interactions at both cellular and molecular levels. Ae. aegypti transcript responses to experimental challenge with mosquitocidal strain B. thuringiensis subsp. israelensis LLP29 and non-mosquitocidal strain B. thuringiensis subsp. kurstaki HD1 have been analyzed with next generation sequencing technology using Illumina/Solexa high-throughput sequencing platform. Significant up- or down-regulation (greater than 2-fold) can be assayed for approximately 2% of the mosquito transcriptome and affected genes represent a variety of functional classes that include immunity,apoptosis,proliferation, detoxification, chitin metabolism and others. Three candidate genes have been selected based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The galectin was selected from the proteins involved in receptor binding; ner was selected as representative of the mitogen-activated protein kinase (MAPK) group, and caspase-18 was selected from the group of proteins involved in cell death. When we affected the expression of galectin and mapk-like by silencing with RNAi the larvae became hypersensitive to toxin action.In addition, we found that mosquito larvae displayed a resistant phenotype when the caspase-18 was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.In transcriptome data, 36 detoxification genes have shown differential expression, which means that the detoxification enzymes in mosquitoes play an important role in xenobiotic metabolism and redox metabolism. Therefore,the resistance to Bti may relate to the detoxification mechanisms. In light of these considerations, we investigated several enzyme activities in Aedes aegypti larvae after Bti treatments. The results revealed that the exposure of Bti significantly increased the level of amylase (183.2%) as well as the activities of cytochromes P450 (177.5%), Na+/K+-ATPase (142.9%). On the contrary, 8.6% and 11.4%decrease in the level of acetylcholinesterase and Glutathione S-transferase were respectively recorded. These results are consistent with the differential expression of each gene families in transcriptome data.There are 5 chitin metabolism related peritrophic matrix protein were up-regulated during Bti infection. It may play a role in the defense against ingested pathogens by function as an immune barrier. Meanwhile, it has been demonstrated that the entomotoxicity and chitinase activity of Bacillus thuringiensis based biopesticides may be increased by fortified with chitin during production. To develop a cost-effective biopesticide, spent mushroom substrate extract was studied as a potential chitin and carbon source for cultivating Bacillus thuringiensis.Several pretreatments were compared to determine the optimal method for degrading cellulose to produce reducing sugars. Pretreatment was followed by standard enzymatic hydrolysis and fermentation. Results showed that the highest cellulose degradation was obtained using 2% dilute sulfuric acid pretreatment at 121? for 1h, resulting in a high yield of reducing sugar (284.24g/kg SMS).Sporulation was also highest using the same pretreatment. Subsequently, Spent mushroom substrate extract (2%H2SO4,121 ?,1h) was selected for Bti LLP29 fermentation, Plackett-Burman design (PB) and response surface methodology(RSM) were employed to optimize the medium components for production of Bti-based biopesticide. The optimal composition was as followes: 54% SMS extract,31.9g/L soybean meal,0.88g/L CaCO3,0.4g/L MnSO4, 0.5g/L K2HPO4, and 0.4g/L Tween 100. Under the optimized conditions, more than 27 times improvement in production of spores was achieved compared with the original SMS medium. These results not only help overcome the problem of SMS waste, but also reduce the cost of Bti fermentation medium when compared with conventional culture medium.To explore a cost-effective method for the mass production of Bacillus thuringiensis by solid-state fermentation, as a locally available agro-industrial by-product, spent mushroom substrate was used as raw material for Bti cultivation,and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32 ?, initial pH value 6, moisture content 50%,the ratio of sieved material to initial material 1:3 and inoculum volume 0.5 ml.Large scale production of B. thuringiensis subsp. israelensis LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1487 ITU/mg) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bti-based biopesticides. The cost of 1 kg Bti production was approximately US $ 0.075. Results from this study suggest that SMS is a highly cost-effective raw material for mass production of Bti.