Effect Of Bt Protein On Security Of Pardosa Pseudoannulata By RNA-seq Technology

Genetically modified plants expressing Bacillus thuringiensis (Bt) proteins have been developed in different crops for insect pest control in recent years, thus reducing environmental contamination caused by the overuse and abuse of broad spectrum insecticides; Rice is the major grain crop in many Asian countries and is facing a serious problem of insect pest resistance and pesticide contamination. To overcome this problem, genetically modified (GM) rice expressing CrylAb Bt protein has been gradually developed to alleviate the situation. Although genetically modified rice offers tremendous benefits and convenience to agricultural production and the environment, potential risks in the utilization of Bt rice lines might exist. In fact, one concern is the potential movements of Bt toxins through the complex natural food webs, including non-target species, in rice cropping systems. This study made an attempt to explain Effect of Bt protein on security of Pardosa pseudoannulata by RNA-seq technology. The results as follows:1. Effect of Bt protein on functional response of Pardosa pseudoannulata by RNA-seq technologyTo explore the mechanism and functional response of P.pseudoannulata under the affect of Bt protein,we compared the functional response and searching behaviour of P.pseudoannulata which fed with Bt protein enriched BPH with normal spiders. And, the ganglion was dissected from P.pseudoannulata to do transcriptome analysis. The result shown that no significant adverse effect on functional response of P.pseudoannulata. However, the spider get more excited under the stimulation of Bt protein. Combined with transcriptome analysis of neural tissue for P.pseudoannulata, we can inferred that oxidative phosphorylation pathway significantly enriched may be the key factor leading to P.pseudo-annulata get excited. Besides, secondary metabolite pathway enrichment may be closely associated with defense mechanism of Bt protein in P.pseudoannulata. Four DEGs were verified by qPCR (comp76553_c0_seq1, comp50268_c0_seq1, comp16097_c0_seq1 and comp50316_c0_seq1).2. Effect of Bt protein on venom gland of Pardosa pseudoannulata by RNA-seq technologyBt protein, which can combine the specific protein of epithelium of insect intestine, resulting in the formation of pits on intestine and then the death of insects, is one kind of insecticidal crystal protein. In our study, by utilizing RNA-seq, transcriptome of Pardosa pseudoannulata venom gland (both Bt and normal) had been sequenced in the normal condition, we acquired 113358 unigenes from the high-throughput sequencing platform. Besides, compared with COG, GO, KEGG etc databases, genes of venom gland were annotated and the different expressions of genes between two samples were analyzed. However, we found 3765 genes up-regulated and 3175 genes down-regulated from the statistics. The different expression of genes mainly involved in the pathways of oxidative phosphorylation and ribosome.Therefore, this research provides fundamental assistance on comprehensively understanding the genome of venom gland and evolvement of genes.3. Effect of Bt protein on growth development of Pardosa pseudoannulata by RNA-seq technologyCry proteins are utilized in transgenic rice lines for protection from lepidopteran pests and can be transferred from transgenic rice plants to non-target arthropods, including planthoppers and then to predatory spiders. Movement of Cry proteins through food webs may reduce fitness of non-target arthropods although no serious effect of Bacillus thuringiensis (Bt) crops on non-target populations have been reported. However, influence of Cry protein on gene expression has recently been reported in Cry-sensitive insects, thus possessing a potential risk on non-target organisms. We hypothesized that Cry protein intoxication influences growth and development of spider by altering enzyme activities. Here we report on the outcomes of experiments designed to test our hypothesis with the wolf spider Pardosa pseudoannulata. The results demonstrate that the CrylAb protein accumulates in the tissues of P. pseudoannulata via its prey brown planthoppers (BPHs) Nilaparvata lugens. Cry protein accumulation in spiders influences the growth, development, and enzymatic activities of two key metabolic enzymes, acetylcholine esterase and peroxidase in the spiders feeding on CrylAb-accumulated BPHs. We infer from these data that Cry proteins originating from transgenic crops can influence the growth and development of spider larva by a minor, sporadic impacts on predators’enzyme activities. We conclude that results of analyses of linear, three-element food chains do not model the complexities of multi-element food webs in agroecosystems. The immediate corollary of future studies should be designed on the platform of multi-dimensional interactions in field circumstances. The transcriptome analysis showed, the change of some biological function may be the main reason for developmental retardation and weight decrease of spiderling, for example, growth hormone receptor signaling pathway, larval development, instar larval development, anterior midgut development, adult somatic muscle development. Besides, there are also some material metabolic pathway had changed in some degree, including glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, fatty acid metabolism and so on, these may affected the detoxifying enzyme activity in spiderling inderectly,then further direct or indirect impacted the development of spiderling. Four DEGs were verified by qPCR。4. Effect of Bt protein on reproductive capability of Pardosa pseudoannulata by RNA-seq technologyA tritrophic bioassay was conducted to evaluate the potential impact of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata. P. pseudoannulata was fed with brown planthopper (BPH) reared on the Bt-rice for 15 days and CrylAb concentrations in BPH and spider were determined using ELISA. The data from the ELISA assay revealed that CrylAb protein level in spider increased dramatically, reached the peak level of 0.396ng/g at days 9 and then decreased gradually, suggesting CrylAb could be transferred from CrylAb expressing rice to BPH and then accumulated in the predator via the food chain. And reproductive parameters (pre-oviposition period, number of cocoons, number of eggsand egg hatching rate) of the spiders fed with the BPH reared on the Bt rice were not different from those fed with the BPHs reared on the non-Bt control rice. Furthermore, transcriptome analysis of gonad from P. pseudoannulata, in total 636 unigenes involved with 23 GO terms directly regulated fecundity of P. pseudoannulata by GO classification while 170 unigenes annotated in 4 reproduction-related metabolic pathways by KEGG classification. These reproduction-related unigenes were not differentially expressed through gene expression analysis. Forty nine unigenes were screened to significant expression with false discovery rate of <0.01 and absolute value of log2 fold change ratio>2, whose gene function were not directly related to reproduction of spider by GO and KEGG classification. Results from quantitative real-time PCR (qPCR) verification confirmed that the expression levels of these genes were not affected by CrylAb expressing rice in ovaries of Bt-BPH fed spiders when compared with that from the non-Bt BPH fed spiders. In conclusion, CrylAb protein has no effect on reproduction-related genes expression and fertility of P. pseudoannulata.