ABSTRACT
Objective: To investigate the effect of sublethal dose of Bacillus sphaericus (Bs) 2 362 strain on the malaria transmission ability of Anopheles (An.) dirus (Hainan strain) and its molecular mechanism. Methods: Firstly, the fourth instar larvae of An. dirus was treated with sublethal dose of Bacillus sphaericus. The surviving larvae were then collected and placed into mosquito cages, where they were hatched into adult mosquitoes. These mosquitoes were recorded as Bs group. Meanwhile, the control group was set without any treatment. Then, for infection with Plasmodium yoelii BY265 RFP, 3- to 5-day old female adults were kept at 24 °C and fed on Plasmodium yoelii BY265 RFP-infected Kunming mice with a gametocytemia above 0.5%. On day 9-11 post infection, mosquitoes were dissected, and the oocysts on the midguts were examined under a fluorescence microscope. Thirdly, total RNA was extracted from mosquitoes of Bs group and the control group at different time-points respectively, and the cDNA were synthesized later. Finally, SYBR quantitative PCR was conducted to investigate the expression of Imd pathway anti-malaria molecules at different time-points, including TEP1 and Rel2, in Bs and control group mosquitoes. Results: Bs treatment remarkably reduced the infection rate of Plasmodium from 23.71% (124/523) to 16.23% (87/536) (Chi-square test, P=0.002 0.05). Additionally, the intensities of melanized oocysts were compared between the two groups, and no significant difference was found, either (P=0.566>0.05). Interestingly, compared with the control group, the expression levels of TEP1 and Rel2 in Bs group were obviously up-regulated in larval, adult and infected mosquitoes. Especially in 3 dpi and 7 dpi, the expression level of TEP1 in Bs group was nearly 4 times higher than that of the control group, while Rel2 reached to approximately 7 times. Conclusions: We firstly found that the sublethal dose of Bs significantly suppressed the vector competence of An. dirus to malaria parasites, which revealed a new important role of Bs on the basis of killing mosquito larvae. Furthermore, the Imd signaling pathway might play an effective way in Bs impacting the vector competence of An. dirus through upregulating the expression of NF-kB transcription factor Rel2, enhancing the expression of TEP1, which killed the Plasmodium, but not through melanization.
ABSTRACT
Objective To analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. Methods Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug adminstration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microcopy. Results TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P