Abstract:

Objective To investigate the ability of Anopheles stephensi at different ages to transmit Plasmodium berghei and elucidate the possible mechanisms. Methods To study and compare the susceptability of A. stephensi of different ages to P. berghei, 4-day and 25-day female A. stephensi were fed with P. berghei infected BALB/c mouse blood with parasitaemia 4%-8%. At 8 days after infection, the mosquitoes were dissected and the number of intestinal Plasmodium oocysts was counted under microscope. Difference in the susceptability to Plasmodium infection was analyzed between the two age groups. To study the intestinal bacteria load in A. stephensi, LB plate culture was used to detect the intestinal bacteria in the mosquitoes of the two groups before infection, and real-time quantitative PCR（qPCR） was performed to check the culturable bacteria and the total bacteria load. The expression levels of major immune response effectors cecropin（CEC1, CEC3）, defensin（DEF）, gambicin（GAM）, attacin（ATT）, nitric oxide synthase（NOS）, dual oxidase（DUOX）, and thioester protein 1（TEP1） in 4-day and 25-day mosquitoes were determined by qPCR. Results At 8 days after infection, the median of oocyst number in 4-day mosquitoes was 139, which was nearly 46 times of that in 25-day mosquitoes（median, 3）（P<0.01）. There was a significant difference in the intestinal bacteria load between 4-day and 25-day mosquitoes. The results of qPCR showed that the total bacteria load in 25-day mosquitoes was 1.5 times of that in 4-day mosquitoes（P<0.05）. By LB plate culture, proliferation of 28 889 colony forming units（cfu） bacteria was found for 25-day mosquitoes, which was 9 times of that for 4-day mosquitoes（3 200 cfu）（P<0.05）. In addition, the NOS expression level in 25-day mosquitoes was 2.4 times of that in 4-day mosquitoes（P<0.01）, while the expression levels of antimicrobiota peptides ATT, DEF, CEC3, and CEC1 in 25-day mosquitoes were only 27%, 48%, 14%, and 61% of those in 4-day mosquitoes, respectively（P<0.05）. The expression levels of GAM, DUOX, TEP1 showed no difference between the two groups. Conclusion The antimicrobiota peptides in A. stephensi are significantly downregulated with age increase, together with increased intestinal bacteria load and NOS expression, resulting in enhanced resistance to Plasmodium.

Key words: Age, Immune response, Microbiota, Plasmodium berghei