Human probing behavior of Aedes aegypti when infected with a life-shortening strain of Wolbachia

Abstract

Background: Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations.

Methodology/principal findings: Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied.

Conclusions/significance: As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.

Figure 1. Pre-probing behavior of A. aegypti mosquitoes.

Comparison of time spent by mosquitoes infected with Wolbachia (black bars) or tetracycline treated counterparts (white bars) of different ages (5, 15, 26 and 35 days) after landing on a human hand until the insertion of mouthparts into the skin (N = 12–40 per group). Bars depict means±S.E.M. * p<0.05; ** p<0.01 by t-test.

Figure 2. Probing behavior of A. aegypti mosquitoes.

Comparison of time spent by mosquitoes infected with Wolbachia (black bars) or tetracycline treated counterparts (white bars) of different ages (5, 15, 26 and 35 days) from the insertion of mouthparts into the skin of a human hand and the first sign of blood within the insect midgut. (N = 12–40 per group). Bars depict means±S.E.M. *** p<0.0001 by t-test.

Figure 3. Percent of A. aegypti mosquitoes that obtained a blood meal.

Percentage of wMelPop-infected (black bars) and tetracycline-treated mosquitoes (white bars), that successfully imbibed blood within 10 minutes of observation, by age class. Bars depict medians±quartiles for four replicate experiments, each based on 10 individual mosquitoes. * p<0.05 by Mann-Whitney U test.

Figure 4. Number of probings in A. aegypti mosquitoes.

Comparison of number of probings of mosquitoes infected with Wolbachia (black bars) or tetracycline treated counterparts (white bars) of different ages (5, 15, 26 and 35 days). (N = 40 per group). Bars depict means±S.E.M. *** p<0.0001 by t-test.

Figure 5. Additional phenotypes observed in Wolbachia-infected A. aegypti.

Proportion of wMelPop-infected mosquitoes exhibiting abnormal pre-probing behavior as: body jittering (“shaky”) or bended proboscis (“bendy”) in mosquitoes from their first occurrence at 15 days of age. Bars depict medians±quartiles for four replicate experiments, each based on 10 individual mosquitoes. Neither of these behaviors was observed in Wolbachia non-infected mosquitoes.

Figure 6. Apyrase content and saliva volume.

Comparisons of apyrase and saliva volume of mosquitoes infected with Wolbachia (black bars) or tetracycline treated counterparts (white bars) of different ages (5, 26 and 35 days). A) Apyrase activity measured through the release of inorganic phosphate from ATP. B) Saliva volume measured through the sphere volume of saliva droplets. Number of replicates in each group and age are represented. Bars depict means±S.E.M. P values relate to univariate tests of significance derived from general linear models. ** indicates P<0.01 from t-tests for the specific age category.