Multiple bloodmeals enhance dissemination of arboviruses in three medically relevant mosquito genera

Abstract

Background: Mosquitoes in nature may acquire multiple bloodmeals (BMs) over the course of their lifetime; however, incorporation of frequent feeding behavior in laboratory vector competence studies is rarely done. We have previously shown that acquisition of a second non-infectious BM can enhance early dissemination of Zika virus (ZIKV), dengue virus, and chikungunya virus in Aedes aegypti and ZIKV in Aedes albopictus mosquitoes, yet it is unknown if other taxonomically-diverse virus-vector pairings show a similar trend under a sequential feeding regimen.

Methods: To test this, we evaluated the impact of a second noninfectious BM on the vector competence of Aedes aegypti and Anopheles quadrimaculatus for Mayaro virus, Culex quinquefasciatus for West Nile virus, Aedes triseriatus for La Crosse virus, and Aedes aegypti for Oropouche virus (OROV). Female mosquitoes were fed BMs containing these viruses and half of them were given a second noninfectious BM at 3 or 4-days post infection. Mosquitoes were harvested at various time points and assayed for virus infection in bodies and disseminated infection in legs by performing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays.

Results: We found that a second noninfectious BM had no impact on midgut infection rates but increased virus dissemination for all but one of the virus-vector pairings- Ae. aegypti and OROV. Unlike the other arboviruses under consideration, which are strictly mosquito-borne, biting midges (Culicoides spp.) serve as the main vector of OROV and this virus rarely disseminated to the mosquito leg tissue in our study.

Conclusions: Taken together, our findings show that sequential blood feeding enhances virus dissemination across diverse arbovirus-vector pairings, representing three mosquito genera and virus families, but a second BM was insufficient to overcome a strong midgut virus escape barrier in a nonnatural virus-vector pairing.

Fig. 1

Comparison of Mayaro virus disseminated infection rates in single-fed (blue) and double-fed (red) mosquitoes for A Ae. aegypti and B Ae. quadrimaculatus. The data for each day post-infection (dpi) were analyzed by a two-sided Fisher’s exact test. Data were also fitted into linear regression lines across all time points and compared by analysis of covariance indicated by brackets. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent the binomial stand error of the mean of sample proportions

Fig. 2

Comparison of virus disseminated infection rates in single-fed (blue) and double-fed (red) mosquitoes for A West Nile virus and Cx. quinquefasciatus and B La Crosse virus and Ae. triseriatus. The data for each day postinfection (dpi) were analyzed by a two-sided Fisher’s exact test. Data were also fitted into linear regression lines across all time points and compared by analysis of covariance indicated by brackets. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent the binomial stand error of the mean of sample proportions