BackgroundDengue fever, caused by the dengue virus (DENV), is now the most common arbovirus transmitted disease globally. One novel approach to control DENV is to use the endosymbiotic bacterium, Wolbachia pipientis, to limit DENV replication inside the primary mosquito vector, Aedes aegypti. Wolbachia that is naturally present in a range of insects reduces the capacity for viruses, bacteria, parasites and fungi to replicate inside insects. Wolbachia’s mode of action is not well understood but may involve components of immune activation or competition with pathogens for limited host resources. The strength of Wolbachia-based anti DENV effects appear to correlate with bacterial density in the whole insect and in cell culture. Here we aimed to determine whether particular tissues, especially those with high Wolbachia densities or immune activity, play a greater role in mediating the anti DENV effect.Methodology/findingsAe. aegypti mosquito lines with and without Wolbachia (Wildtype) were orally fed DENV 3 and their viral loads subsequently measured over two time points post infection in the midgut, head, salivary glands, Malpighian tubules, fat body and carcass. We did not find correlations between Wolbachia densities and DENV loads in any tissue, nor with DENV loads in salivary glands, the endpoint of infection. This is in contrast with strong positive correlations between DENV loads in a range of tissues and salivary gland loads for Wildtype mosquitoes. Lastly, there was no evidence of a heightened role for tissues with known immune function including the fat body and the Malpighian tubules in Wolbachia’s limitation of DENV.Conclusion/significanceWe conclude that the efficacy of DENV blocking in Wolbachia infected mosquitoes is not reliant on any particular tissue. This work therefore suggests that the mechanism of Wolbachia-based antiviral effects is either systemic or acts locally via processes that are fundamental to diverse cell types. We further conclude that the relationship between DENV blocking and Wolbachia density is not linear in mosquito tissues

Mosquitoes transmit a diverse group of human flaviviruses including West Nile, dengue, yellow fever, and Zika viruses. Mosquitoes are also naturally infected with insect‐specific flaviviruses (ISFs), a subgroup of the family not capable of infecting vertebrates. Although ISFs are not medically important, they are capable of altering the mosquito's susceptibility to flaviviruses and may alter host fitness. Wolbachia is an endosymbiotic bacterium of insects that when present in mosquitoes limits the replication of co‐infecting pathogens, including flaviviruses. Artificially created Wolbachia‐infected Aedes aegypti mosquitoes are being released into the wild in a series of trials around the globe with the hope of interrupting dengue and Zika virus transmission from mosquitoes to humans. Our work investigated the effect of Wolbachia on ISF infection in wild‐caught Ae. aegypti mosquitoes from field release zones. All field mosquitoes were screened for the presence of ISFs using general degenerate flavivirus primers and their PCR amplicons sequenced. ISFs were found to be common and widely distributed in Ae. aegypti populations. Field mosquitoes consistently had higher ISF infection rates and viral loads compared to laboratory colony material indicating that environmental conditions may modulate ISF infection in Ae. aegypti. Surprisingly, higher ISF infection rates and loads were found in Wolbachia‐infected mosquitoes compared to the Wolbachia‐free mosquitoes. Our findings demonstrate that the symbiont is capable of manipulating the mosquito virome and that Wolbachia‐mediated viral inhibition is not universal for flaviviruses. This may have implications for the Wolbachia‐based DENV control strategy if ISFs confer fitness effects or alter mosquito susceptibility to other flaviviruses.

BackgroundTwo lymphatic filariasis endemic communities Mampong and Hwida in Ghana have been regularly monitored for impact on transmission after annual mass drug administration (MDA) with albendazole and ivermectin. After six MDAs even though the ABR for Mampong was 55883/person/year and that of Hwida was 2494/person/year, they both had ATPs of 15.21 infective larvae/person/year. Interestingly the human microfilaraemia levels had reduced significantly from 14% to 0% at Mampong and 12% to 3% at Hwida. In an attempt to understand this anomaly, we collected mosquitoes over a 5-month period using human landing catches to determine the species composition, the number of cibarial teeth, the lengths and widths of the cibarium and the cibarial dome of the vector populations.ResultsOut of 2553 mosquitoes caught at Mampong, 42.6% were An. gambiae s.l. All 280 identified further by PCR were An. gambiae s.s (275 M and 5 S molecular forms). At Hwida, 112 mosquitoes were obtained; 67 (59.8%) were An. gambiae s.l, comprised of 40 (59.7%) An. melas, 24 (35.8%) An. gambiae s.s (17 and 5 M and S molecular forms respectively) and 3 (4.5%) unidentified. The mean number of teeth for An. melas was 14.1 (median = 14, range = 12-15), An. gambiae s.s., 15.7 (median = 15, range = 13-19) M form 15.5 (median = 15 range = 13-19) and S form 16 (median = 16, range 15-17). The observed differences in teeth numbers were significantly different between An. melas and An. gambiae s.s (p = 0.004), and the M form (p = 0.032) and the S form (p = 0.002).ConclusionsIn this study, An. gambiae s.s was the main vector at Mampong and was found to possess significantly more cibarial teeth than An. melas, the principal vector at Hwida. We postulate that the different impact observed after 6 MDAs may be due to An. gambiae s.s exhibiting 'facilitation' at Mampong and at Hwida An. melas the main vector exhibits 'limitation'. Thus it may be necessary to compliment MDA with vector control to achieve interruption of transmission in areas where An. melas may exhibit limitation.

BackgroundThe introduction of the endosymbiotic bacterium, Wolbachia into Aedes aegypti populations is a novel approach to reduce disease transmission. The presence of Wolbachia limits the ability of the mosquito to transmit dengue virus (DENV) and the strength of this effect appears to correlate with Wolbachia densities in the mosquito. There is also some evidence that Wolbachia densities may increase following the consumption of a bloodmeal. Here we have examined whether multiple blood feeds lead to increases in density or associated changes in Wolbachia-mediated blocking of DENV.MethodsThe Wolbachia infected Aedes aegypti mosquito line was used for the study. There were three treatment groups; a non-blood fed control, a second group fed once and a third group fed twice on human blood. All groups were orally infected with DENV-2 and then their midguts and salivary glands were dissected 10–11 days post infection. RNA/DNA was simultaneously extracted from each tissue and subsequently used for DENV RNA copies and Wolbachia density quantification, respectively.ResultsWe found variation between replicate vector competence experiments and no clear evidence that Wolbachia numbers increased in either the salivary glands or remainder of the body with feeding and hence saw no corresponding improvements in DENV blocking.ConclusionsAedes aegypti are “sip” feeders returning often to obtain bloodmeals and hence it is important to assess whether repeat blood feeding improved the efficacy of Wolbachia-based DENV blocking. Our work suggests in the laboratory context when Wolbachia densities are high that repeat feeding does not improve blocking and hence this ability should likely be stable with respect to feeding cycle in the field.