Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa

Abstract

Background: The mosquito vectors of Plasmodium spp. have largely been overlooked in studies of ecology and evolution of avian malaria and other vertebrates in wildlife.

Methods: Plasmodium DNA from wild-caught Coquillettidia spp. collected from lowland forests in Cameroon was isolated and sequenced using nested PCR. Female Coquillettidia aurites were also dissected and salivary glands were isolated and microscopically examined for the presence of sporozoites.

Results: In total, 33% (85/256) of mosquito pools tested positive for avian Plasmodium spp., harbouring at least eight distinct parasite lineages. Sporozoites of Plasmodium spp. were recorded in salivary glands of C. aurites supporting the PCR data that the parasites complete development in these mosquitoes. Results suggest C. aurites, Coquillettidia pseudoconopas and Coquillettidia metallica as new and important vectors of avian malaria in Africa. All parasite lineages recovered clustered with parasites formerly identified from several bird species and suggest the vectors capability of infecting birds from different families.

Conclusion: Identifying the major vectors of avian Plasmodium spp. will assist in understanding the epizootiology of avian malaria, including differences in this disease distribution between pristine and disturbed landscapes.

Figure 1

Sampling locations of mosquitoes in the lowland forest areas of Cameroon. Areas where Coquillettidia sp were recorded are indicated by filled triangles.

Figure 2

Mosquito trapping methods used in this study. A – CDC Light trap hung from dead tree in grassland along Nyong River, Ndibi; B – Net trap placed in grassland along Nyong River; C – Collecting mosquitoes resting in grass and on tree branches by sweep net. Mosquitoes were aspirated out from the sweep net and then placed into holding cages for identification and preservation. D – Ehrenberg bird trap hung in branches of dead tree in along edge of Nyong River grassland.

Figure 3

Habitat preferences for Coquillettidia aurites. Overall, there was a significant preference for shaded areas (p = 0.04). No C. pseudoconopas was collected in Ndibi and Plasmodium DNA isolations were not associated with any of the four microhabitats. Collections from the other sites represent less than 5% of the total and are not included in the analysis.

Figure 4

Proportion of infected pools of the eight Plasmodium lineages of Coquillettidia sppin Ndibi and Nkouak. Because sites sampled are more than 100 km apart, populations are considered independent. * indicates significant differences in proportion of infected pools between both habitats. Lineages PV11 and PV12 were the most common while the others were relatively rare.

Figure 5

Giemsa-stained sporozoite of Plasmodium spp. from the salivary glands of Coquillettidia aurites. Note a prominent centrally located nucleus. (Scale bar = 5 μm).

Figure 6

Bayesian phylogeny of the 8 lineages of Plasmodium mitochondrial cytochrome b gene obtained from Coquillettidia species, 4 lineages from published sequences of avian Plasmodium and three lineages of Haemoproteus spp. as outgroups. Names of the lineages and GenBank accession numbers of the sequences are given after the species names of parasites. Bayesian support are indicated above the branches while ML Bootstrap support, based on 100 replications are shown below the branches. The vector species in which the parasites were found (including parasites already known) is indicated under 'Vector Species.'