Vector species-specific association between natural Wolbachia infections and avian malaria in black fly populations

Abstract

Artificial infection of mosquitoes with the endosymbiont bacteria Wolbachia can interfere with malaria parasite development. Therefore, the release of Wolbachia-infected mosquitoes has been proposed as a malaria control strategy. However, Wolbachia effects on vector competence are only partly understood, as indicated by inconsistent effects on malaria infection reported under laboratory conditions. Studies of naturally-occurring Wolbachia infections in wild vector populations could be useful to identify the ecological and evolutionary conditions under which these endosymbionts can block malaria transmission. Here we demonstrate the occurrence of natural Wolbachia infections in three species of black fly (genus Simulium), which is a main vector of the avian malaria parasite Leucocytozoon. Prevalence of Leucocytozoon was high (25%), but the nature and magnitude of its association with Wolbachia differed between black fly species. Wolbachia infection was positively associated with avian malaria infection in S. cryophilum, negatively associated in S. aureum, and unrelated in S. vernum. These differences suggest that Wolbachia interacts with the parasite in a vector host species-specific manner. This provides a useful model system for further study of how Wolbachia influences vector competence. Such knowledge, including the possibility of undesirable positive association, is required to guide endosymbiont based control methods.

Figure 1

Species composition of the most abundant Simulium species over the season and phylogenetic analysis of the Leucocytozoon haplotypes detected in each species. (A) The percentage of each Simulium species collected each month, based on RFLP analysis and identification. N = X at the bottom of the x-axis indicates the total number of samples collected per month. (B) Phylogenetic analysis of all Leucocytozoon sequences from black flies and blue tits. Branches are labelled based on the infected host (Simulium species or C. caeruleus) and which parasite haplotype they are grouped in (Supplementary Table S1). (C) Phylogenetic analysis of Wolbachia MLST sequences identified in the three main black fly species in relation with other Wolbachia strains.

Figure 2

Variation in Leucocytozoon and Wolbachia infectivity of black flies based on month collected and black fly species identified. (A) shows the percentage of black fly individuals which tested positive for Leucocytozoon per month. On top, the average daily temperature between May and October from the nearest Met Office weather station (Ardtalnaig) taken between 9.00 am-9.00 pm. (B) shows the proportion of black fly species infected with Leucocytozoon among the overall infected samples per month. (C) shows the percentage of individuals which tested positive for Wolbachia per month and (D) shows the proportion of black fly species infected with Wolbachia among the overall infected samples per month. Figures in bars of A and C indicate the total number of samples collected and tested per month. N = X shown below B and D indicate the number of samples positive for either Leucocytozoon or Wolbachia, respectively. Error bars in A and C represent the standard errors of the mean.

Figure 3

Variation of Leucocytozoon prevalence in black flies in the presence of Wolbachia. (A) The proportion of Leucocytozoon-infected black flies in the presence and absence of Wolbachia based on PCR results for each of the three major black fly species identified, S. aureum, S. vernum and S. cryophilum. Error bars represent the standard error calculated from the mean value of each data point and N = X represents the number of samples in each set. (B) Leucocytozoon prevalence in the presence and absence of Wolbachia for each black fly species based on the coefficients of the significant interaction between Wolbachia and black fly species from the best fitting model.