Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes

Abstract

Wolbachia strain wMelPop reduces the longevity of its Drosophila melanogaster host and, when introduced into the mosquito Aedes aegypti, halves its life span. We show that wMelPop induces up-regulation of the mosquito's innate immune system and that its presence inhibits the development of filarial nematodes in the mosquito. These data suggest that wMelPop could be used in the global effort to eliminate lymphatic filariasis and possibly for the control of other mosquito-borne parasites where immune preactivation inhibits their development. The cost of constitutive immune up-regulation may contribute to the life-shortening phenotype.

Figure 1. Induction of immune gene transcription in wMelPop-infected female Ae. aegypti

A summary of data from four replicate Ae. aegypti microarrays is shown as gene transcripts significantly upregulated in wMelPop-infected versus uninfected female mosquitoes. Of 199 upregulated gene transcripts with fold change (FC) ≥ 2 (p ≤ 0.05), a considerable proportion had a putative immune function (Immunity) compared with genes of other known function (Other) and unknown function (Unknown). Gene transcripts with immune-related function were categorized into effectors, signal modulation and pattern recognition. Gene transcripts encoding immune effectors dominated the list of highly upregulated genes (FC ≥ 10).

Figure 2. Microarray and qRT-PCR analyses of differentially regulated genes

The differential regulation of transcript levels in wMelPop-infected Aedes aegypti versus a genetically identical uninfected Aedes aegypti line was examined for a selection of nine upregulated genes corresponding to two cecropins (CECE, AAEL000611; CECD, AAEL000598), a fibrinogen/fibronectin related protein (FREP38, AAEL0015428), a C-type galactose specific lectin (AAEL005641), a transferrin (AAEL0015458), two CLIP domain serine proteases (CLIPB46 AAEL005431, CLIPB37 AAEL005093), a Peptidoglycan recognition protein (PGRPS1, AAEL009474), a Thio-ester containing protein (TEP20, AAEL001794) and a conserved hypothetical protein that was downregulated (CHP, AAEL003467). The values shown are the mean (± SEM) of three different qRT-PCR experiments using independent samples, at two and fifteen days post eclosion.

Figure 3. wMelPop-infected mosquitoes show a reduction in filarial infection prevalence and intensity

The mean numbers (± SEM) of infective third (L3) stage Brugia pahangi larvae are shown at 10-13 days post microfilarial challenge in Ae. aegypti wMelPop infected (Ae_Pop) versus uninfected (Ae_Tet) lines, that are filaria-susceptible following backcrossing with the Ref^m strain. Four independent challenge experiments are shown, using microfilarial densities of 11 (experiments A and B), 13 (C) and 23 (D) microfilariae / μl blood. Values above bars show the prevalence of filarial infection as a proportion of mosquitoes that contained at least one L3 Brugia larva over the total number of mosquitoes dissected in each category. Differences were significant at *P<0.01 or **P<0.001 (Mann-Whitney U test).

Figure 4. Increased resistance to a pathogenic bacterium in wMelPop-infected mosquitoes

The daily survival rates (%) of Aedes aegypti carrying wMelPop (Ae_Pop) was compared to the Wolbachia free line (Ae_Tet) after infection with Erwinia carotovora 15 (Ecc15), a pathogenic Gram-negative bacterium. The horizontal axis represents the incubation time after infection in hours. Control infections with the Gram-positive Micrococcus luteus demonstrate that the early death / protection effects are not due to septic wounding alone.