Figure 1.
Immune gene expression in An. gambiae somatically infected with wMelPop.
The expression of six immune genes were analyzed by qRT-PCR: leucine-rich repeat immune protein, LRIM1; thioester-containing protein, TEP1; cecropin, CEC1; defensin, DEF1; C-type lectin, CTL4; and clip-domain serine protease, CLIPB3. Adult An. gambiae females were injected with E. coli, wMelPop or the buffer alone, 2–3 days post-eclosion, and RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition).
Figure 2.
Immune gene expression in the An. gambiae wMelPop-infected MOS55 cell line.
The expression of six immune genes as described for Figure 1 were analyzed by qRT-PCR, for the An. gambiae MOS55 cell culture infected with wMelPop, normalized to expression of these genes in a tetracycline treated, wMelPop free, genetically identical, MOS55 cell culture. Three samples of cells were taken from the cultures at different times; error bars show the SEM of these three samples.
Figure 3.
An. gambiae somatically infected with wMelPop: challenges with Plasmodium berghei.
Each panel represents an independent experiment showing mean numbers of oocysts per midgut (parasite intensities), comparing An. gambiae challenged with P. berghei eight (A–C) or five (D) days after intrathoracic innoculation with, in A–C, Wolbachia wMelPop compared to buffer (BI) and non-injected (NI) controls plus in C E. coli (EI); and in (D) Wolbachia+dsLacZ (WLI), Wolbachia+dsTEP1 (WTI) and NI. Parasite survival was determined by oocyst counting on day 10 post infection. In A–C significant reductions in intensity were observed in WI females compared to the NI, BI and EI controls: ***P<0.001; ** P<0.01. P. berghei prevalence was also significantly reduced (P<0.05) in WI compared to one or more of the controls: expt. A. NI = 78.5% (33/42); BI = 81.8% (27/33), WI = 60.0% (27/45); expt. B NI = 88.4% (23/26), BI = 92.3% (12/13), WI = 57.1% (12/21; expt. C NI = 90.3% (28/31), BI = 96.0% (24/25), WI = 63.1% (12/19), EI = 81.2% (13/16). In experiment D intensity was significantly lower in the WLI group compared to WTI and NI, *P<0.05. Prevalence was 81% (39/48) for NI, 81% (13/16) for WTI and 50% (6/12) for WLI.
Figure 4.
Immune gene expression and challenges with Brugia pahangi in Ae. aegypti somatically infected with wMelPop, and effects of immune knockdown on Wolbachia density.
A) The expression of four immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, PGRPS1; cecropin D, CECD; CLIP-domain serine protease, CLIPB37; and a C-type galactose-specific lectin. Adult females were injected with wMelPop or the buffer alone, approximately seven days post-eclosion. RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition). B) The mean numbers of L3 stage (infective) larvae per mosquito are shown following B. pahangi challenge in Ae. aegypti Refm strain previously injected with wMelPop or buffer; * P<0.05. Numbers 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. C) We measured the levels Wolbachia ftsZ gene expression as a proxy for Wolbachia density and normalized the qRT-PCR data to the mosquito Actin5C gene. Two sets of three females per time point injected with either dsLacZ or dsRel2 were assayed. ftsZ gene expression was found to be higher in dsRel2-injected mosquitoes than in dsLacZ-injected mosquitoes at both six and ten days post injection. The mean level of Rel2 transcript in dsRel2-injected mosquitoes was confirmed to be approximately 40% of that in dsLacZ injected mosquitoes at both time points. These data suggest that the immune effectors controlled by the Imd pathway (Rel2-controlled) can influence Wolbachia densities.
Figure 5.
Model of possible effects of wMelPop on malaria vectorial capacity.
Vectorial capacity is a measure that describes the transmission potential of a mosquito population and is independent of Plasmodium prevalence. It can be thought of as proportional to the number of infectious bites that occur per day after a single infectious human arrives in a previously malaria-free area. If we assume recruitment to the adult mosquito stage is constant then vectorial capacity can be written (A b (1−μ)τ)/μ where b is the ability of the mosquito to transmit Plasmodium, μ is adult daily survival, τ is the length of the intrinsic incubation period of the Plasmodium and all other parameters are combined in A [42]. The figure plots vectorial capacity as transmission (b) and daily survival (μ) are each reduced because of the presence of Wolbachia by a multiplicative factor (1−x) where x varies in the range 0 to 1 (parameters: b = 1; μ = 0.1; τ = 1; A = 1). A more advanced analysis tailored to a specific system might want to include age-specific adult mortality, the effect of Wolbachia on mosquito population dynamics and seasonality.
Table 1.
Oligonucleotide primers used in quantitative PCR experiments and dsRNA synthesis.