Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management.

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40-75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential

Novel <i>Wolbachia</i>-transinfected <i>Aedes aegypti</i> mosquitoes possess diverse fitness and vector competence phenotypes

<div><p><i>Wolbachia pipientis</i> from <i>Drosophila melanogaster</i> (<i>w</i>Mel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector <i>Aedes aegypti</i>. To date, <i>w</i>Mel-infected <i>Ae</i>. <i>aegypti</i> have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing <i>w</i>Mel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of <i>w</i>Mel, transinfecting different or additional <i>Wolbachia</i> strains into <i>Ae</i>. <i>aegypti</i> may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of <i>Ae</i>. <i>aegypti</i> with the <i>Wolbachia</i> strains <i>w</i>MelCS (<i>D</i>. <i>melanogaster</i>), <i>w</i>Ri (<i>D</i>. <i>simulans</i>) and <i>w</i>Pip (<i>Culex quinquefasciatus)</i> and assess the effects on <i>Ae</i>. <i>aegypti</i> fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that <i>w</i>MelCS provides a similar degree of protection against dengue virus as <i>w</i>Mel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of <i>w</i>Mel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by <i>w</i>Ri is markedly weaker than that of <i>w</i>MelCS, consistent with previous characterisations of these lines in <i>Drosophila</i>, while <i>w</i>Pip was found to substantially reduce the fitness of <i>Ae</i>. <i>aegypti</i>. Thus, we determine <i>w</i>MelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative <i>Wolbachia</i> strain with more desirable attributes than <i>w</i>Mel for future field testing.</p></div

Maternal transmission of <i>Wolbachia</i> assessed from progeny of crosses between infected females and uninfected males.

<p>Maternal transmission of <i>Wolbachia</i> assessed from progeny of crosses between infected females and uninfected males.</p

<i>Wolbachia</i> density and distribution in transinfected <i>Ae</i>. <i>aegypti</i> lines.

<p>(A) Density of <i>Wolbachia</i> within 5-, 10- and 15-day old whole female mosquitoes was determined by qPCR using primers directed to the conserved <i>16S</i> rRNA gene. Density is expressed as the mean ratio between <i>16S</i> and the <i>Ae</i>. <i>aegypti</i> host <i>rps17 gene</i>. Data are the mean and SEM of 24 mosquitoes. Asterisks indicate significance compared to <i>w</i>Mel at each time point (Kruskal-Wallis, Dunn's test with multiple test corrections; *p<0.05, **p<0.01, ***p<0.001, n.s. not significant). (B) The distribution of <i>w</i>MelCS, <i>w</i>Ri and <i>w</i>Pip <i>Wolbachia</i> strains in mosquitoes was determined in sections of paraffin-embedded female mosquitoes (5 to 7-day old) using fluorescence <i>in situ</i> hybridisation (FISH). The fluorescently labelled 16S probe detects the <i>16S</i> rRNA gene from all four <i>Wolbachia</i> strains. Total DNA was stained in blue using DAPI and a green filter was included to increase contrast with surrounding tissues. <i>Sg</i> indicates salivary gland tissue, <i>m</i> indicates muscle, and <i>c</i> indicates cardia. White arrows identify select regions of <i>Wolbachia</i> staining.</p

Longevity in transinfected <i>Ae</i>. <i>aegypti</i> lines.

<p>Triplicate cages of age-controlled (emergence within 24 h) adults (~150 males and ~150 females/cage) were maintained at 26°C, 65% relative humidity and a 12:12 h light:dark cycle in a climate controlled room. The number of dead males and/or females was recorded and carcasses removed daily until all mosquitoes in the cages were dead. Data are the total % survival from the three cages/line. Significant differences were observed for <i>w</i>Ri females (p<0.0001), <i>w</i>Mel (p<0.05) and <i>w</i>MelCS (p<0.05) males, relative to their respective Tet control line. <i>w</i>Pip had a significantly shorter lifespan for both males and females compared its matched Tet-control (p<0.0001). Statistical analysis was performed using a Log-rank (Mantel-Cox) test.</p

CI, fecundity and egg diapause viability in <i>w</i>MelCS, <i>w</i>Ri, and <i>w</i>Pip transinfected <i>Ae</i>. <i>aegypti</i>.

<p>(A) Fecundity was determined as a measure of eggs laid per female. Bars are the mean number of eggs laid ± SEM from >40 females (individual data points are superimposed). Symbols for tetracycline-treated mosquitoes (uninfected) are in black, <i>Wolbachia</i>-infected are in red. Asterisks indicate significance compared to Tet x Tet controls (Kruskal-Wallis, Dunn's test, * for <0.05, ** for <0.01, *** for <0.001, **** for <0.0001). (B) Hatch rates for crosses between infected and uninfected mosquitoes show CI and successful hatching. Symbol codes and statistics are as per (A). Data are the mean ± SEM from >40 females (individual data points are superimposed). (C) Eggs from gravid females were collected over 72 h, from 3-days post blood meal. Eggs were dried slowly over 3–5 days then stored in a humid, airtight container. Batches of 100–500 eggs were hatched after 1, 2, 3, 4, 6, 8, 10 and 12 weeks. Hatched larvae were counted at 2nd instar stage until no hatch was observed for a week then the percent hatch calculated. Statistical analysis was performed using 2way ANOVA Sidak's test (n = 4 at each time point). <i>w</i>Pip hatch rate was significantly reduced at all weeks compared to <i>w</i>Pip.Tet (p<0.0001). <i>w</i>Mel hatch rate was significantly reduced at weeks 4, 8, 10, (p<0.05) and 12 (p<0.0001) compared to <i>w</i>Mel.Tet. <i>w</i>MelCS had a significantly reduced hatch rate at weeks 10 and 12 compared to <i>w</i>MelCS.Tet (p<0.01 and p<0.0001, respectively). <i>w</i>Ri had a significantly reduced hatch rate at week 12 only, compared to <i>w</i>Ri.Tet (p<0.0001).</p

<i>w</i>MelCS provides superior blocking of DENV-3 genome replication in a mosquito injection challenge model.

<p>(A) DENV-3 was injected into the thorax of 6 or 7-day old female mosquitoes at 2.5 x10⁶ TCID₅₀/ml (undiluted) or 10, 100 or 1000-fold dilutions thereof. RNA was extracted from whole mosquito bodies 7-days post infection and virus replication was quantified by qRT-PCR. Data are the mean number of genome copies per mosquito ± SEM. Number of DENV-3 positive mosquitoes/total <i>n</i> are indicated above each bar. ** p < 0.01, ****p<0.0001, Mann-Whitney test. (B) The mean DENV genome copies and SEM from (A) are replotted as a function of virus concentration injected. Significant differences in the mean RNA copies of <i>w</i>Mel and <i>w</i>MelCS lines are indicated by ** (p<0.01). Significant differences in the mean RNA copies of <i>w</i>Mel.Tet and <i>w</i>MelCS.Tet lines are indicated by <b>‡</b> (p<0.05), Mann-Whitney test. Injection dilutions were performed as independent experiments, with the data combined to produce the final data series.</p

Maternal transmission rates of <i>w</i>Mel and <i>w</i>AlbB in the superinfected <i>w</i>Mel<i>w</i>AlbB <i>Ae</i>. <i>aegypti</i> line.

<p>Maternal transmission rates of <i>w</i>Mel and <i>w</i>AlbB in the superinfected <i>w</i>Mel<i>w</i>AlbB <i>Ae</i>. <i>aegypti</i> line.</p

Proportion of mosquitoes infected with DENV after blood-feeding on 36 dengue patient blood samples, as a function of plasma viremia, and stratified by serotype.

<p>Lines represent fits from logistic regression to the data. All three strains were assessed for every feed; for DENV-1 there were 23 blood feeds, and 13 for DENV-4. DENV-2 and DENV-3 are not represented due to small sample sizes (n = 3 and n = 2 respectively). (A) Each point represents the proportion of mosquitoes with a DENV-infected abdomen, stratified by strain, and pooled across all time points. (B) The proportion of mosquitoes, pooled across all time points, that expectorated infectious DENV in their saliva.</p