Diet-Induced Nutritional Stress and Pathogen Interference in <i>Wolbachia</i>-Infected <i>Aedes aegypti</i>

<div><p>The pathogen interference phenotype greatly restricts infection with dengue virus (DENV) and other pathogens in <i>Wolbachia</i>-infected <i>Aedes aegypti</i>, and is a vital component of <i>Wolbachia</i>-based mosquito control. Critically, the phenotype’s causal mechanism is complex and poorly understood, with recent evidence suggesting that the cause may be species specific. To better understand this important phenotype, we investigated the role of diet-induced nutritional stress on interference against DENV and the avian malarial parasite <i>Plasmodium gallinaceum</i> in <i>Wolbachia</i>-infected <i>Ae</i>. <i>aegypti</i>, and on physiological processes linked to the phenotype. <i>Wolbachia</i>-infected mosquitoes were fed one of four different concentrations of sucrose, and then challenged with either <i>P</i>. <i>gallinaceum</i> or DENV. Interference against <i>P</i>. <i>gallinaceum</i> was significantly weakened by the change in diet however there was no effect on DENV interference. Immune gene expression and H₂O₂ levels have previously been linked to pathogen interference. These traits were assayed for mosquitoes on each diet using RT-qPCR and the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, and it was observed that the change in diet did not significantly affect immune expression, but low carbohydrate levels led to a loss of ROS induction in <i>Wolbachia</i>-infected mosquitoes. Our data suggest that host nutrition may not influence DENV interference for <i>Wolbachia</i>-infected mosquitoes, but <i>Plasmodium</i> interference may be linked to both nutrition and oxidative stress. This pathogen-specific response to nutritional change highlights the complex nature of interactions between <i>Wolbachia</i> and pathogens in mosquitoes.</p></div

Stage-, sex-and diet-specific density of <i>w</i>Flu in <i>Ae. fluviatilis</i>.

<p>Graphs showing the absolute (A) and relative (B) densities of <i>w</i>Flu throughout the life cycle of the wildtype strain (<i>wolb⁺</i>) of the mosquito <i>Ae. fluviatilis</i>. The density of <i>w</i>Flu was estimated using real-time quantitative PCR of the <i>Wolbachia</i>-specific <i>wsp</i> gene and the mosquito-specific <i>actin</i> gene (see <i>Materials and Methods</i> for details). Each circle represents a single, whole individual, while the blue horizontal bars indicate either the median number of <i>wsp</i> copies (Graph A) or the median <i>wsp</i>/<i>actin</i> ratio (Graph B) per individual. The data shown are from three independent biological replicates (i.e., three different cohorts – generations – of the laboratory colony of <i>Ae. fluviatilis</i>). For each life cycle stage/sex/diet type, 4 individuals were assayed from each of the three cohorts, so that in total 12 individuals were used. For each cohort, adult females were separated into two groups 6 days after eclosion from pupae, and one group was blood-fed on the same day, such that 7, 8, 9 and 20 day-old adults are, respectively, 24, 48, 72 and 336 hours after blood-feeding, while the other group of age-matched adult females was maintained on sugar only. After day 9, blood-fed females were allowed to oviposit, so that fully-developed eggs would not be retained. As the sex of larvae cannot currently be unambiguously determined for aedine mosquitoes, only a single group representing an unknown mix of randomly selected male and female 4^th instar individuals was assayed. Comparisons marked with an asterisk (*) were significantly different between sugar- and blood-fed females using a Mann-Whitney <i>U</i> test, while comparisons marked with “NS” were not significantly different between sugar- and blood-fed females. Statistically significant differences were also observed between different life cycle stages and sexes as described in the main text.</p

<i>w</i>Flu has no effect on the longevity of adult <i>Ae. fluviatilis</i>.

<p>Graphs showing the Kaplan-Meier survival curves for sugar-fed adult males (♂, top graph) and females (♀, bottom graph) of the wildtype (<i>wolb⁺</i>) and antibiotic-treated (<i>wolb⁻</i>) strains of the mosquito <i>Ae. fluviatilis</i>. The data shown were pooled from two independent biological replicates (i.e., two different generations of the laboratory colony of <i>Ae. fluviatilis</i>), and analysed together (see <i>Materials and Methods</i> for details of the experimental design). The survival curves for each sex did not differ significantly between wildtype (<i>wolb⁺</i>) and antibiotic-treated (<i>wolb⁻</i>) individuals (log-rank (Mantel-Cox) test: males, χ² = 0.6743, <i>P</i> = 0.4116; and females, χ² = 0.5850, <i>P</i> = 0.4444; and Mantel-Haenszel hazard ratios: males, ratio = 0.9046, 95% CI 0.7121 to 1.1490; and females, ratio = 0.9103, 95% CI 0.7154 to 1.1580).</p

Immune activation in <i>w</i>Mel-infected <i>Ae</i>. <i>aegypti</i> fed on different carbohydrate regimes.

<p>Levels of 4 key immune genes, Cecropin E (<i>cece</i>) <b>(A)</b>, Defensin C (<i>defc</i>) <b>(B)</b>, C-type lectin, galactose binding 5 (<i>ctlga5</i>) <b>(C)</b>, and Transferrin (<i>tsf</i>) <b>(D)</b>, were quantified through RT-qPCR for <i>w</i>Mel (+<i>Wolb</i>) and Tet (-<i>Wolb</i>) mosquitoes, after 7 days feeding on their respective carbohydrate diets. Expression levels were normalized against host <i>rps17</i> expression levels. Each circle represents one pair of either Tet (black circles) or <i>w</i>Mel (green circles) mosquitoes, with 12 samples examined for each treatment. Solid black lines represent mean expression (± s.e.m.). <i>P</i> values: Student’s <i>t</i> tests, * < 0.05, ** < 0.01 *** < 0.001.</p

<i>w</i>Flu causes incomplete unidirectional cytoplasmic incompatibility in <i>Ae. fluviatilis</i>.

<p>Graph showing the percentage of eggs hatching in reciprocal crosses between the wildtype (<i>wolb⁺</i>) and antibiotic-treated (<i>wolb⁻</i>) strains of the mosquito <i>Ae. fluviatilis</i> (see <i>Materials and Methods</i> for details of the experimental design). Each circle represents a single adult female mosquito, while the red horizontal bars indicate the median number of hatched eggs per female. The data shown are pooled from two independent biological replicates (i.e., two different generations of the laboratory colony of <i>Ae. fluviatilis</i>). The total number of females (<i>n</i>_♀) and the total number of eggs (<i>n</i>_eggs) examined are indicated in the figure, above the data for each cross. The smallest group within either biological replicate comprised 16 females, which laid a total of 1109 eggs. All data from both biological replicates were analysed together using a Kruskal-Wallis test (<i>P</i><0.0001), followed by pairwise comparison using Dunn’s test to determine which crosses differed significantly from one another. The letters (a, b) at the top of the figure, above the data for each cross, indicate the results of the Dunn’s test (Dt). Only the ♀<i>^wolb−</i>×♂<i>^wolb+</i> cross (highlighted in yellow) differed significantly from the other three crosses (b: in all three comparisons, <i>P</i><0.001), which did not differ significantly from one another (a: in all three comparisons, <i>P</i>>0.05).</p

<i>w</i>Flu: Characterization and Evaluation of a Native <i>Wolbachia</i> from the Mosquito <i>Aedes fluviatilis</i> as a Potential Vector Control Agent

<div><p>There is currently considerable interest and practical progress in using the endosymbiotic bacteria <i>Wolbachia</i> as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different <i>Wolbachia</i> strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of <i>w</i>Flu, a native <i>Wolbachia</i> from the neotropical mosquito <i>Aedes fluviatilis</i>, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of <i>Ae. fluviatilis</i> cured of its native <i>Wolbachia</i> by antibiotic treatment, we show that in its natural host <i>w</i>Flu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, <i>w</i>Flu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite <i>Plasmodium gallinaceum</i>. The stage- and sex-specific density of <i>w</i>Flu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of <i>w</i>Flu was also shown to be specifically-reduced in the ovaries after bloodfeeding <i>Ae. fluviatilis</i>. Overall, our observations indicate that the <i>Wolbachia</i> strain <i>w</i>Flu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether <i>w</i>Flu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.</p> </div

Interference against DENV-3 in <i>w</i>Mel-infected <i>Ae</i>. <i>aegypti</i> fed on different carbohydrate regimes.

<p>DENV-3 prevalence and intensity data for <i>w</i>Mel (+<i>Wolb</i>) and Tet (-<i>Wolb</i>) mosquitoes fed on one of four carbohydrate diets after experimental oral infection (R₁—<b>A</b> - 7dpi, <b>B</b> - 14dpi; R₂—<b>C</b> - 7dpi, <b>D</b> - 14dpi), as determined by RT-qPCR quantification using a DENV-specific TaqMan probe. Pie charts represent prevalence of infection (dark blue—proportion infected, light blue—proportion uninfected), and dot plots represent viral load in infected mosquitoes. Horizontal lines in each treatment represent mean viral load. <i>P</i> values: ** < 0.01, *** < 0.001, Prevalence—Fisher’s exact test.</p

Mathematical modelling of the ability of <i>w</i>Flu to invade host populations.

<p>Theoretical prediction of the ability of the <i>Wolbachia</i> strain <i>w</i>Flu to invade uninfected host populations using the empirically-determined laboratory-based parameter estimates observed in this study for <i>w</i>Flu in its native host <i>Ae. fluviatilis</i>, and equation (1) from Dobson et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone.0059619-Dobson4" target="_blank">[62]</a>, modified from Turelli & Hoffmann <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone.0059619-Turelli3" target="_blank">[130]</a>. Graph A shows three different predictions of the rate of spread of <i>w</i>Flu based upon three different initial prevalences of <i>w</i>Flu in the host population (5, 10 and 20%), which can be interpreted as the size of released <i>Wolbachia</i>-infected seed populations relative to the uninfected host population during a vector control programme. Graph B shows the general relationship between the initial prevalence of <i>w</i>Flu and the number of host generations required for <i>w</i>Flu to attain 100% prevalence in the host population. Coloured circles indicate values for the initial prevalences used in Graph A. The following parameter values were used to calculate the prevalence of infection (<i>p</i>) at generation time (<i>t</i>) by iteration: μ, the maternal transmission efficiency (the proportion of uninfected offspring produced by infected mothers) = 0.0 (i.e., complete maternal transmission was assumed; see main text for justification); <i>H</i>, the relative egg hatching rate (the ratio of hatched eggs from infected versus uninfected mothers) = 0.071; α, the relative fitness of infected versus uninfected females = 1.0 (i.e., no difference in fitness was inferred based on the survival and fecundity data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone-0059619-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone-0059619-g003" target="_blank">3</a>, respectively). <i>H</i> was calculated using pooled total egg counts for the compatible and incompatible crosses shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone-0059619-g001" target="_blank">Figure 1</a>, rather than the average hatch rate per female, in order to provide a more conservative estimate of the strength of cytoplasmic incompatibility (i.e., to account for the variation in the expression of cytoplasmic incompatibility observed with <i>w</i>Flu – see main text for detailed explanation).</p

Tissue-specific density of <i>w</i>Flu in sugar- and blood-fed adult female <i>Ae. fluviatilis</i>.

<p>Graphs showing the absolute (A) and relative (B) densities of <i>w</i>Flu in different tissues of adult females of the wildtype strain (<i>wolb⁺</i>) of the mosquito <i>Ae. fluviatilis</i>. The density of <i>w</i>Flu was estimated using real-time quantitative PCR of the <i>Wolbachia</i>-specific <i>wsp</i> gene and the mosquito-specific <i>actin</i> gene (see <i>Materials and Methods</i> for details). Each circle represents a single pool of 5 individual organs taken from different age- and cohort-matched individuals, while the blue horizontal bars indicate either the median number of <i>wsp</i> copies (Graph A) or the median <i>wsp</i>/<i>actin</i> ratio (Graph B) per individual. The data shown are from two independent biological replicates (i.e., two different generations of the laboratory colony of <i>Ae. fluviatilis</i>). Three to 5 day-old adult females were separated into two groups after eclosion from pupae, and one group was blood-fed, while the other was maintained on sugar only. Twenty-four hours later (i.e., after blood-feeding, when the females were 4 to 6 days old), both sugar-fed and blood-fed individuals were dissected, and their organs harvested. In graph A, the absolute density of <i>w</i>Flu per <i>individual</i> organ was estimated by dividing the calculated number of <i>wsp</i> copies for each sample (i.e., pool of organs) by the number of organs in each pool (i.e., 5 organs). The cohorts (i.e., generations) of mosquitoes assayed were different from those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone-0059619-g007" target="_blank">Figure 7</a>, such that the data presented in the two figures are not directly comparable, although they give consistent results. Comparisons marked with an asterisk (*) were significantly different between sugar- and blood-fed females using a Mann-Whitney <i>U</i> test, while unmarked comparisons were not significantly different between sugar- and blood-fed females. Statistically significant differences were also observed between some of the different tissues as described in the main text.</p

<i>w</i>Flu does not inhibit <i>Plasmodium</i> in <i>Ae. fluviatilis</i>.

<p>Graphs showing the number of oocyst stage malaria parasites observed on the midguts of wildtype (<i>wolb⁺</i>) and antibiotic-treated (<i>wolb⁻</i>) strains of the mosquito <i>Ae. fluviatilis</i> 7 days after infection with the avian malaria parasite <i>P. gallinaceum</i>. Each circle represents a single midgut from an adult female mosquito, while the red horizontal bars indicate the median number of oocysts per midgut. The data shown are from four independent biological replicates (i.e., four different generations, after antibiotic treatment, of the laboratory colony of <i>Ae. fluviatilis</i>). The numbers of oocysts per midgut were compared separately for each biological replicate (i.e., generation) using a Mann-Whitney <i>U</i> test. * = significantly different; NS = not significantly different. The dashed blue lines indicate the threshold used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059619#pone-0059619-g006" target="_blank">Figure 6</a> to classify mosquitoes as having either relatively low or high <i>P. gallinaceum</i> oocyst infections.</p