The modulation of the symbiont/host interaction between wolbachia pipientis and aedes fluviatilis embryos by glycogen metabolism

Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W2) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism

Avaliação da influência da Wolbachia na infecção e transmissão vertical do vírus dengue em mosquitos Aedes aegypti

Submitted by Nuzia Santos ([email protected]) on 2015-11-20T17:24:52Z No. of bitstreams: 1 PACIDÔNIO.pdf: 32476477 bytes, checksum: a96cf41a7daea427bc40612b36dfc340 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-11-20T17:25:04Z (GMT) No. of bitstreams: 1 PACIDÔNIO.pdf: 32476477 bytes, checksum: a96cf41a7daea427bc40612b36dfc340 (MD5)Made available in DSpace on 2015-11-20T17:25:04Z (GMT). No. of bitstreams: 1 PACIDÔNIO.pdf: 32476477 bytes, checksum: a96cf41a7daea427bc40612b36dfc340 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.A dengue é um problema médico crescente em países subtropicais e tropicais. A dengue é uma infecção viral que apresentem quatro sorotipos distintos, DENV-1 a - 4, sendo recentemente relatado o quinto sorotipo, DENV-5, na Malásia. Estes são transmitidos pela picada de mosquitos infectados do gênero Aedes, sendo o Aedes aegypti o principal vetor. Atualmente, a prevenção e controle da dengue dependem exclusivamente de medidas de combate ao vetor, e estas apresentam-se ineficientes, principalmente em países em desenvolvimento. Nesse contexto, há a necessidade da busca de novas estratégias que possam ser utilizadas concomitantemente com as formas de controle já existentes. A Wolbachia é uma bactéria intracelular, amplamente conhecida por promover o fenótipo de bloqueio do vírus dengue em mosquitos A. aegypti. Apesar disto, não se conhece se a Wolbachia é capaz de interferir na infecção da progênie de mosquitos infectados com o vírus dengue, fenômeno conhecido como transmissão vertical. A transmissão vertical pode estar associada com a manutenção do vírus em períodos interepidêmicos ou em locais nos quais a transmissão ativa está diminuída. Assim, o objetivo deste trabalho foi avaliar se a Wolbachia introduzida na linhagem brasileira de A. aegypti influencia a transmissão vertical da dengue. Duas metodologias de infecção foram utilizadas: infecção oral e nanoinjeção intratorácica, onde utilizou-se o DENV-4 e DENV-1,-3 e -4, respectivamente. Para estes experimentos, foram comparados mosquitos com Wolbachia (Mel) e uma linhagem de mosquitos tratada com antibiótico e portanto, livre da bactéria (Tet). Os ovos dos mosquitos infectados foram coletados em um período de 20 e 11 dias após a infecção, oral e injeções, respectivamente. A fecundidade e fertilidade, de forma geral, foram diminuídas na co-infecção da Wolbachia com o vírus dengue. Em todos os mosquitos infectados, não se observou diferenças na suscetibilidade ao vírus, porém a carga viral foi diminuída pela Wolbachia no DENV-4, em ambas as formas de infecção. Foi possível a detecção de pools positivos independente da forma de infecção, demonstrando que a transmissão vertical do vírus dengue ocorreu, porém a proporção de pools positivos entre os grupos Mel e Tet não foi alterada. Entretanto, pudemos observar uma diminuição da carga viral nos pools positivos Mel. Os ovários na infecção oral, interessantemente, tiveram uma forte diminuição da suscetibilidade conferida pela Wolbachia. A carga viral dos ovários foi diminuída em DENV-1 e DENV-4, em ambas as formas de infecção. Os dados obtidos nestes experimentos apontam que a transmissão vertical do vírus dengue ocorreu em pequenas taxas. Independente da forma de infecção utilizada, as taxas não variaram, mostrando que os mecanismos que regulam a ocorrência da transmissão vertical precisam ser explorados. Concluímos que, a Wolbachia pode potencialmente ser responsável pela redução da transmissão vertical em campo. Isto é considerado um grande avanço no controle da dengue, se realmente for comprovada que a transmissão vertical é a responsável pela manutenção do vírus em períodos inter-epidêmicos.Dengue is a growing medical problem in subtropical and tropical countries. Dengue is a viral infection which is known four distinct serotypes, DENV-1 to -4, recently being reported the fifth serotype DENV-5 in Malaysia. These are transmitted by the bite of infected mosquitoes of the genus Aedes, being the main vector Aedes aegypti. Currently, prevention and control of dengue rely solely on anti-vector measures, and these have to be inefficient, especially in developing countries. In this context, there is the need to search for new strategies that can be used concurrently with the forms of existing control. Wolbachia is an intracellular bacterium The widely known for promoting the virus lock phenotype in dengue mosquitoes A. aegypti. Despite this, it is not known if the Wolbachia is able to interfere in the progeny of infected mosquitoes, known as vertical transmission. Vertical transmission may be associated with the maintenance of the virus in inter-epidemic periods or active sites that transmission is decreased. The objective of this study was to evaluate whether Wolbachia introduced in the Brazilian strain of A. aegypti influences the vertical transmission of dengue. Two methods were used for infection: Oral nanoinjeção intrathoracic infection and, where used DENV-4 and DENV-1, -3 and -4 respectively. For these experiments, mosquitoes were compared with Wolbachia (Mel) and a strain of mosquitoes treated with antibiotics and therefore free of bacteria (Tet). The eggs of infected mosquitoes were collected over a period of 20 and 11 days after infection, orally and injections, respectively. The fecundity and fertility, in general, were reduced in the co-infection of Wolbachia with the dengue virus. In all infected mosquitoes, no differences were observed in susceptibility to the virus, but the viral load was reduced by Wolbachia in DENV-4 in both forms of the infection. Independent positive pools of detection was possible the form of infection, demonstrating that vertical transmission of dengue virus occurred, but the proportion of positive pools between Mel and Tet group was unchanged. However, we observed a decrease in viral load in positive pools Mel. Ovaries in the oral infection, interestingly, had a strong decrease in susceptibility conferred by Wolbachia. The viral load in the ovaries was decreased DENV-1 and DENV-4 in both forms of the infection. The data from these experiments indicate that vertical transmission of dengue virus occurred in small fees. Independent of the infection used, the rates did not change, showing that the mechanisms that regulate the occurrence of vertical transmission need to be explored. We conclude that the Wolbachia could potentially be responsible for the reduction of vertical transmission in the field. This is considered a major breakthrough in dengue control, if it is really proven that vertical transmission is responsible for virus maintenance in inter-epidemic period

<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

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

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

<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

<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

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