16S rRNA gene-based identification of microbiota associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga (Diptera, Psychodidae) from central Amazon, Brazil

Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria. © 2013, Sociedade Brasileira de Microbiologia

Occurrence records and metadata for sand flies (Diptera, Psychodidae, Phlebotominae) collected in the lands of indigenous people in the Brazilian Amazon

In order to contribute to knowledge of the epidemiology of American cutaneous leishmaniasis (ACL) among indigenous people living in sylvatic regions, we studied the sand fly fauna collected in areas of disease transmission in the Brazilian Amazon. Our two datasets reported here are comprised of occurrence data for sand flies from the Suruwaha Indigenous Land in the state of Amazonas collected between 2012-1013, and the Wajãpi Indigenous Land in the state of Amapá collected between 2013-2014. Sand flies were collected using unbaited CDC-like light traps at various sites within each study area and were identified to species-level by taxonomists with expertise in Amazonian fauna. A total of 4,646 records are reported: 1,428 from the Suruwaha and 3,218 from the Wajãpi. These records will contribute to a better understanding of ACL transmission dynamics, as well as the distribution of insect vectors, in these areas

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of São Paulo, Brazil

<p>Abstract</p> <p>Background</p> <p>American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of São Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of São Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area.</p> <p>Results</p> <p>A database containing 910 individual records of sand fly occurrence in the state of São Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (<it>Nyssomyia intermedia</it>, <it>N. neivai</it>, <it>N. whitmani</it>, <it>Pintomyia fischeri</it>, <it>P. pessoai </it>and <it>Migonemyia migonei</it>). The distribution of each of the six incriminated or suspected vector species of ACL in the state of São Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - <it>Nyssomyia intermedia </it>and <it>N. neivai </it>- are highly localized, while the other sand fly species - <it>N. whitmani, M. migonei, P. fischeri </it>and <it>P. pessoai </it>- are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL.</p> <p>Conclusions</p> <p>The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of São Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of São Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.</p

Comparative infectivity of Plasmodium falciparum to Anopheles albimanus and Anopheles stephensi

The infectivity of three different clones of the human malaria parasite Plasmodium falciparum to two different natural vector mosquito species, Anopheles albimanus and Anopheles stephensi was investigated. Two of the P. falciparum clones studied (HB3B-B2 and 7G8) established relatively low levels of mature oocyst infection in both mosquito species. In contrast, the third P. falciparum clone investigated (3D7A) did not produce mature oocyst infections in An. albimanus but infected stephensi at a relatively high level. These observations demonstrate the existence of differences between the three malaria parasite clones iii the ability to infect the mosquitoes, and the two mosquito species in their susceptibility to malaria parasite infection. Direct immunofluorescence microscopy and examination of Giemsa-stained histological sections by light microscopy were used to investigate further the development of the P. falciparum 3D7A clone in An. albimanus and An. stephensi. The P. falciparum 3D7A clone formed mature ookinetes within the bloodmeals of both albimanus and An. stephensi. In An. albimanus, these malaria parasite stages subsequently failed to migrate from the bloodmeal and invade the surrounding midgut epithelium suggesting that ookinetes were destroyed within the endoperitrophic space of this mosquito species. The reasons for the disappearance of ookinetes within the endoperitrophic space of An. albimanus were uncertain but were possibly related to the faster time to completion of bloodmeal digestion in this mosquito species compared to An. stephensi. Simultaneous investigation of P. falciparum 3D7A development within An. Stephensi revealed that in this mosquito species ookinetes entered the midgut epithelium via an intracellular route, causing destruction of invaded midgut epithelial cells, and subsequently exited the midgut epithelium by an intercellular route. A general model for the route of ookinete invasion across the midgut epithelium is proposed based upon these observations. Examination of the Giemsa-stained histological sections also provided evidence that regenerative cells within the midgut epithelium of An. stephensi imdergo proliferation and differentiation into midgut epithelial cells following infection with P. falciparum 3D7A, presumably as a mechanism to replace the midgut epithelial cells destroyed as a consequence of ookinete invasion of the midgut epithelium

<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

<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

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

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