Wolbachia wAlbB inhibits bluetongue and epizootic hemorrhagic fever viruses in Culicoides midge cells.

Culicoides midges are hematophagous insects that transmit arboviruses of veterinary importance. These viruses include bluetongue virus (BTV) and epizootic hemorrhagic fever virus (EHDV). The endosymbiont Wolbachia pipientis Hertig spreads rapidly through insect host populations and has been demonstrated to inhibit viral pathogen transmission in multiple mosquito vectors. Here, we have demonstrated a replication inhibitory effect on BTV and EHDV in a Wolbachia (wAlbB strain)-infected Culicoides sonorensis Wirth and Jones W8 cell line. Viral replication was significantly reduced by day 5 for BTV and by day 2 for EHDV as detected by real-time polymerase chain reaction (RT-qPCR) of the non-structural NS3 gene of both viruses. Evaluation of innate cellular immune responses as a cause of the inhibitory effect showed responses associated with BTV but not with EHDV infection. Wolbachia density also did not play a role in the observed pathogen inhibitory effects, and an alternative hypothesis is suggested. Applications of Wolbachia-mediated pathogen interference to impact disease transmission by Culicoides midges are discussed.

The impact of non-lethal doses of pyriproxyfen on male and female Aedes albopictus reproductive fitness.

Introduction: Control of the mosquito Aedes albopictus is confounded by its behavior due to females preferring to oviposition in small natural and artificial containers that are often difficult to remove or treat with insecticides. Autodissemination strategies utilizing highly potent insect growth regulators (IGRs) have emerged as promising tools for the control of this container-inhabiting species. The intended goal of autodissemination approaches is to use mosquitoes to self-deliver an IGR to these cryptic oviposition locations. Previous studies have focused on the efficacy of these approaches to impact natural populations, but little focus has been placed on the impacts on mosquitoes when exposed to non-lethal doses of IGRs similar to the levels they would be exposed to with autodissemination approaches. Methods: In this study, the impact of non-lethal doses of pyriproxyfen (PPF) on the reproductive fitness of Ae. albopictus was investigated. Female and male Ae. albopictus mosquitoes were exposed to non-lethal doses of PPF and their fecundity and fertility were measured. To examine the impact of non-lethal doses of PPF, the expression of the ecdysone-regulated genes USP, HR3, and Vg, which are involved in vitellogenesis, was determined. Results: Our results demonstrated a significant reduction in female fecundity and in the blood feeding and egg hatching rates upon exposure to non-lethal doses of PPF. Oocyte development was also delayed in PPF-treated females. Furthermore, exposure to non-lethal doses of PPF altered the expression of the genes involved in vitellogenesis, indicating disruption of hormonal regulation. Interestingly, PPF exposure also reduced the sperm production in males, suggesting a potential semi-sterilization effect. Discussion: These findings suggest that non-lethal doses of PPF could enhance the efficacy of autodissemination approaches by impacting the reproductive fitness of both males and females. However, further research is needed to validate these laboratory findings in field settings and to assess their practical implications for vector control strategies.

The effects of nano- and microplastic ingestion on the survivorship and reproduction of Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Microplastics (MPs) and nanoplastics (NPs) are pervasive environmental pollutants that are commonly ingested by organisms at different trophic levels. While the effects of MPs on aquatic organisms have been extensively studied, the impacts of MP ingestion on the host fitness of terrestrial organisms, mainly insects, have been relatively unexplored. This study investigates the effects of MP and NP ingestion on the survivorship and reproduction of 2 medically important mosquito species, Aedes aegypti Linnaeus (Diptera: Culicidae) and Aedes albopictus Skuse (Diptera: Culicidae). Larval and pupal survivorship of Ae. albopictus were not significantly affected by particle size or concentration, but there was a reduction of Ae. aegypti pupal survivorship associated with the ingestion of 0.03 µm NPs. In addition, there was little observed impact of 0.03 µm NP and 1.0 µm MP ingestion on adult survivorship, fecundity, and longevity. To further investigate the effects of MP ingestion on mosquito fitness, we also examined the effects of MPs of varying shape, size, and plastic polymer type on Ae. aegypti immature and adult survivorship. The data suggest that the polymer type and shape did not impact Ae. aegypti immature or adult survivorship. These findings highlight that understanding the effects of microplastic ingestion by mosquitoes may be complicated by the size, composition, and amount ingested.

Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics.

BACKGROUND: Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60). RESULTS: MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17). CONCLUSIONS: High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda.

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans.

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.

Population impacts of Wolbachia on Aedes albopictus.

Prior studies have demonstrated that Wolbachia, a commonly occurring bacterium capable of manipulating host reproduction, can affect life history traits in insect hosts, which in turn can have population-level effects. Effects on hosts at the individual level are predicted to impact population dynamics, but the latter has not been examined empirically. Here, we describe a biological model system based on Aedes albopictus (Asian tiger mosquito) that allows for measurement of population dynamics, which has not been accomplished in prior field trials or laboratory designs. The results demonstrate the studied populations to be robust and allow for persistent, closed populations with overlapping generations, which are regulated solely through density-dependent, intraspecific competition for limited resources. Using a novel experimental design, we compare populations that are either uninfected or infected with Wolbachia. The results show differences that include population size, eclosion rates, adult survivorship, and fecundity. The aposymbiotic populations were generally larger and adults longer lived relative to the infected populations. The outcome is discussed in context with naturally occurring Wolbachia invasions, proposed autocidal strategies, and the utility of the developed system as a biological platform for hypothesis testing and improved parameterization.

Intercommunity effects on microbiome and GpSGHV density regulation in tsetse flies.

Tsetse flies have a highly regulated and defined microbial fauna made of 3 bacterial symbionts (obligate Wigglesworthia glossinidia, commensal Sodalis glossinidius and parasitic Wolbachia pipientis) in addition to a DNA virus (Glossina pallidipes Salivary gland Hypertrophy Virus, GpSGHV). It has been possible to rear flies in the absence of either Wigglesworthia or in totally aposymbiotic state by dietary supplementation of tsetse's bloodmeal. In the absence of Wigglesworthia, tsetse females are sterile, and adult progeny are immune compromised. The functional contributions for Sodalist are less known, while Wolbachia cause reproductive manupulations known as cytoplasmic incompatibility (CI). High GpSGHV virus titers result in reduced fecundity and lifespan, and have compromised efforts to colonize flies in the insectary for large rearing purposes. Here we investigated the within community effects on the density regulation of the individual microbiome partners in tsetse lines with different symbiotic compositions. We show that absence of Wigglesworthia results in loss of Sodalis in subsequent generations possibly due to nutritional dependancies between the symbiotic partners. While an initial decrease in Wolbachia and GpSGHV levels are also noted in the absence of Wigglesworthia, these infections eventually reach homeostatic levels indicating adaptations to the new host immune environment or nutritional ecology. Absence of all bacterial symbionts also results in an initial reduction of viral titers, which recover in the second generation. Our findings suggest that in addition to the host immune system, interdependencies between symbiotic partners result in a highly tuned density regulation for tsetse's microbiome.

Tsetse-Wolbachia symbiosis: comes of age and has great potential for pest and disease control.

Tsetse flies (Diptera: Glossinidae) are the sole vectors of African trypanosomes, the causative agent of sleeping sickness in human and nagana in animals. Like most eukaryotic organisms, Glossina species have established symbiotic associations with bacteria. Three main symbiotic bacteria have been found in tsetse flies: Wigglesworthia glossinidia, an obligate symbiotic bacterium, the secondary endosymbiont Sodalis glossinidius and the reproductive symbiont Wolbachia pipientis. In the present review, we discuss recent studies on the detection and characterization of Wolbachia infections in Glossina species, the horizontal transfer of Wolbachia genes to tsetse chromosomes, the ability of this symbiont to induce cytoplasmic incompatibility in Glossina morsitans morsitans and also how new environment-friendly tools for disease control could be developed by harnessing Wolbachia symbiosis.

Microplastic ingestion perturbs the microbiome of Aedes albopictus (Diptera: Culicidae) and Aedes aegypti.

Microplastics (MPs) are common environmental pollutants; however, little is known about their effects after ingestion by insects. Here we fed Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) mosquito larvae 1 µm polystyrene MPs and examined the impacts of ingestion on adult emergence rates, gut damage, and fungal and bacterial microbiota. Results show that MPs accumulate in the larval guts, resulting in gut damage. However, little impact on adult emergence rates was observed. MPs are also found in adult guts postemergence from the pupal stage, and adults expel MPs in their frass after obtaining sugar meals. Moreover, MPs effects on insect microbiomes need to be better defined. To address this knowledge gap, we investigated the relationship between MP ingestion and the microbial communities in Ae. albopictus and Ae. aegypti. The microbiota composition was altered by the ingestion of increasing concentrations of MPs. Amplicon sequence variants (ASVs) that contributed to differences in the bacterial and fungal microbiota composition between MP treatments were from the genera Elizabethkingia and Aspergillus, respectively. Furthermore, a decrease in the alpha diversity of the fungal and bacterial microbiota was observed in treatments where larvae ingested MPs. These results highlight the potential for the bacterial and fungal constituents in the mosquito microbiome to respond differently to the ingestion of MPs. Based on our findings and the effects of MP ingestion on the mosquito host micro- and mycobiome, MP pollution could impact the vector competence of important mosquito-transmitted viruses and parasites that cause human and animal diseases.

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda.

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.

Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina).

BACKGROUND: Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. RESULTS: In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome. CONCLUSIONS: Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.

Nectar Source and Pollinator Cross Contamination and Persistence of Pyriproxyfen Associated with the Use of Autodissemination Techniques for Mosquito Control.

Controlling container inhabiting mosquitoes such as Aedes aegypti and Ae. albopictus is often difficult because of the requirement to treat small and inaccessible cryptic sources of water where larvae are located. Autodissemination approaches based on the dissemination of insect growth regulators (IGRs) have been demonstrated as an effective means to treat these cryptic larval habitats and provide population control. Autodissemination approaches are attractive because they are based on the mosquitoes disseminating small amounts of IGRs compared to more traditional insecticide applications. While dissemination of small amounts of IGRs seems like an advantage, these approaches could lead to unintended transfer and effects on nontarget insect pollinators by delivering highly potent IGRs to nectar sources. Here we looked for the indirect and direct transfer of pyriproxyfen (PPF) to natural and artificial nectar sources and painted lady butterflies, Vanessa cardui, in semifield cages using the release of treated Ae. albopictus males or an autodissemination station. We also performed persistence tests of PPF in oviposition containers and natural and artificial nectar sources when exposed to laboratory and natural conditions. The data suggest that there is direct and indirect transfer to nectar sources and V. cardui associated with the use of autodissemination approaches. We discuss the results in the context of using autodissemination approaches for mosquito control and the potential risks these approaches may pose to nontarget insect pollinators.

Wolbachia Impacts Anaplasma Infection in Ixodes scapularis Tick Cells.

The specific interactions of members of tick bacterial microbiota and their effects on pathogen transmission remains relatively unexplored. Here, we introduced a novel Wolbachia infection type into Ixodes scapularis tick cells and examined the antipathogenic effects on the intracellular pathogen Anaplasma phagocytophilum. An increase in A. phagocytophilum replication was observed in Wolbachia-infected tick cells. However, Wolbachia infection densities decreased when cells were serially passaged and ultimately the infection was lost. Host-cell immune response was also examined as an additional factor that could have affected A. phagocytophilum replication in Wolbachia-infected cells. In early passages post-Wolbachia infection, a decreased immune response was observed, but in later passages of cells with low Wolbachia densities, there was no change in the immune response. The results are discussed in relation to the importance of studying the interactions of the tick microbiota, the host cell, and the pathogen and the development of novel tick and tick-borne disease-control approaches.

Next-generation tools to control biting midge populations and reduce pathogen transmission.

Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches.

Indirect transfer of pyriproxyfen to European honeybees via an autodissemination approach.

The frequency of arboviral disease epidemics is increasing and vector control remains the primary mechanism to limit arboviral transmission. Container inhabiting mosquitoes such as Aedes albopictus and Aedes aegypti are the primary vectors of dengue, chikungunya, and Zika viruses. Current vector control methods for these species are often ineffective, suggesting the need for novel control approaches. A proposed novel approach is autodissemination of insect growth regulators (IGRs). The advantage of autodissemination approaches is small amounts of active ingredients compared to traditional insecticide applications are used to impact mosquito populations. While the direct targeting of cryptic locations via autodissemination seems like a significant advantage over large scale applications of insecticides, this approach could actually affect nontarget organisms by delivering these highly potent long lasting growth inhibitors such as pyriproxyfen (PPF) to the exact locations that other beneficial insects visit, such as a nectar source. Here we tested the hypothesis that PPF treated male Ae. albopictus will contaminate nectar sources, which results in the indirect transfer of PPF to European honey bees (Apis mellifera). We performed bioassays, fluorescent imaging, and mass spectrometry on insect and artificial nectar source materials to examine for intra- and interspecific transfer of PPF. Data suggests there is direct transfer of PPF from Ae. albopictus PPF treated males and indirect transfer of PPF to A. mellifera from artificial nectar sources. In addition, we show a reduction in fecundity in Ae. albopictus and Drosophila melanogaster when exposed to sublethal doses of PPF. The observed transfer of PPF to A. mellifera suggests the need for further investigation of autodissemination approaches in a more field like setting to examine for risks to insect pollinators.

Phenotypic Response of Wolbachia pipientis in a Cell-Free Medium.

Wolbachia, an obligate intracellular bacterium estimated to infect millions of arthropod species worldwide, is currently being utilized in novel control strategies to limit the transmission of Dengue and Zika viruses. A limitation for Wolbachia-based control approaches is the difficulty of transferring Wolbachia to novel hosts and the lack of tools for the genetic transformation of Wolbachia due to the inability to culture Wolbachia outside the insect host cell in an axenic media. Here, we applied extracellular Wolbachia to phenotypic microarrays to measure the metabolic response of Wolbachia in media formulations with different pH levels and supplementation with Casamino acids. Results suggested a pH of 6.5-6.8 and showed that the supplementation of 1 mg/mL casamino acids increased the survival and longevity of Wolbachia in an axenic medium. In addition, phenotypic microarrays are a useful tool to measure the phenotypic response of Wolbachia under different media conditions, as well as determine specific components that may be required for an axenic medium. This study is an initial step toward the development of a potential Wolbachia axenic culture system.

Cryptic Wolbachia (Rickettsiales: Rickettsiaceae) Detection and Prevalence in Culicoides (Diptera: Ceratopogonidae) Midge Populations in the United States.

Culicoides midges vector numerous veterinary and human pathogens. Many of these diseases lack effective therapeutic treatments or vaccines to limit transmission. The only effective approach to limit disease transmission is vector control. However, current vector control for Culicoides midges is complicated by the biology of many Culicoides species and is not always effective at reducing midge populations and impacting disease transmission. The endosymbiont Wolbachia pipientis Hertig may offer an alternative control approach to limit disease transmission and affect Culicoides populations. Here the detection of Wolbachia infections in nine species of Culicoides midges is reported. Infections were detected at low densities using qPCR. Wolbachia infections were confirmed with the sequencing of a partial region of the 16S gene. Fluorescence in situ hybridization of Culicoides sonorensis Wirth and Jones adults and dissected ovaries confirm the presence of Wolbachia infections in an important vector of Bluetongue and Epizootic hemorrhagic disease viruses. The presence of Wolbachia in Culicoides populations in the United States suggests the need for further investigation of Wolbachia as a strategy to limit transmission of diseases vectored by Culicoides midges.

Transfection of Culicoides sonorensis biting midge cell lines with Wolbachia pipientis.

BACKGROUND: Biting midges of the genus Culicoides vector multiple veterinary pathogens and are difficult to control. Endosymbionts particularly Wolbachia pipientis may offer an alternative to control populations of Culicoides and/or impact disease transmission in the form of population suppression or replacement strategies. METHODS: Culicoides sonorensis cell lines were transfected with a Wolbachia infection using a modified shell vial technique. Infections were confirmed using PCR and cell localization using fluorescent in situ hybridization (FISH). The stability of Wolbachia infections and density was determined by qPCR. qPCR was also used to examine immune genes in the IMD, Toll and JACK/STAT pathways to determine if Wolbachia were associated with an immune response in infected cells. RESULTS: Here we have transfected two Culicoides sonorensis cell lines (W3 and W8) with a Wolbachia infection (walbB) from donor Aedes albopictus Aa23 cells. PCR and FISH showed the presence of Wolbachia infections in both C. sonorensis cell lines. Infection densities were higher in the W8 cell lines when compared to W3. In stably infected cells, genes in the immune Toll, IMD and JAK/STAT pathways were upregulated, along with Attacin and an Attacin-like anti-microbial peptides. CONCLUSIONS: The successful introduction of Wolbachia infections in C. sonorensis cell lines and the upregulation of immune genes, suggest the utility of using Wolbachia for a population replacement and/or population suppression approach to limit the transmission of C. sonorensis vectored diseases. Results support the further investigation of Wolbachia induced pathogen inhibitory effects in Wolbachia-infected C. sonorensis cell lines and the introduction of Wolbachia into C. sonorensis adults via embryonic microinjection to examine for reproductive phenotypes and host fitness effects of a novel Wolbachia infection.

Aedes aegypti Males as Vehicles for Insecticide Delivery.

Aedes aegypti continues to spread globally and remains a challenge to control, in part due to its 'cryptic behavior' in that it often deposits eggs (oviposits) in larval habitats that are difficult to find and treat using traditional methods. Auto-dissemination strategies target these cryptic breeding sites by employing mosquitoes to deliver lethal doses of insecticide. This report describes the initial field trials of an application known as Autodissemination Augmented by Males (ADAM), utilizing A. aegypti males dusted with pyriproxyfen (PPF). Findings presented here are drawn from both caged and field trial studies. Together, these trials examined for the ability of A. aegypti males to disseminate PPF and to impact field populations. PPF-dusted males were able to effectively deliver lethal doses of PPF to oviposition sites under the conditions tested. Results from field trials in Florida and California demonstrated reduced A. aegypti populations in treated areas, compared to areas where PPF-treated males were not released. These results indicate that the release of PPF-dusted A. aegypti males can impact A. aegypti populations as measured by both reduced larval survival and lower numbers of adult female A. aegypti. We propose the ADAM approach as an addition to existing mosquito control techniques targeting A. aegypti and other mosquitoes that utilize cryptic larval habitats.

Sequence analysis of the rDNA internal transcribed spacer 2 and polymerase chain reaction identification of Anopheles fluminensis (Diptera: Culicidae: Anopheles) in Bolivia.

Anopheles fluminensis Root is a member of the Arribalzagia Series in the subgenus Anopheles. We report the first record of this species in the department of Cochabamba, Bolivia. This species was sampled from two locations in the foothills of the eastern Andes Mountains within the Chapare Valley. Larvae were collected in fast-flowing, shaded streams at the edges of rocky pools. We provide the first sequence data for the rDNA of An. fluminensis, a partial sequence of the 5.8S and the internal transcribed spacer 2 (ITS2). The ITS2 of An. fluminensis, sequenced from two individuals at one site, was at least 596 bp, had 56.5% GC, and included three large repeats (approximately equal to 125 bp each). We describe a polymerase chain reaction protocol and species-specific primers for identifying this species in the Chapare Valley, Bolivia.