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.

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.

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 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.

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.

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.

Female Adult Aedes albopictus Suppression by Wolbachia-Infected Male Mosquitoes.

Dengue, chikungunya and zika viruses are pathogens with an increasing global impact. In the absence of an approved vaccine or therapy, their management relies on controlling the mosquito vectors. But traditional controls are inadequate, and the range of invasive species such as Aedes albopictus (Asian Tiger Mosquito) is expanding. Genetically modified mosquitoes are being tested, but their use has encountered regulatory barriers and public opposition in some countries. Wolbachia bacteria can cause a form of conditional sterility, which can provide an alternative to genetic modification or irradiation. It is unknown however, whether openly released, artificially infected male Ae. albopictus can competitively mate and sterilize females at a level adequate to suppress a field population. Also, the unintended establishment of Wolbachia at the introduction site could result from horizontal transmission or inadvertent female release. In 2014, an Experimental Use Permit from the United States Environmental Protection Agency approved a pilot field trial in Lexington, Kentucky, USA. Here, we present data showing localized reduction of both egg hatch and adult female numbers. The artificial Wolbachia type was not observed to establish in the field. The results are discussed in relation to the applied use of Wolbachia-infected males as a biopesticide to suppress field populations of Ae. albopictus.

Male mosquitoes as vehicles for insecticide.

BACKGROUND: The auto-dissemination approach has been shown effective at treating cryptic refugia that remain unaffected by existing mosquito control methods. This approach relies on adult mosquito behavior to spread larvicide to breeding sites at levels that are lethal to immature mosquitoes. Prior studies demonstrate that 'dissemination stations,' deployed in mosquito-infested areas, can contaminate adult mosquitoes, which subsequently deliver the larvicide to breeding sites. In some situations, however, preventative measures are needed, e.g., to mitigate seasonal population increases. Here we examine a novel approach that combines elements of autocidal and auto-dissemination strategies by releasing artificially reared, male mosquitoes that are contaminated with an insecticide. METHODOLOGY: Laboratory and field experiments examine for model-predicted impacts of pyriproxyfen (PPF) directly applied to adult male Aedes albopictus, including (1) the ability of PPF-treated males to cross-contaminate females and to (2) deliver PPF to breeding sites. PRINCIPAL FINDINGS: Similar survivorship was observed in comparisons of PPF-treated and untreated males. Males contaminated both female adults and oviposition containers in field cage tests, at levels that eliminated immature survivorship. Field trials demonstrate an ability of PPF-treated males to transmit lethal doses to introduced oviposition containers, both in the presence and absence of indigenous females. A decline in the Ae. albopictus population was observed following the introduction of PPF-treated males, which was not observed in two untreated field sites. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that, in cage and open field trials, adult male Ae. albopictus can tolerate PPF and contaminate, either directly or indirectly, adult females and immature breeding sites. The results support additional development of the proposed approach, in which male mosquitoes act as vehicles for insecticide delivery, including exploration of the approach with additional medically important mosquito species. The novelty and importance of this approach is an ability to safely achieve auto-dissemination at levels of intensity that may not be possible with an auto-dissemination approach that is based on indigenous females. Specifically, artificially-reared males can be released and sustained at any density required, so that the potential for impact is limited only by the practical logistics of mosquito rearing and release, rather than natural population densities and the self-limiting impact of an intervention upon them.

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.

Open release of male mosquitoes infected with a wolbachia biopesticide: field performance and infection containment.

BACKGROUND: Lymphatic filariasis (LF) is a globally significant disease, with 1.3 billion persons in 83 countries at risk. A coordinated effort of administering annual macrofilaricidal prophylactics to the entire at-risk population has succeeded in impacting and eliminating LF transmission in multiple regions. However, some areas in the South Pacific are predicted to persist as transmission sites, due in part to the biology of the mosquito vector, which has led to a call for additional tools to augment drug treatments. Autocidal strategies against mosquitoes are resurging in the effort against invasive mosquitoes and vector borne disease, with examples that include field trials of genetically modified mosquitoes and Wolbachia population replacement. However, critical questions must be addressed in anticipation of full field trials, including assessments of field competitiveness of transfected males and the risk of unintended population replacement. METHODOLOGY/PRINCIPAL FINDINGS: We report the outcome of field experiments testing a strategy that employs Wolbachia as a biopesticide. The strategy is based upon Wolbachia-induced conditional sterility, known as cytoplasmic incompatibility, and the repeated release of incompatible males to suppress a population. A criticism of the Wolbachia biopesticide approach is that unintended female release or horizontal Wolbachia transmission can result in population replacement instead of suppression. We present the outcome of laboratory and field experiments assessing the competitiveness of transfected males and their ability to transmit Wolbachia via horizontal transmission. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that Wolbachia-transfected Aedes polynesiensis males are competitive under field conditions during a thirty-week open release period, as indicated by mark, release, recapture and brood-hatch failure among females at the release site. Experiments demonstrate the males to be 'dead end hosts' for Wolbachia and that methods were adequate to prevent population replacement at the field site. The findings encourage the continued development and extension of a Wolbachia autocidal approach to additional medically important mosquito species.

Population genetic structure of Aedes polynesiensis in the Society Islands of French Polynesia: implications for control using a Wolbachia-based autocidal strategy.

BACKGROUND: Aedes polynesiensis is the primary vector of Wuchereria bancrofti in the South Pacific and an important vector of dengue virus. An improved understanding of the mosquito population genetics is needed for insight into the population dynamics and dispersal, which can aid in understanding the epidemiology of disease transmission and control of the vector. In light of the potential release of a Wolbachia infected strain for vector control, our objectives were to investigate the microgeographical and temporal population genetic structure of A. polynesiensis within the Society Islands of French Polynesia, and to compare the genetic background of a laboratory strain intended for release into its population of origin. METHODS: A panel of eight microsatellite loci were used to genotype A. polynesiensis samples collected in French Polynesia from 2005-2008 and introgressed A. polynesiensis and Aedes riversi laboratory strains. Examination of genetic differentiation was performed using F-statistics, STRUCTURE, and an AMOVA. BAYESASS was used to estimate direction and rates of mosquito movement. RESULTS: FST values, AMOVA, and STRUCTURE analyses suggest low levels of intra-island differentiation from multiple collection sites on Tahiti, Raiatea, and Maupiti. Significant pair-wise FST values translate to relatively minor levels of inter-island genetic differentiation between more isolated islands and little differentiation between islands with greater commercial traffic (i.e., Tahiti, Raiatea, and Moorea). STRUCTURE analyses also indicate two population groups across the Society Islands, and the genetic makeup of Wolbachia infected strains intended for release is similar to that of wild-type populations from its island of origin, and unlike that of A. riversi. CONCLUSIONS: The observed panmictic population on Tahiti, Raiatea, and Moorea is consistent with hypothesized gene flow occurring between islands that have relatively high levels of air and maritime traffic, compared to that of the more isolated Maupiti and Tahaa. Gene flow and potential mosquito movement is discussed in relation to trials of applied autocidal strategies.

Integration of irradiation with cytoplasmic incompatibility to facilitate a lymphatic filariasis vector elimination approach.

BACKGROUND: Mass drug administration (MDA) is the emphasis of an ongoing global lymphatic filariasis (LF) elimination program by the World Health Organization, in which the entire 'at risk' human population is treated annually with anti-filarial drugs. However, there is evidence that the MDA strategy may not be equally appropriate in all areas of LF transmission, leading to calls for the augmentation of MDA with anti-vector interventions. One potential augmentative intervention is the elimination of vectors via repeated inundative releases of male mosquitoes made cytoplasmically incompatible via an infection with Wolbachia bacteria. However, with a reduction in the vector population size, there is the risk that an accidental female release would permit the establishment of the incompatible Wolbachia infection type, resulting in population replacement instead of population elimination. To avoid the release of fertile females, we propose the exposure of release individuals to low doses of radiation to sterilize any accidentally released females, reducing the risk of population replacement. RESULTS: Aedes polynesiensis pupae of differing ages were irradiated to determine a radiation dose that results in sterility but that does not affect the survival and competitiveness of males. Laboratory assays demonstrate that males irradiated at a female sterilizing dosage of 40 Gy are equally competitive with un-irradiated males. No effect of irradiation on the ability of Wolbachia to affect egg hatch was observed. CONCLUSION: An irradiation dose of 40 Gy is sufficient to cause female sterility, but has no observed negative effect on male fitness. The results support further development of this approach as a preventative measure against accidental population replacement.

Interspecific hybridization yields strategy for South Pacific filariasis vector elimination.

BACKGROUND: Lymphatic filariasis (LF) is a leading cause of disability in South Pacific regions, where >96% of the 1.7 million population are at risk of LF infection. As part of current global campaign, mass drug administration (MDA) has effectively reduced lymphatic filiariasis prevalence, but mosquito vector biology can complicate the MDA strategy. In some regions, there is evidence that the goal of LF elimination cannot be attained via MDA alone. Obligate vector mosquitoes provide additional targets for breaking the LF transmission cycle, but existing methods are ineffective for controlling the primary vector throughout much of the South Pacific, Aedes polynesiensis. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that interspecific hybridization and introgression results in an A. polynesiensis strain ('CP' strain) that is stably infected with the endosymbiotic Wolbachia bacteria from Aedes riversi. The CP strain is bi-directionally incompatible with naturally infected mosquitoes, resulting in female sterility. Laboratory assays demonstrate that CP males are equally competitive, resulting in population elimination when CP males are introduced into wild type A. polynesiensis populations. CONCLUSIONS/SIGNIFICANCE: The findings demonstrate strategy feasibility and encourage field tests of the vector elimination strategy as a supplement to ongoing MDA efforts.

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.