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The current review of the Sterile Insect Technique (SIT) is motivated by new technologies and the recent renaissance of male release field trials, which is driving an evolution in mosquito control and regulation. Practitioners that are releasing male mosquitoes would do well to learn from past successes and failures, including political and public engagement complications. With examples that include nuanced integrations of the different technologies, e.g., combinations of Wolbachia and irradiation, it is critical that scientists understand and communicate accurately about the technologies, including their evolving management by different regulatory agencies in the USA. Some male release approaches are considered 'pesticides' and regulated by federal and state agencies, while other male release approaches are unregulated. It is important to consider how the new technologies fit with the more 'traditional' chemical applications of adulticides and larvicides. The economics of male release programs are substantially different from traditional control costs, which can be a challenge to their adoption by abatement districts. However, there is substantial need to overcome these complications and challenges, because the problem with invasive mosquitoes grows ever worse with factors that include insecticide resistance, globalization and climate change., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6-96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs.

As part of the response to autochthonous Zika transmission in the United States, the City of South Miami implemented a 6-mo period in which Wolbachia-infected WB1 Aedes aegypti (L.) males were released into an ~170-acre area. Intracellular Wolbachia bacteria infections in Ae. aegypti cause early embryonic arrest (known as cytoplasmic incompatibility [CI]) and egg hatch failure, and inundative introductions have been suggested as a potential control tool. Throughout the release period, the Ae. aegypti population was monitored within both the release area and an equivalent area that did not receive WB1 male releases. The results show a significant reduction in egg hatch at the area receiving WB1 males, which is consistent with expectations for CI. Similarly, the number of Ae. aegypti was significantly reduced at the area receiving WB1 males, relative to the untreated area. The observed population reduction and results encourage additional work and replication of the Wolbachia biopesticide approach against Ae. aegypti, as an additional tool to be integrated with existing control tools for the control of this medically important vector and nuisance pest., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.

Recent efforts in controlling mosquito-borne diseases focus on biocontrol strategies that incapacitate pathogens inside mosquitoes by altering the mosquito's microbiome. A case in point is the introduction of Wolbachia into natural mosquito populations in order to eliminate Dengue virus. However, whether this strategy can successfully control vector-borne diseases is debated; particularly, how artificial infection affects population dynamics of hosts remains unclear. Here, we show that natural Wolbachia infections are associated with unstable mosquito population dynamics by contrasting Wolbachia-infected versus uninfected cage populations of the Asian tiger mosquito (Aedes albopictus). By analyzing weekly data of adult mosquito abundances, we found that the variability of the infected populations is significantly higher than that of the uninfected. The elevated population variability is explained by increased instability in dynamics, as quantified by system nonlinearity (i.e., state-dependence). In addition, predictability of infected populations is substantially lower. A mathematical model analysis suggests that Wolbachia may alter mosquito population dynamics by modifying larval competition of hosts. These results encourage examination for effects of artificial Wolbachia establishment on mosquito populations, because an enhancement of population variability with reduced predictability could pose challenges in management. Our findings have implications for application of microbiome alterations in biocontrol programs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Wolbachia bacteria are being introduced into natural populations of vector mosquitoes, with the goal of reducing the transmission of human diseases such as Zika and dengue fever. The successful establishment of Wolbachia infection is largely dependent on the effects of Wolbachia infection to host fitness, but the effects of Wolbachia infection on the individual life-history traits of immature mosquitoes can vary. Here, the effects of life-shortening Wolbachia (wMelPop) on population growth of infected individuals were evaluated by measuring larval survival, developmental time and adult size of Aedes aegypti in intra- (infected or uninfected only) and inter-group (mixed with infected and uninfected) larval competition assays. At low larval density conditions, the population growth of wMelPop infected and uninfected individuals was similar. At high larval densities, wMelPop infected individuals had a significantly reduced population growth rate relative to uninfected individuals, regardless of competition type. We discuss the results in relation to the invasion of the wMelPop Wolbachia infection into naturally uninfected populations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Wolbachia are maternally transmitted obligate bacteria that occur naturally in many arthropods. The phenotype observed in mosquitoes is known as cytoplasmic incompatibility (CI), which results in reduced or absent egg hatch in crosses between individuals with different infection types. Applied mosquito control strategies propose that by releasing individuals infected with a Wolbachia strain that differs from that in the natural host population, CI could be used to suppress or replace mosquito populations. Here, using tetracycline treatment and embryonic microinjection, Aedes albopictus (Skuse) was cleared of its natural Wolbachia infection and artificially infected with a Wolbachia strain originating from Aedes riversi Bohart & Ingram. Crossing experiments were carried out to determine whether CI could be observed between the artificially infected strain (UC), naturally infected (wild type), and uninfected strains of Ae. albopictus. Crosses between UC males and uninfected females resulted in no egg hatch, a classic unidirectional CI pattern. Crosses between the wild-type and UC strain also exhibited a unidirectional pattern of CI, demonstrating that the UC strain is compatible with both of the Wolbachia types that occur within Ae. albopictus and that wild-type Wolbachia infections are unable to fully rescue the UC Wolbachia type. Crosses between the UC strain and another artificially infected Ae. albopictus strain (ARwP), were bidirectionally incompatible, demonstrating that the UC strain is not compatible with all Wolbachia types. The CI patterns observed in this study were atypical and the opposite of that typically observed with superinfections., (© 2014 Entomological Society of America.)

Most human filarial nematode parasites and arthropods are hosts for a bacterial endosymbiont, Wolbachia. In filaria, Wolbachia are required for normal development, fertility and survival, whereas in arthropods, they are largely parasitic and can influence development and reproduction, but are generally not required for host survival. Due to their obligate nature in filarial parasites, Wolbachia have been a target for drug discovery initiatives using several approaches including diversity and focused library screening and genomic sequence analysis. In vitro and in vivo anti-Wolbachia antibiotic treatments have been shown to have adulticidal activity, a long sought goal of filarial parasite drug discovery. In mosquitoes, it has been shown that the presence of Wolbachia can inhibit the transmission of certain viruses, such as Dengue, Chikungunya, Yellow Fever, West Nile, as well as the infectivity of the malaria-causing protozoan, Plasmodium and filarial nematodes. Furthermore, Wolbachia can cause a form of conditional sterility that can be used to suppress populations of mosquitoes and additional medically important insects. Thus Wolbachia, a pandemic endosymbiont offers great potential for elimination of a wide-variety of devastating human diseases., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Mosquito species, members of the genera Aedes, Anopheles and Culex, are the major vectors of human pathogens including protozoa (Plasmodium sp.), filariae and of a variety of viruses (causing dengue, chikungunya, yellow fever, West Nile). There is lack of efficient methods and tools to treat many of the diseases caused by these major human pathogens, since no efficient vaccines or drugs are available; even in malaria where insecticide use and drug therapies have reduced incidence, 219 million cases still occurred in 2010. Therefore efforts are currently focused on the control of vector populations. Insecticides alone are insufficient to control mosquito populations since reduced susceptibility and even resistance is being observed more and more frequently. There is also increased concern about the toxic effects of insecticides on non-target (even beneficial) insect populations, on humans and the environment. During recent years, the role of symbionts in the biology, ecology and evolution of insect species has been well-documented and has led to suggestions that they could potentially be used as tools to control pests and therefore diseases. Wolbachia is perhaps the most renowned insect symbiont, mainly due to its ability to manipulate insect reproduction and to interfere with major human pathogens thus providing new avenues for pest control. We herein present recent achievements in the field of mosquito-Wolbachia symbiosis with an emphasis on Aedes albopictus. We also discuss how Wolbachia symbiosis can be harnessed for vector control as well as the potential to combine the sterile insect technique and Wolbachia-based approaches for the enhancement of population suppression programs., (Copyright © 2013 International Atomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved.)

Wolbachia are maternally inherited intracellular bacteria that frequently infect a diverse range of arthropod species. Empirical and theoretical studies examining Wolbachia invasiveness have emphasized Wolbachia effects on adult hosts, but recent studies show that Wolbachia impacts on immature hosts can be important also. Here, we have examined for effects of Wolbachia infection in Aedes aegypti. Specifically, differential survivorship is observed when young larvae (1st instar) are exposed to older Aedes albopictus larvae (4th instar) or con-specific larvae. In an additional experiment, we have examined for differential behavior and observed that Wolbachia-infected larvae differ from uninfected larvae in their reaction to light stimulation. Our results support a hypothesized effect of Wolbachia on A. aegypti larval behavior. The results are discussed in relation to the ability of Wolbachia to invade natural populations and recently applied public health strategies that target the replacement or suppression of this important disease vector., (Published by Elsevier Inc.)

Recent releases of mosquitoes infected with different Wolbachia types into Australia have led to conditions that have not been observed naturally and that have not been considered theoretically. In an ongoing public health campaign, two Wolbachia infection types have been introduced, and the infections differ in their fitness cost imposed on the host mosquito. The dynamics of each infection type as it interacts with the indigenous uninfected host population have been previously examined via models and empirical tests. Here we conduct a modeling analysis to predict the dynamics when the two infection types do not remain geographically isolated. The results demonstrate the potential replacement and loss of the more costly infection type, which may not be the desired public health outcome.

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.

Background: Cytoplasmic incompatibility (CI) is induced in nature by Wolbachia bacteria, resulting in conditional male sterility. Previous research demonstrated that the two Wolbachia strains (wAlbA and wAlbB) that naturally co-infect the disease vector mosquito Aedes albopictus (Asian tiger mosquito) can be replaced with the wPip Wolbachia strain from Culex pipiens. Since Wolbachia-based vector control strategies depend upon the strength and consistency of CI, a greater understanding is needed on the CI relationships between wPip, wAlbA and wAlbB Wolbachia in Ae. albopictus., Methods: This work consisted of a collaborative series of crosses carried out in Italy and in US to study the CI relationships between the "wPip" infected Ae. albopictus strain (ARwP) and the superinfected SR strain. The Ae. albopictus strains used in Italian tests are the wPip infected ARwP strain (ARwPIT), the superinfected SR strain and the aposymbiotic AR strain. To understand the observed pattern of CI, crossing experiments carried out in USA focused on the study of the CI relationships between ARwP (ARwPUS) and artificially-generated single infected lines, in specific HTA and HTB, harbouring only wAlbA and wAlbB Wolbachia respectively., Results: The paper reports an unusual pattern of CI observed in crossing experiments between ARwP and SR lines. Specifically, ARwP males are able to induce full sterility in wild type females throughout most of their lifetime, while crosses between SR males and ARwP females become partially fertile with male aging. We demonstrated that the observed decrease in CI penetrance with SR male age, is related to the previously described decrease in Wolbachia density, in particular of the wAlbA strain, occurring in aged superinfected males., Conclusions: The results here reported support the use of the ARwP Ae. albopictus line as source of "ready-made sterile males", as an alternative to gamma radiation sterilized males, for autocidal suppression strategies against the Asian tiger mosquito. In addition, the age dependent CI weakening observed in the crosses between SR males and ARwP females simplifies the downstream efforts to preserve the genetic variability within the laboratory ARwP colonies, to date based on the antibiotic treatment of wild captured superinfected mosquitoes, also reducing the costs.

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.

Heterologous transinfection with the endosymbiotic bacterium Wolbachia has been shown previously to induce pathogen interference phenotypes in mosquito hosts. Here we examine an artificially infected strain of Aedes polynesiensis, the primary vector of Wuchereria bancrofti, which is the causative agent of Lymphatic filariasis (LF) throughout much of the South Pacific. Embryonic microinjection was used to transfer the wAlbB infection from Aedes albopictus into an aposymbiotic strain of Ae. polynesiensis. The resulting strain (designated "MTB") experiences a stable artificial infection with high maternal inheritance. Reciprocal crosses of MTB with naturally infected wild-type Ae. polynesiensis demonstrate strong bidirectional incompatibility. Levels of reactive oxygen species (ROS) in the MTB strain differ significantly relative to that of the wild-type, indicating an impaired ability to regulate oxidative stress. Following a challenge with Brugia pahangi, the number of filarial worms achieving the infective stage is significantly reduced in MTB as compared to the naturally infected and aposymbiotic strains. Survivorship of MTB differed significantly from that of the wild-type, with an interactive effect between survivorship and blood feeding. The results demonstrate a direct correlation between decreased ROS levels and decreased survival of adult female Aedes polynesiensis. The results are discussed in relation to the interaction of Wolbachia with ROS production and antioxidant expression, iron homeostasis and the insect immune system. We discuss the potential applied use of the MTB strain for impacting Ae. polynesiensis populations and strategies for reducing LF incidence in the South Pacific.

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.

Background: The evolutionary success of Wolbachia bacteria, infections of which are widespread in invertebrates, is largely attributed to an ability to manipulate host reproduction without imposing substantial fitness costs. Here, we describe a stage-structured model with deterministic immature lifestages and a stochastic adult female lifestage. Simulations were conducted to better understand Wolbachia invasions into uninfected host populations. The model includes conventional Wolbachia parameters (the level of cytoplasmic incompatibility, maternal inheritance, the relative fecundity of infected females, and the initial Wolbachia infection frequency) and a new parameter termed relative larval viability (RLV), which is the survival of infected larvae relative to uninfected larvae., Results: The results predict the RLV parameter to be the most important determinant for Wolbachia invasion and establishment. Specifically, the fitness of infected immature hosts must be close to equal to that of uninfected hosts before population replacement can occur. Furthermore, minute decreases in RLV inhibit the invasion of Wolbachia despite high levels of cytoplasmic incompatibility, maternal inheritance, and low adult fitness costs., Conclusions: The model described here takes a novel approach to understanding the spread of Wolbachia through a population with explicit dynamics. By combining a stochastic female adult lifestage and deterministic immature/adult male lifestages, the model predicts that even those Wolbachia infections that cause minor decreases in immature survival are unlikely to invade and spread within the host population. The results are discussed in relation to recent theoretical and empirical studies of natural population replacement events and proposed applied research, which would use Wolbachia as a tool to manipulate insect populations.

Background: Lymphatic filariasis (LF), a global public health problem affecting approximately 120 million people worldwide, is a leading cause of disability in the developing world including the South Pacific. Despite decades of ongoing mass drug administration (MDA) in the region, some island nations have not yet achieved the threshold levels of microfilaremia established by the World Health Organization for eliminating transmission. Previously, the generation of a novel Aedes polynesiensis strain (CP) infected with an exogenous type of Wolbachia has been described. The CP mosquito is cytoplasmically incompatible (i.e., effectively sterile) when mated with wildtype mosquitoes, and a strategy was proposed for the control of A. polynesiensis populations by repeated, inundative releases of CP males to disrupt fertility of wild females. Such a strategy could lead to suppression of the vector population and subsequently lead to a reduction in the transmission of filarial worms., Methodology/principal Findings: CP males and F1 male offspring from wild-caught A. polynesiensis females exhibit near equal mating competitiveness with F1 females under semi-field conditions., Conclusions/significance: While laboratory experiments are important, prior projects have demonstrated the need for additional testing under semi-field conditions in order to recognize problems before field implementation. The results reported here from semi-field experiments encourage forward progression toward small-scale field releases.

Bacterial endosymbionts induce various effects on hosts and can dramatically impact host fitness and development. An example is provided by obligate, maternally-inherited Wolbachia, which infect a broad range of invertebrates. Wolbachia are capable of altering host reproduction, thereby promoting infection spread. Wolbachia also pose direct physiological costs and benefits to hosts, complicating their categorization as parasites or mutualists. This study examines for an effect of Wolbachia infection in intra-specific larval competition by Aedes albopictus mosquitoes, with the goal of examining for an impact of Wolbachia infection in mixed populations. Similar to prior work examining for an influence of Wolbachia infection on the fitness of A. albopictus in adults, the results presented here support the hypothesized impact of Wolbachia across all life stages, including immatures. The differential competitiveness of infected larvae detected in our experiments indicates that Wolbachia infected A. albopictus females are less competitive relative to uninfected females when competing under highly competitive conditions. In contrast, under low competitive pressures, infected females experience higher survivorship. Thus, Wolbachia infection shifts from parasitism to mutualism as a function of developmental conditions. Results are discussed in relation to the invasion and persistence of Wolbachia in A. albopictus populations. The results are important to the evolution of stable Wolbachia symbioses, including Wolbachia invasion of an uninfected population. The resulting infection dynamics that occur in an infected population are discussed., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Obligately intracellular Wolbachia bacteria infect numerous invertebrates and often manipulate host reproduction to facilitate the spread of infection. An example of reproductive manipulation is Wolbachia-induced cytoplasmic incompatibility (CI), which occurs commonly in insects. This CI has been the focus both of basic scientific studies of naturally occurring invasion events and of applied investigations on the use of Wolbachia as a vehicle to drive desired genotypes into insect populations ("gene drive" or "population replacement" strategies). The latter application requires an ability to generate artificial infections that cause a pattern of unidirectional incompatibility with the targeted host population. A suggested target of population replacement strategies is the mosquito Aedes albopictus (Asian tiger mosquito), an important invasive pest and disease vector. Aedes albopictus individuals are naturally "superinfected" with two Wolbachia types: wAlbA and wAlbB. Thus, generating a strain that is unidirectionally incompatible with field populations requires the introduction of an additional infection into the preexisting superinfection. Although prior reports demonstrate an ability to transfer Wolbachia infections to A. albopictus artificially, including both intra- and interspecific Wolbachia transfers, previous efforts have not generated a strain capable of invading natural populations. Here we describe the generation of a stable triple infection by introducing Wolbachia wRi from Drosophila simulans into a naturally superinfected A. albopictus strain. The triple-infected strain displays a pattern of unidirectional incompatibility with the naturally infected strain. This unidirectional CI, combined with a high fidelity of maternal inheritance and low fecundity effects, suggests that the artificial cytotype could serve as an appropriate vehicle for gene drive.

Wolbachia is a maternally inherited endosymbiont inducing various effects in insects and other invertebrate hosts that facilitate the invasion of naive host populations. One of the effects is a form of sterility known as cytoplasmic incompatibility (CI) through which females are effectively sterilized when they mate with males harboring a different Wolbachia strain. The repeated mass release of cytoplasmically incompatible males can be a tool to suppress insect populations. Here, we attempt to infect an Aedes albopictus (Skuse) (Diptera: Culicidae) strain, artificially deprived of the natural Wolbachia infection, with a new Wolbachia strain from Culex pipiens (L.) (Diptera: Culicidae). Further experiments were designed to study the effects of the new infection on Ae. albopictus fitness and evaluate key parameters that affect infection dynamics, including CI level and maternal inheritance. Using embryonic microinjection, the new Wolbachia strain was successfully established in Ae. albopictus. Crosses demonstrated a pattern of bidirectional CI between naturally infected and transinfected individuals. Specifically, egg hatch was essentially absent (i.e., CI was very high) in all crosses between the transinfected males and females with a different infection status. Furthermore, naturally infected Ae. albopictus males were incompatible with the transinfected females. Maternal inheritance was close to 100%. Moreover, the new infection did not affect immature and adult survivorship, but it significantly reduced female fecundity and egg hatch rate. The results are discussed in relation to the potential use of the new Ae. albopictus-Wolbachia symbiotic association as a suitable system for the study and development of CI-based strategies for suppressing populations of this important pest and disease vector.

Maternally inherited Wolbachia bacteria have evolved mechanisms to manipulate the reproduction of their invertebrate hosts, promoting infection spread. A high fitness cost to the host is maladaptive for obligate endosymbionts, and prior studies show rapid selection of new Wolbachia associations toward commensal or mutualistic symbioses. Here, wMelPop Wolbachia is transferred from Drosophila melanogaster into the mosquito Aedes albopictus. Characterization of the resulting strain provides an extreme example of Wolbachia as a pathogen. In addition to reduced longevity and fecundity, abnormally high Wolbachia density is associated with embryonic mortality that masks the typical pattern of cytoplasmic incompatibility. The results are consistent with earlier reports that show unpredictable shifts in the Wolbachia phenotype after interspecific transfer, which can complicate proposed strategies to modify the age structure of medically important vector populations.

Wolbachia pipientis Hertig and Wolbach (Rickettsiales: Rickettsiaceae) are intracellular alpha-proteobacteria that occur naturally in Aedes albopictus (Skuse) (Diptera: Culicidae) and numerous other invertebrates. These endosymbionts can invade host populations by manipulating host reproduction. Wolbachia infections have been shown to impart both costs and benefits to hosts in terms of development, survival, and fecundity. Here, we monitor intraspecific competition among independent cohorts of infected or uninfected larvae. Levels of competition are manipulated by varying initial larval densities and food levels. Although larval density is observed to have major impacts on immature survivorship, sex ratio of eclosing adults, and developmental rates, the Wolbachia infection status had minimal impact on male immatures and no effect on immature females under these experimental conditions. Female and male immatures were observed to respond differently to competitive pressure, with the functional relationships of females and males consistent with scramble and contest competition, respectively. The results are discussed in relation to the evolution of naturally occurring Wolbachia infections in Ae. albopictus (i.e., natural population replacement events) and public health strategies that propose the manipulation of Wolbachia infections in Ae. albopictus populations.

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.

Background: Intracellular Wolbachia bacteria are obligate, maternally-inherited, endosymbionts found frequently in insects and other invertebrates. The success of Wolbachia can be attributed in part to an ability to alter host reproduction via mechanisms including cytoplasmic incompatibility (CI), parthenogenesis, feminization and male killing. Despite substantial scientific effort, the molecular mechanisms underlying the Wolbachia/host interaction are unknown., Results: Here, an in vitro Wolbachia infection was generated in the Drosophila S2 cell line, and transcription profiles of infected and uninfected cells were compared by microarray. Differentially-expressed patterns related to reproduction, immune response and heat stress response are observed, including multiple genes that have been previously reported to be involved in the Wolbachia/host interaction. Subsequent in vivo characterization of differentially-expressed products in gonads demonstrates that Angiotensin Converting Enzyme (Ance) varies between Wolbachia infected and uninfected flies and that the variation occurs in a sex-specific manner. Consistent with expectations for the conserved CI mechanism, the observed Ance expression pattern is repeatable in different Drosophila species and with different Wolbachia types. To examine Ance involvement in the CI phenotype, compatible and incompatible crosses of Ance mutant flies were conducted. Significant differences are observed in the egg hatch rate resulting from incompatible crosses, providing support for additional experiments examining for an interaction of Ance with the CI mechanism., Conclusion: Wolbachia infection is shown to affect the expression of multiple host genes, including Ance. Evidence for potential Ance involvement in the CI mechanism is described, including the prior report of Ance in spermatid differentiation, Wolbachia-induced sex-specific effects on Ance expression and an Ance mutation effect on CI levels. The results support the use of Wolbachia infected cell cultures as an appropriate model for predicting in vivo host/Wolbachia interactions.

Wolbachia is a genus of obligate intracellular Alpha-Proteobacteria represented by the type species Wolbachia pipientis (Dumler et al., 2001). Wolbachia commonly reside within cytoplasmic vacuoles of arthropods and helminths (Werren and Windsor, 2000; Casiraghi et al., 2004); vertebrate infections have not been identified. Wolbachia are maternally transmitted from mothers to offspring though the embryonic cytoplasm. Wolbachia are able to induce a diverse range of phenotypes in their invertebrate hosts, ranging from classical mutualism to reproductive parasitism. Examples of the latter include male killing, host feminization, parthenogenesis, and cytoplasmic incompatibility (reviewed in Dobson, 2003a). Current Wolbachia research foci include examining the impacts of Wolbachia infection on host evolution, characterizing the mechanisms by which Wolbachia manipulate invertebrate hosts, and developing applied strategies that employ Wolbachia for pest and disease control. Wolbachia transfection has proven a useful technique for addressing questions within each of these research foci. This unit describes a method for Wolbachia transfection via embryonic microinjection.

Wolbachia bacteria manipulate the reproduction of mosquito hosts via a form of sterility known as cytoplasmic incompatibility (CI), promoting the spread of infections into host populations. The rate at which an infection invades is affected by host fitness costs associated with the Wolbachia infection. Here, we examine for an effect of Wolbachia infection on the immature fitness of the Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae). In two experiments, we examine for a Wolbachia effect on immature survivorship and developmental rate, adult size, and an effect of larval nutrition on CI level. The highest survivorship can be observed in uninfected larvae, primarily because of reduced survivorship of Wolbachia-infected males. Although differences in the developmental rates are observed between the examined strains, the differences cannot be readily attributed to Wolbachia. An effect of Wolbachia on adult size is not observed. Poor male nutrition is associated with reduced fecundity and egg hatch of mates. The latter is hypothesized to explain the reduced egg hatch observed in CI crosses of malnourished males relative to well fed males. We discuss the results in relation to previously identified differences in adult fitness, naturally occurring invasions of Wolbachia, applied strategies of population replacement, and the need for additional modeling effort.

Intracellular Wolbachia bacteria are obligate, maternally inherited endosymbionts found frequently in insects and other invertebrates. The evolutionary success of Wolbachia is due in part to an ability to manipulate reproduction. In mosquitoes and many other insects, Wolbachia causes a form of sterility known as cytoplasmic incompatibility (CI). Wolbachia-induced CI has attracted interest as a potential agent for affecting medically important disease vectors. However, application of the approach has been restricted by an absence of appropriate, naturally occurring Wolbachia infections. Here, we report the interspecific transfer of Wolbachia infection into a medically important mosquito. Using embryonic microinjection, Wolbachia is transferred from Drosophila simulans into the invasive pest and disease vector: Aedes albopictus (Asian tiger mosquito). The resulting infection is stably maintained and displays a unique pattern of bidirectional CI in crosses with naturally infected mosquitoes. Laboratory population cage experiments examine a strategy in which releases of Wolbachia-infected males are used to suppress mosquito egg hatch. We discuss the results in relation to developing appropriate Wolbachia-infected mosquito strains for population replacement and population suppression strategies.

Bacteriophages are commonly found in association with free-living bacteria, both as exogenic phages (virions) and as prophages integrated into the bacterial genome. In contrast, the observation of bacteriophages associated with obligate intracellular bacteria has been described infrequently. An exception is provided by Wolbachia endosymbionts, which harbor multiple phage elements that have been designated as WO phage. Wolbachia are maternally inherited bacteria that occur in the cytoplasm of many invertebrates, where they often manipulate host reproduction. Previously, the WO phage orf7 locus and ankyrin repeat-encoding genes have been observed to represent sources of genetic diversity between Wolbachia (wPip) strains infecting mosquitoes of the Culex pipiens complex and have been suggested as potential participants in the reproductive manipulations. We have characterized WO phage associated with multiple Wolbachia-infected Culex strains and an uninfected strain using electron microscopy and RT-PCR. For each strain, different developmental stages were examined for transcription of three WO phage orf7 genes. The results provide evidence for the presence of both actively transcribed virions and inactive prophages. Variable orf7 transcription patterns are observed in comparisons of differing Cx. pipiens strains. Variability includes both mosquito stage-specific and sexually dimorphic orf7 expression patterns. This report provides additional support for the hypothesis that bacteriophages play an important role in Wolbachia and host evolution.

A proposed strategy to aid in controlling the growing burden of vector-borne disease is population replacement, in which a natural vector population is replaced by a population with a reduced capacity for disease transmission. An important component of such a strategy is the drive system, which serves to spread a desired genotype into the targeted field population. Endosymbiotic Wolbachia bacteria are potential transgene drivers, but infections do not naturally occur in some important mosquito vectors, notably Aedes aegypti. In this work, stable infections of wAlbB Wolbachia were established in A. aegypti and caused high rates of cytoplasmic incompatibility (that is, elimination of egg hatch). Laboratory cage tests demonstrated the ability of wAlbB to spread into an A. aegypti population after seeding of an uninfected population with infected females, reaching infection fixation within seven generations.

Gene flow between populations of Culex pipiens L. is relevant to observed differences in disease transmission, insecticide resistance, behavior, and physiology. Intracellular Wolbachia bacteria have been hypothesized to affect gene flow in insects. Specifically, Wolbachia cause a form of embryonic mortality known as cytoplasmic incompatibility (CI) in crosses between individuals with different Wolbachia types. Incompatibility in Culex is exceptional in that it represents the most complex CI pattern known, and yet Culex populations are not infected with divergent Wolbachia strains. This has led to the hypothesis that extrachromosomal factors such as phages or mobile genetic elements may be involved in determining CI phenotype. Recent molecular characterization of Culex laboratory strains has identified variation in the orf7 locus of the Wolbachia-associated bacteriophage WO. Here, crosses between eight Culex strains differing in their orf7 type were conducted to examine for the hypothesized involvement of bacteriophage WO in determining CI in Culex. Although crossing results show examples of compatibility, partial compatibility, and incompatibility, the results fail to show a correlation between the CI phenotypes and orf7 type. Specific examples include high egg hatch resulting in crosses between Culex strains that differ significantly in their orf7 type and low egg hatch resulting in crosses between Culex strains with similar orf7 types. Thus, the phage orf7 locus alone cannot predict CI type in the Culex strains examined in this study. However, rejection of the hypothesized role of WO phage in Culex CI will require the characterization of additional phage loci.

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.

Wolbachia spp. are intracellular alpha proteobacteria closely related to Rickettsia. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of Wolbachia between hosts (transfection) is used both for basic research examining the Wolbachia-host interaction and for applied strategies that use Wolbachia infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer Wolbachia-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in Wolbachia quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared Wolbachia bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for Wolbachia during homogenization and injection: phosphate-buffered saline, Drosophila Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that Wolbachia bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific Wolbachia transfection and the future adaptation of the microinjection technique for additional insects.

The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.

Prior studies have identified a complicated pattern of interspecific hybridization between members of the Aedes (Stegomyia) scutellaris (Walker) mosquito group, which includes medically important vectors of bancroftian filariasis and dengue. Here, we report that two members of the group, Aedes polynesiensis Marks and Aedes riversi Bohart & Ingram, are both infected with intracellular Wolbachia bacteria. Sequencing of the Wolbachia wsp gene demonstrates that the infections differ from each other and from Wolbachia infections previously reported in mosquitoes. Aedes polynesiensis is the first mosquito identified with a wMel Wolbachia type. Intraspecific crosses of infected and aposymbiotic lines generated via antibiotic treatment show that the Wolbachia infections in both species cause high levels of cytoplasmic incompatibility. Interspecific crosses show that the two species are reproductively isolated. However, repeating the interspecific crosses with aposymbiotic mosquito strains demonstrates that the Wolbachia infections play a role in preventing hybrid offspring. We discuss Wolbachia infections in relation to better defining the evolutionary relationships and causes of speciation within the group, understanding the basis for the observed east-to-west gradient in filarial refractoriness, and developing novel genetic control measures.

Genetic manipulation that reduces the competence of a vector population to transmit pathogens would provide a useful tool to complement current control strategies, which are based primarily on the reduction/exclusion of vector populations and the prophylactic/therapeutic treatment of the vertebrate host population. Genetic drive is an important component of vector population replacement strategies, facilitating the replacement of natural populations with a genetically modified population. Genetic drive is reviewed here, emphasizing strategies that would employ infections of intracellular Wolbachia bacteria as a vehicle for population replacement. Also discussed are strategies for the retarding, arresting or reversing of Wolbachia-based population replacement. These strategies are based upon altering the conditions required for transgene invasion and are a prudent safeguard, should unexpected detrimental effects become associated with transgene spread.

Obligate, intracellular bacteria of the genus Wolbachia often behave as reproductive parasites by manipulating host reproduction to enhance their vertical transmission. One of these reproductive manipulations, cytoplasmic incompatibility, causes a reduction in egg-hatch rate in crosses between individuals with differing infections. Applied strategies based upon cytoplasmic incompatibility have been proposed for both the suppression and replacement of host populations. As Wolbachia infections occur within a broad range of invertebrates, these strategies are potentially applicable to a variety of medically and economically important insects. Here, we examine the interaction between Wolbachia infection frequency and host population size. We use a model to describe natural invasions of Wolbachia infections, artificial releases of infected hosts and releases of sterile males, as part of a traditional sterile insect technique programme. Model simulations demonstrate the importance of understanding the reproductive rate and intraspecific competition type of the targeted population, showing that releases of sterile or incompatible individuals may cause an undesired increase in the adult number. In addition, the model suggests a novel applied strategy that employs Wolbachia infections to suppress host populations. Releases of Wolbachia-infected hosts can be used to sustain artificially an unstable coexistence of multiple incompatible infections within a host population, allowing the host population size to be reduced, maintained at low levels, or eliminated.

Maternally inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods, often behaving as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. One manipulation is cytoplasmic incompatibility (CI), which causes a significant reduction in brood hatch and promotes the spread of the maternally inherited Wolbachia infection into the host population (i.e., cytoplasmic drive). Here, we have examined a Wolbachia superinfection in the mosquito Aedes albopictus and found the infection to be associated with both cytoplasmic incompatibility and increased host fecundity. Relative to uninfected females, infected females live longer, produce more eggs, and have higher hatching rates in compatible crosses. A model describing Wolbachia infection dynamics predicts that increased fecundity will accelerate cytoplasmic drive rates. To test this hypothesis, we used population cages to examine the rate at which Wolbachia invades an uninfected Ae. albopictus population. The observed cytoplasmic drive rates were consistent with model predictions for a CI-inducing Wolbachia infection that increases host fecundity. We discuss the relevance of these results to both the evolution of Wolbachia symbioses and proposed applied strategies for the use of Wolbachia infections to drive desired transgenes through natural populations (i.e., population replacement strategies).

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.

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. [ABSTRACT FROM AUTHOR]

Background: Intracellular Wolbachia bacteria are obligate, maternally-inherited, endosymbionts found frequently in insects and other invertebrates. The success of Wolbachia can be attributed in part to an ability to alter host reproduction via mechanisms including cytoplasmic incompatibility (CI), parthenogenesis, feminization and male killing. Despite substantial scientific effort, the molecular mechanisms underlying the Wolbachia/host interaction are unknown. Results: Here, an in vitro Wolbachia infection was generated in the Drosophila S2 cell line, and transcription profiles of infected and uninfected cells were compared by microarray. Differentially-expressed patterns related to reproduction, immune response and heat stress response are observed, including multiple genes that have been previously reported to be involved in the Wolbachia/host interaction. Subsequent in vivo characterization of differentially-expressed products in gonads demonstrates that Angiotensin Converting Enzyme (Ance) varies between Wolbachia infected and uninfected flies and that the variation occurs in a sex-specific manner. Consistent with expectations for the conserved CI mechanism, the observed Ance expression pattern is repeatable in different Drosophila species and with different Wolbachia types. To examine Ance involvement in the CI phenotype, compatible and incompatible crosses of Ance mutant flies were conducted. Significant differences are observed in the egg hatch rate resulting from incompatible crosses, providing support for additional experiments examining for an interaction of Ance with the CI mechanism. Conclusion: Wolbachia infection is shown to affect the expression of multiple host genes, including Ance. Evidence for potential Ance involvement in the CI mechanism is described, including the prior report of Ance in spermatid differentiation, Wolbachia-induced sex-specific effects on Ance expression and an Ance mutation effect on CI levels. The results support the use of Wolbachia infected cell cultures as an appropriate model for predicting in vivo host/Wolbachia interactions. [ABSTRACT FROM AUTHOR]

Simple Summary: The release of Wolbachia trans-infected mosquitoes to the environment has demonstrated promising results in reducing the target mosquito populations. The use of boric acid toxic sugar bait is another successful and upcoming technique in mosquito control. However, the potential complementary use of the two techniques had not been evaluated. This study demonstrates the significant mortality impact of boric acid toxic sugar bait on Wolbachia trans-infected Aedes albopictus Skuse mosquitoes, thus giving important insight to program planners. The field release of Wolbachia trans-infected male mosquitoes, as well as the use of toxic sugar baits, is a novel and promising candidate technique for integrated mosquito management programs. However, the methods of action of the two techniques may not be complementary, because the Wolbachia method releases mosquitoes into the environment expecting a wild population reduction in subsequent generations while the toxic baits are intended to reduce the wild population by killing mosquitoes. This laboratory study was conducted to evaluate the effectiveness of boric acid toxic sugar baits on Wolbachia trans-infected male Aedes albopictus, relative to wild-type Ae. albopictus males. Wolbachia trans-infected (ZAP male®) and the wild-type Ae. albopictus males were exposed separately to 1% boric acid in a 10% sucrose solution in BugDorms. In the control test, the two groups were exposed to 10% sucrose solution without boric acid. Percent mortalities were counted for 24 h, 48 h and 72 h post exposure periods. The results show that 1% boric acid toxic sugar bait can effectively kill ZAP males under laboratory conditions, and the effectiveness was significantly higher after 24 h and 48 h, compared to wild-type male Ae. albopictus. This finding will help in planning and coordinating integrated mosquito management programs, including both Wolbachia trans-infected mosquito releases and the use of toxic sugar baits against Ae. albopictus. [ABSTRACT FROM AUTHOR]

Obligately intracellular Wolbachia bacteria infect numerous invertebrates and often manipulate host reproduction to facilitate the spread of infection. An example of reproductive manipulation is Wolbachia-induced cytoplasmic incompatibility (CI), which occurs commonly in insects. This CI has been the focus both of basic scientific studies of naturally occurring invasion events and of applied investigations on the use of Wolbachia as a vehicle to drive desired genotypes into insect populations ("gene drive" or "population replacement" strategies). The latter application requires an ability to generate artificial infections that cause a pattern of unidirectional incompatibility with the targeted host population. A suggested target of population replacement strategies is the mosquito Aedes albopictus (Asian tiger mosquito), an important invasive pest and disease vector. Aedes albopictus individuals are naturally "superinfected" with two Wolbachia types: wAlbA and wAlbB. Thus, generating a strain that is unidirectionally incompatible with field populations requires the introduction of an additional infection into the preexisting superinfection. Although prior reports demonstrate an ability to transfer Wolbachia infections to A. albopictus artificially, including both intra- and interspecific Wolbachia transfers, previous efforts have not generated a strain capable of invading natural populations. Here we describe the generation of a stable triple infection by introducing Wolbachia wRi from Drosophila simulans into a naturally superinfected A. albopictus strain. The triple-infected strain displays a pattern of unidirectional incompatibility with the naturally infected strain. This unidirectional CI, combined with a high fidelity of maternal inheritance and low fecundity effects, suggests that the artificial cytotype could serve as an appropriate vehicle for gene drive. [ABSTRACT FROM AUTHOR]

Maternally inherited Wolbachia bacteria have evolved mechanisms to manipulate the reproduction of their invertebrate hosts, promoting infection spread. A high fitness cost to the host is maladaptive for obligate endosymbionts, and prior studies show rapid selection of new Wolbachia associations toward commensal or mutualistic symbioses. Here, wMelPop Wolbachia is transferred from Drosophila melanogaster into the mosquito Aedes albopictus. Characterization of the resulting strain provides an extreme example of Wolbachia as a pathogen. In addition to reduced longevity and fecundity, abnormally high Wolbachia density is associated with embryonic mortality that masks the typical pattern of cytoplasmic incompatibility. The results are consistent with earlier reports that show unpredictable shifts in the Wolbachia phenotype after interspecific transfer, which can complicate proposed strategies to modify the age structure of medically important vector populations. [ABSTRACT FROM AUTHOR]