Identification of Wolbachia Strains in Mosquito Disease Vectors

Wolbachia bacteria are common endosymbionts of insects, and some strains are known to protect their hosts against RNA viruses and other parasites. This has led to the suggestion that releasing Wolbachia-infected mosquitoes could prevent the transmission of arboviruses and other human parasites. We have identified Wolbachia in Kenyan populations of the yellow fever vector Aedes bromeliae and its relative Aedes metallicus, and in Mansonia uniformis and Mansonia africana, which are vectors of lymphatic filariasis. These Wolbachia strains cluster together on the bacterial phylogeny, and belong to bacterial clades that have recombined with other unrelated strains. These new Wolbachia strains may be affecting disease transmission rates of infected mosquito species, and could be transferred into other mosquito vectors as part of control programs.     

Simple,sensitive, and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes,using loop mediated isothermal amplification combined with the electrochemical biosensing method

BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia-based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity and cost issues.Methodology/Principal findingsWe describe here the development of a LAMP assay combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers used a low-cost dye detecting system and 4 oligo nucleotide primers which can reduce the cost of analysis while the specificity is comparable to the previous methods. The detection capacity of our LAMP technique was 1.4 nM and the detection limit reduced to 2.2 fM when combined with the BIOSENSOR. Our study demonstrates that a BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.     

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.     

The Potential Use of Wolbachia-Based Mosquito Biocontrol Strategies for Japanese Encephalitis

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by the infectious bite of Culex mosquitoes. The virus causes the development of the disease Japanese encephalitis (JE) in a small proportion of those infected, predominantly affecting children in eastern and southern Asia. Annual JE incidence estimates range from 50,000–175,000, with 25%–30% of cases resulting in mortality. It is estimated that 3 billion people live in countries in which JEV is endemic. The virus exists in an enzootic transmission cycle, with mosquitoes transmitting JEV between birds as reservoir hosts and pigs as amplifying hosts. Zoonotic infection occurs as a result of spillover events from the main transmission cycle. The reservoir avian hosts include cattle egrets, pond herons, and other species of water birds belonging to the family Ardeidae. Irrigated rice fields provide an ideal breeding ground for mosquitoes and attract migratory birds, maintaining the transmission of JEV. Although multiple vaccines have been developed for JEV, they are expensive and require multiple doses to maintain efficacy and immunity. As humans are a “dead-end” host for the virus, vaccination of the human population is unlikely to result in eradication. Therefore, vector control of the principal mosquito vector, Culex tritaeniorhynchus, represents a more promising strategy for reducing transmission. Current vector control strategies include intermittent irrigation of rice fields and space spraying of insecticides during outbreaks. However, Cx. Tritaeniorhynchus is subject to heavy exposure to pesticides in rice fields, and as a result, insecticide resistance has developed. In recent years, significant advancements have been made in the potential use of the bacterial endosymbiont Wolbachia for mosquito biocontrol. The successful transinfection of Wolbachia strains from Drosophila flies to Aedes (Stegomyia) mosquitoes has resulted in the generation of “dengue-refractory” mosquito lines. The successful establishment of Wolbachia in wild Aedes aegypti populations has recently been demonstrated, and open releases in dengue-endemic countries are ongoing. This review outlines the current control methods for JEV in addition to highlighting the potential use of Wolbachia-based biocontrol strategies to impact transmission. JEV and dengue virus are both members of the Flavivirus genus, and the successful establishment of Drosophila Wolbachia strains in Cx. Tritaeniorhynchus, as the principal vector of JEV, is predicted to significantly impact JEV transmission.     

Arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing

Background

Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown.

Methodology/Principal Findings

In this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing.

Conclusions/Significance

In two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (p^IP), which ranged from 2.75×10⁻⁸ to 1.08×10⁻⁷. DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus.     

Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia

Background

Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations.

Methodology/Principal Findings

Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied.

Conclusions/Significance

As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.     

Arbovirus detection in synanthropic mosquitoes from the Brazilian Amazon and in mosquito saliva using Flinders Technology Associates cards

Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs.     

Wolbachia Infection Reduces Blood-Feeding Success in the Dengue Fever Mosquito, Aedes aegypti

Background

The mosquito Aedes aegypti was recently transinfected with a life-shortening strain of the endosymbiont Wolbachia pipientis (wMelPop) as the first step in developing a biocontrol strategy for dengue virus transmission. In addition to life-shortening, the wMelPop-infected mosquitoes also exhibit increased daytime activity and metabolic rates. Here we sought to quantify the blood-feeding behaviour of Wolbachia-infected females as an indicator of any virulence or energetic drain associated with Wolbachia infection.

Methodology/Principal Findings

In a series of blood-feeding trials in response to humans, we have shown that Wolbachia-infected mosquitoes do not differ in their response time to humans, but that as they age they obtain fewer and smaller blood meals than Wolbachia-uninfected controls. Lastly, we observed a behavioural characteristic in the Wolbachia infected mosquitoes best described as a “bendy” proboscis that may explain the decreased biting success.

Conclusions/Significance

Taken together the evidence suggests that wMelPop infection may be causing tissue damage in a manner that intensifies with mosquito age and that leads to reduced blood-feeding success. These behavioural changes require further investigation with respect to a possible physiological mechanism and their role in vectorial capacity of the insect. The selective decrease of feeding success in older mosquitoes may act synergistically with other Wolbachia-associated traits including life-shortening and viral protection in biocontrol strategies.     

Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.     

Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.     

Modelling the Use of Wolbachia to Control Dengue Fever Transmission

Experiments and field trials have shown that the intracellular bacterium Wolbachia may be introduced into populations of the mosquito Aedes aegypti, the primary vector for dengue fever. In the absence of Wolbachia, a mosquito acquiring the dengue virus from an infected human enters an exposed (infected but not infectious) period before becoming infectious itself. A Wolbachia-infected mosquito that acquires dengue (i) may have a reduced lifespan, so that it is less likely to survive the exposed period and become infectious, and (ii) may have a reduced ability to transmit dengue, even if it has survived the exposed period. Wolbachia introduction has therefore been suggested as a potential dengue control measure. We set up a mathematical model for the system to investigate this suggestion and to evaluate the desirable properties of the Wolbachia strain to be introduced. We show that Wolbachia has excellent potential for dengue control in areas where R ₀ is not too large. However, if R ₀ is large, Wolbachia strains that reduce but do not eliminate dengue transmission have little effect on endemic steady states or epidemic sizes. Unless control measures to reduce R ₀ by reducing mosquito populations are also put in place, it may be worth the extra effort in such cases to introduce Wolbachia strains that eliminate dengue transmission completely.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

Constraints on the use of lifespan-shortening Wolbachia to control dengue fever

Dengue fever, a viral disease spread by the mosquito Aedes aegypti, affects 50–100 million people a year in many tropical countries. Because the virus must incubate within mosquito hosts for two weeks before being able to transmit the infection, shortening the lifespan of mosquitoes may curtail dengue transmission. We developed a continuous time reaction-diffusion model of the spatial spread of Wolbachia through a population of A. aegypti. This model incorporates the lifespan-shortening effects of Wolbachia on infected A. aegypti and the fitness advantage to infected females due to cytoplasmic incompatibility (CI). We found that local establishment of the Wolbachia infection can occur if the fitness advantage due to CI exceeds the fitness reduction due to lifespan-shortening effects, in accordance with earlier results concerning fecundity reduction. However, spatial spread is possible only if the fitness advantage due to CI is twice as great as the fitness reduction due to lifespan shortening effects. Moreover, lifespan-shortening and fecundity-reduction can have different effects on the speed of wave-retreat. Using data from the literature, we estimated all demographic parameters for infected and uninfected mosquitoes and computed the velocities of spread of infection. Our most optimistic estimates suggest that the spatial spread of lifespan-shortening Wolbachia may be so slow that efficient spatial spread would require a prohibitively large number of point releases. However, as these estimates of demographic parameters may not accurately reflect natural conditions, further research is necessary to corroborate these predictions.     

Replacing a Native Wolbachia with a Novel Strain Results in an Increase in Endosymbiont Load and Resistance to Dengue Virus in a Mosquito Vector

Wolbachia is a maternally transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce pathogen interference and spread into mosquito vector populations makes it possible to develop Wolbachia as a biological control agent for vector-borne disease control. Although Wolbachia induces resistance to dengue virus (DENV), filarial worms, and Plasmodium in mosquitoes, species like Aedes polynesiensis and Aedes albopictus, which carry native Wolbachia infections, are able to transmit dengue and filariasis. In a previous study, the native wPolA in Ae. polynesiensis was replaced with wAlbB from Ae. albopictus, and resulted in the generation of the transinfected “MTB” strain with low susceptibility for filarial worms. In this study, we compare the dynamics of DENV serotype 2 (DENV-2) within the wild type “APM” strain and the MTB strain of Ae. polynesiensis by measuring viral infection in the mosquito whole body, midgut, head, and saliva at different time points post infection. The results show that wAlbB can induce a strong resistance to DENV-2 in the MTB mosquito. Evidence also supports that this resistance is related to a dramatic increase in Wolbachia density in the MTB''s somatic tissues, including the midgut and salivary gland. Our results suggests that replacement of a native Wolbachia with a novel infection could serve as a strategy for developing a Wolbachia-based approach to target naturally infected insects for vector-borne disease control.     

Impact of Wolbachia on Infection with Chikungunya and Yellow Fever Viruses in the Mosquito Vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10⁴ times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.     

Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40–75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.     

Competition for Amino Acids Between Wolbachia and the Mosquito Host,Aedes aegypti

The endosymbiont Wolbachia represents a promising method of dengue control, as it reduces the ability of the primary vector, the mosquito Aedes aegypti, to transmit viruses. When mosquitoes infected with the virulent Wolbachia strain wMelPop are fed non-human blood, there is a drastic reduction in mosquito fecundity and egg viability. Wolbachia has a reduced genome and is clearly dependent on its host for a wide range of nutritional needs. The fitness defects seen in wMelPop-infected A. aegypti could be explained by competition between the mosquito and the symbiont for essential blood meal nutrients, the profiles of which are suboptimal in non-human blood. Here, we examine cholesterol and amino acids as candidate molecules for competition, as they have critical roles in egg structural development and are known to vary between blood sources. We found that Wolbachia infection reduces total cholesterol levels in mosquitoes by 15–25 %. We then showed that cholesterol supplementation of a rat blood meal did not improve fecundity or egg viability deficits. Conversely, amino acid supplementation of sucrose before and after a sheep blood meal led to statistically significant increases in fecundity of approximately 15–20 eggs per female and egg viability of 30–40 %. This mosquito system provides the first empirical evidence of competition between Wolbachia and a host over amino acids and may suggest a general feature of Wolbachia–insect associations. These competitive processes could affect many aspects of host physiology and potentially mosquito fitness, a key concern for Wolbachia-based mosquito biocontrol.     

Lab-scale characterization and semi-field trials of Wolbachia Strain wAlbB in a Taiwan Wolbachia introgressed Ae. aegypti strain

Dengue fever is one of the most severe viral diseases transmitted by Aedes mosquitoes, with traditional approaches of disease control proving insufficient to prevent significant disease burden. Release of Wolbachia-transinfected mosquitoes offers a promising alternative control methodologies; Wolbachia-transinfected female Aedes aegypti demonstrate reduced dengue virus transmission, whilst Wolbachia-transinfected males cause zygotic lethality when crossed with uninfected females, providing a method for suppressing mosquito populations. Although highly promising, the delicate nature of population control strategies and differences between local species populations means that controlled releases of Wolbachia-transinfected mosquitoes cannot be performed without extensive testing on specific local Ae. aegypti populations. In order to investigate the potential for using Wolbachia to suppress local Ae. aegypti populations in Taiwan, we performed lab-based and semi-field fitness trials. We first transinfected the Wolbachia strain wAlbB into a local Ae. aegypti population (wAlbB-Tw) and found no significant changes in lifespan, fecundity and fertility when compared to controls. In the laboratory, we found that as the proportion of released male mosquitoes carrying Wolbachia was increased, population suppression could reach up to 100%. Equivalent experiments in semi-field experiments found suppression rates of up to 70%. The release of different ratios of wAlbB-Tw males in the semi-field system provided an estimate of the optimal size of male releases. Our results indicate that wAlbB-Tw has significant potential for use in vector control strategies aimed at Ae. aegypti population suppression in Taiwan. Open field release trials are now necessary to confirm that wAlbB-Tw mediated suppression is feasible in natural environments.     

Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus

Species of the genus Wolbachia are a group of Rickettsia-like, maternally-inherited bacteria (gram negative), which cause various reproductive alterations in their arthropod and nematode hosts including cytoplasmic incompatibility (CI), male-killing, parthenogenesis and feminization. They can be divided into supergroups such as A and B based on phylogenetic analysis of 16S rDNA sequences. In this study, we examined the relative infection densities of Wolbachia strains among life cycle stages in the mosquito, Aedes albopictus in terms of crowding effect and temperature effect. A. albopictus is known to be superinfected with both A- and B-supergroup Wolbachia which cause CI. The relative Wolbachia densities within each individual mosquito were determined and quantified by using real-time quantitative PCR assay based on the wsp gene. We found that B-supergroup Wolbachia strain densities in this host species were consistently and significantly higher than in the A-supergroup. Larval crowding also reduced adult size of mosquitoes. Our results show clearly that the higher densities of mosquito larvae cause lower densities of Wolbachia strains. Examination of the effect of temperature on Wolbachia density in each stage of the mosquito clearly revealed a significant decrease in bacterial density following exposure to elevated temperature (37 °C) in both males and females.