A Wolbachia wMel Transinfection in Aedes albopictus Is Not Detrimental to Host Fitness and Inhibits Chikungunya Virus

Background

Wolbachia inherited intracellular bacteria can manipulate the reproduction of their insect hosts through cytoplasmic incompatibility (CI), and certain strains have also been shown to inhibit the replication or dissemination of viruses. Wolbachia strains also vary in their relative fitness effects on their hosts and this is a particularly important consideration with respect to the potential of newly created transinfections for use in disease control.

Methodology/Principal Findings

In Aedes albopictus mosquitoes transinfected with the wMel strain from Drosophila melanogaster, which we previously reported to be unable to transmit dengue in lab challenges, no significant detrimental effects were observed on egg hatch rate, fecundity, adult longevity or male mating competitiveness. All these parameters influence the population dynamics of Wolbachia, and the data presented are favourable with respect to the aim of taking wMel to high population frequency. Challenge with the chikungunya (CHIKV) virus, for which Ae. albopictus is an important vector, was conducted and the presence of wMel abolished CHIKV dissemination to the saliva.

Conclusions/significance

Taken together, these data suggest that introducing wMel into natural Ae. albopictus populations using bidirectional CI could be an efficient strategy for preventing or reducing the transmission of arboviruses by this species.     

Open Release of Male Mosquitoes Infected with a Wolbachia Biopesticide: Field Performance and Infection Containment

Background

Lymphatic filariasis (LF) is a globally significant disease, with 1.3 billion persons in 83 countries at risk. A coordinated effort of administering annual macrofilaricidal prophylactics to the entire at-risk population has succeeded in impacting and eliminating LF transmission in multiple regions. However, some areas in the South Pacific are predicted to persist as transmission sites, due in part to the biology of the mosquito vector, which has led to a call for additional tools to augment drug treatments. Autocidal strategies against mosquitoes are resurging in the effort against invasive mosquitoes and vector borne disease, with examples that include field trials of genetically modified mosquitoes and Wolbachia population replacement. However, critical questions must be addressed in anticipation of full field trials, including assessments of field competitiveness of transfected males and the risk of unintended population replacement.

Methodology/Principal Findings

We report the outcome of field experiments testing a strategy that employs Wolbachia as a biopesticide. The strategy is based upon Wolbachia-induced conditional sterility, known as cytoplasmic incompatibility, and the repeated release of incompatible males to suppress a population. A criticism of the Wolbachia biopesticide approach is that unintended female release or horizontal Wolbachia transmission can result in population replacement instead of suppression. We present the outcome of laboratory and field experiments assessing the competitiveness of transfected males and their ability to transmit Wolbachia via horizontal transmission.

Conclusions/Significance

The results demonstrate that Wolbachia-transfected Aedes polynesiensis males are competitive under field conditions during a thirty-week open release period, as indicated by mark, release, recapture and brood-hatch failure among females at the release site. Experiments demonstrate the males to be ‘dead end hosts’ for Wolbachia and that methods were adequate to prevent population replacement at the field site. The findings encourage the continued development and extension of a Wolbachia autocidal approach to additional medically important mosquito species.     

Male mating competitiveness of a Wolbachia-introgressed Aedes polynesiensis strain under semi-field conditions

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.     

The Effect of Virus-Blocking Wolbachia on Male Competitiveness of the Dengue Vector Mosquito,Aedes aegypti

Background

The bacterial endosymbiont Wolbachia blocks the transmission of dengue virus by its vector mosquito Aedes aegypti, and is currently being evaluated for control of dengue outbreaks. Wolbachia induces cytoplasmic incompatibility (CI) that results in the developmental failure of offspring in the cross between Wolbachia-infected males and uninfected females. This increases the relative success of infected females in the population, thereby enhancing the spread of the beneficial bacterium. However, Wolbachia spread via CI will only be feasible if infected males are sufficiently competitive in obtaining a mate under field conditions. We tested the effect of Wolbachia on the competitiveness of A. aegypti males under semi-field conditions.

Methodology/Principal Findings

In a series of experiments we exposed uninfected females to Wolbachia-infected and uninfected males simultaneously. We scored the competitiveness of infected males according to the proportion of females producing non-viable eggs due to incompatibility. We found that infected males were equally successful to uninfected males in securing a mate within experimental tents and semi-field cages. This was true for males infected by the benign wMel Wolbachia strain, but also for males infected by the virulent wMelPop (popcorn) strain. By manipulating male size we found that larger males had a higher success than smaller underfed males in the semi-field cages, regardless of their infection status.

Conclusions/Significance

The results indicate that Wolbachia infection does not reduce the competitiveness of A. aegypti males. Moreover, the body size effect suggests a potential advantage for lab-reared Wolbachia-males during a field release episode, due to their better nutrition and larger size. This may promote Wolbachia spread via CI in wild mosquito populations and underscores its potential use for disease control.     

Genetic Structure and Wolbachia Genotyping in Naturally Occurring Populations of Aedes albopictus across Contiguous Landscapes of Orissa,India

Background

Aedes albopictus has recently been implicated as a major vector in the emergence of dengue and chikungunya in several parts of India, like Orissa, which is gradually gaining endemicity for arboviral diseases. Ae. albopictus is further known to be naturally infected with Wolbachia (maternally inherited bacterium), which causes cytoplasmic incompatibility (CI) in mosquitoes leading to sperm-egg incompatibility inducing the death of embryo. Knowledge of genetic diversity of Ae. albopictus, along with revealing the type of Wolbachia infection in Ae. albopictus is important to explore the genetic and biological characteristics of Ae. albopictus, prior to exploring the uses of CI-based vector control strategies. In this study, we assessed the population genetic structure and the pattern of Wolbachia infection in Ae. albopictus mosquitoes of Orissa.

Methods and Results

Ae. albopictus mosquitoes were collected from 15 districts representing the four physiographical regions of Orissa from 2010–2012, analyzed for genetic variability at seven microsatellite loci and genotyped for Wolbachia strain detection using wsp gene primers. Most microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structure among all geographic populations (F_ST = 0.088). Genetic diversity was high (F_ST = 0.168) in Coastal Plains populations when compared with other populations, which was also evident from cluster analyses that showed most Coastal Plains populations consisted of a separate genetic cluster. Genotyping analyses revealed that Wolbachia-infected Ae. albopictus field populations of Orissa were mostly superinfected with wAlbA and wAlbB strains. Wolbachia superinfection was more pronounced in the Coastal Plain populations.

Conclusion

High genetic structure and Wolbachia superinfection, observed in the Coastal Plain populations of Orissa suggested it to be genetically and biologically more unique than other populations, and hence could influence their vectorial attributes. Such high genetic diversity observed among Coastal Plains populations could be attributed to multiple introductions of Ae. albopictus in this region.     

Wolbachia Age-Sex-Specific Density in Aedes albopictus: A Host Evolutionary Response to Cytoplasmic Incompatibility?

Background

Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI). These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework.

Methodology/Principal Findings

Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only.

Conclusions/Significance

Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.     

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.     

Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila

Background

Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI). CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown.

Methodology/Principal Findings

Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI.

Conclusions/Significance

Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role Hira plays in the interaction of Wolbachia and its insect host.     

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

Introduction

Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.

Methodology/Principal Findings

Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.

Conclusions/Significance

Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.     

Interspecific hybridization yields strategy for South Pacific filariasis vector elimination

Background

Lymphatic filariasis (LF) is a leading cause of disability in South Pacific regions, where >96% of the 1.7 million population are at risk of LF infection. As part of current global campaign, mass drug administration (MDA) has effectively reduced lymphatic filiariasis prevalence, but mosquito vector biology can complicate the MDA strategy. In some regions, there is evidence that the goal of LF elimination cannot be attained via MDA alone. Obligate vector mosquitoes provide additional targets for breaking the LF transmission cycle, but existing methods are ineffective for controlling the primary vector throughout much of the South Pacific, Aedes polynesiensis.

Methodology/Principal Findings

Here we demonstrate that interspecific hybridization and introgression results in an A. polynesiensis strain (‘CP’ strain) that is stably infected with the endosymbiotic Wolbachia bacteria from Aedes riversi. The CP strain is bi-directionally incompatible with naturally infected mosquitoes, resulting in female sterility. Laboratory assays demonstrate that CP males are equally competitive, resulting in population elimination when CP males are introduced into wild type A. polynesiensis populations.

Conclusions/Significance

The findings demonstrate strategy feasibility and encourage field tests of the vector elimination strategy as a supplement to ongoing MDA efforts.     

A re-examination of Wolbachia-induced cytoplasmic incompatibility in California Drosophila simulans

Background

In California Drosophila simulans, the maternally inherited Riverside strain Wolbachia infection (wRi) provides a paradigm for rapid spread of Wolbachia in nature and rapid evolutionary change. wRi induces cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females produce reduced egg-hatch. The three parameters governing wRi infection-frequency dynamics quantify: the fidelity of maternal transmission, the level of cytoplasmic incompatibility, and the relative fecundity of infected females. We last estimated these parameters in nature in 1993. Here we provide new estimates, under both field and laboratory conditions. Five years ago, we found that wRi had apparently evolved over 15 years to enhance the fecundity of infected females; here we examine whether CI intensity has also evolved.

Methodology/Principal Findings

New estimates using wild-caught flies indicate that the three key parameters have remained relatively stable since the early 1990s. As predicted by our three-parameter model using field-estimated parameter values, population infection frequencies remain about 93%. Despite this relative stability, laboratory data based on reciprocal crosses and introgression suggest that wRi may have evolved to produce less intense CI (i.e., higher egg hatch from incompatible crosses). In contrast, we find no evidence that D. simulans has evolved to lower the susceptibility of uninfected females to CI.

Conclusions/Significance

Evolution of wRi that reduces CI is consistent with counterintuitive theoretical predictions that within-population selection on CI-causing Wolbachia does not act to increase CI. Within taxa, CI is likely to evolve mainly via pleiotropic effects associated with the primary targets of selection on Wolbachia, i.e., host fecundity and transmission fidelity. Despite continuous, strong selection, D. simulans has not evolved appreciably to suppress CI. Our data demonstrate a lack of standing genetic variation for CI resistance in the host.     

Monitoring of Insecticide Resistance in Field-Collected Populations of Culex pipiens pallens (Diptera: Culicidae)

A susceptible strain and six field populations of the northern house mosquito, Culex pipiens pallens Coquillett, were collected in Seoul, Paju, Daejeon, Heongseong, Jinhae, and Buan, and tested for their susceptibility to seven insecticides by topical application. In tests with female Cx. pipiens pallens, marked regional variations of insecticide susceptibility were observed. Five field-collected populations of Cx. pipiens pallens, except for the Seoul colony, exhibitedhightoveryhighlevelsofresistanceto cyfluthrin [resistance ratio (RR), 96–554] and deltamethrin (RR, 192–959). Low to moderate levels of resistance were observed in bendiocarb (RR, 14–46), chlorpyrifos [RR, 7–38; exceptions: Buan (RR, 63) and Jinhae (RR, 80) colonies], λ-cyhalothrin (RR, 5–23), S-bioallethrin (RR, 1–47), and permethrin [RR, 2–27; exception: Jinhae colony (RR, 247)]. These results indicate that careful selection and rotational use of these insecticides should result in continued satisfactory control against field populations of Cx. pipiens pallens.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

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.     

Wolbachia induces male-specific mortality in the mosquito Culex pipiens (LIN strain)

Background

Wolbachia are maternally inherited endosymbionts that infect a diverse range of invertebrates, including insects, arachnids, crustaceans and filarial nematodes. Wolbachia are responsible for causing diverse reproductive alterations in their invertebrate hosts that maximize their transmission to the next generation. Evolutionary theory suggests that due to maternal inheritance, Wolbachia should evolve toward mutualism in infected females, but strict maternal inheritance means there is no corresponding force to select for Wolbachia strains that are mutualistic in males.

Methodology/Principal findings

Using cohort life-table analysis, we demonstrate that in the mosquito Culex pipiens (LIN strain), Wolbachia-infected females show no fitness costs due to infection. However, Wolbachia induces up to a 30% reduction in male lifespan.

Conclusions/significance

These results indicate that the Wolbachia infection of the Culex pipiens LIN strain is virulent in a sex-specific manner. Under laboratory situations where mosquitoes generally mate at young ages, Wolbachia strains that reduce male survival could evolve by drift because increased mortality in older males is not a significant selective force.     

Wolbachia-Associated Bacterial Protection in the Mosquito Aedes aegypti

Background

Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.

Methodology/Findings

Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.

Conclusions/Significance

This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.     

The Significance of Genetic Polymorphisms within and between Founder Populations of Ceratitis capitata (Wied.) from Argentina

Background

The Mediterranean fruit fly Ceratitis Capitata (DIPTERA: Tephritidae) is a major agricultural pest in Argentina. One main cause for the success of non-contaminant control programs based on genetic strategies is compatibility between natural and laboratory germplasms.A comprehensive characterization of the fruit fly based on genetic studies and compatibility analysis was undertaken on two founder populations from the provinces of Buenos Aires and Mendoza, used in pioneering sterile male technique control programmes in our country. The locations are 1,000 km apart from each other.

Methodology/Principal Findings

We compared the genetic composition of both populations based on cytological, physiological and morphological characterization. Compatibility studies were performed in order to determine the presence of isolation barriers. Results indicate that the Buenos Aires germplasm described previously is partially different from that of the Mendoza population. Both laboratory colonies are a reservoir of mutational and cytological polymorphisms. Some sexual chromosome variants such as the XL and the YL resulting from attachment of a B-chromosome to the X-chromosome or Y-chromosome behave as a lethal sex-linked factor. Our results also show incompatibility between both germplasms and pre-zygotic isolation barriers between them. Our evidence is consistent with the fact that polymorphisms are responsible for the lack of compatibility.

Conclusions

The genetic control mechanism should be directly produced in the germplasm of the target population in order to favour mating conditions. This is an additional requirement for the biological as well as economic success of control programs based on genetic strategies such as the sterile insect technique. The analysis of representative samples also revealed natural auto-control mechanisms which could be used in modifying pest population dynamics.