Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions.

The global expansion of Aedes albopictus has stimulated the development of environmentally friendly methods aiming to control disease transmission through the suppression of natural vector populations. Sterile male release programmes are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation which can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic Wolbachia-free Ae. albopictus line. We then measured the penetrance of cytoplasmic incompatibility and life-history traits of both transinfected lines, selected the wPip-IV line and combined it with the genetic sexing strain. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Optimization of Dieldrin Selection for the Genetic Sexing of Aedes albopictus.

The mass production of mosquitoes at an industrial scale requires efficient sex separation, which can be achieved through mechanical, genetic or artificial intelligence means. Compared with other methods, the genetic sexing approach offers the advantage of limiting costs and space by removing females at the larval stage. We recently developed a Genetic Sexing Strain (GSS) in Aedes albopictus based on the sex linkage of the rdlR allele, conferring resistance to dieldrin, to the male (M) locus. It has been previously reported that dieldrin ingested by larvae can be detected in adults and bioaccumulated in predators, raising the question of its use at a large scale. In this context, we performed several experiments aiming at optimizing dieldrin selection by decreasing both dieldrin concentration and exposure time while maintaining a stable percentage of contaminating females averaging 1%. We showed that the previously used dieldrin exposure induced an important toxicity as it killed 60% of resistant males at the larval stage. We lowered this toxicity by reducing the dose and/or the exposure time to recover nearly all resistant males. We then quantified the residues of dieldrin in resistant male adults and showed that dieldrin toxicity in larvae was positively correlated with dieldrin concentrations detected in adults. Interestingly, we showed that the use of reduced dieldrin exposure led to a dieldrin quantification in adult males that was below the quantity threshold of the Gas Chromatography-Mass Spectrometry detection method. Presented data show that dieldrin exposure can be adjusted to suppress toxicity in males while achieving efficient sexing and lowering the levels of dieldrin residues in adults to barely quantifiable levels.

Genomic and cytogenetic analysis of the Ceratitis capitata temperature-sensitive lethal region.

Genetic sexing strains (GSS) are an important tool in support of sterile insect technique (SIT) applications against insect pests and disease vectors. The yet unknown temperature-sensitive lethal (tsl) gene and the recently identified white pupae (wp) gene have been used as selectable markers in the most successful GSS developed so far, the Ceratitis capitata (medfly) VIENNA 8 GSS. The molecular identification of the tsl gene may open the way for its use as a marker for the development of GSS in other insect pests and disease vectors of SIT importance. Prior studies have already shown that the tsl gene is located on the right arm of chromosome 5, between the wp and Zw loci (tsl genomic region). In the present study, we used genomic, transcriptomic, bioinformatic, and cytogenetic approaches to characterize and analyze this genomic region in wild-type and tsl mutant medfly strains. Our results suggested the presence of 561 genes, with 322 of them carrying SNPs and/or insertion-deletion (indel) mutations in the tsl genomic region. Furthermore, comparative transcriptomic analysis indicated the presence of 32 differentially expressed genes, and bioinformatic analysis revealed the presence of 33 orthologs with a described heat-sensitive phenotype of Drosophila melanogaster in this region. These data can be used in functional genetic studies to identify the tsl gene(s) and the causal mutation(s) responsible for the temperature-sensitive lethal phenotype in medfly, and potentially additional genes causing a similar phenotype.

A Comparison of Feathers and Oral Swab Samples as DNA Sources for Molecular Sexing in Companion Birds.

The early age determinism of the sex in case of monomorphic birds is very important, because most companion birds have no distinct sexual dimorphic traits. Molecular genetic sexing was proved to be one of the most accurate sex determinations in monomorphic birds. The aim of this study was to compare the results obtained by PCR performed on isolate genomic DNA from paired samples of feathers and oral swabs collected from the same individuals. Samples of oral swabs (n = 101) and feathers (n = 74) were collected from 101 companion birds from four different species (Columba livia domestica, Psittacula krameri, Neophema splendida and Agapornis spp.). The PCR was performed for the amplification of the CHD1W and CHD1Z genes in females and the CHD1Z gene in males. The overall PCR success rate of sex determination was significantly higher from oral swabs than from feathers. The PCR success rate from oral swabs was higher in juveniles and from feathers was significantly higher in adults. The similarity between the oral swab and feathers was obtained in 78.38% of the birds. Oral swabs proved to be a more reliable sample for genetic sex determination in the species tested in this study.

New self-sexing Aedes aegypti strain eliminates barriers to scalable and sustainable vector control for governments and communities in dengue-prone environments.

For more than 60 years, efforts to develop mating-based mosquito control technologies have largely failed to produce solutions that are both effective and scalable, keeping them out of reach of most governments and communities in disease-impacted regions globally. High pest suppression levels in trials have yet to fully translate into broad and effective Aedes aegypti control solutions. Two primary challenges to date-the need for complex sex-sorting to prevent female releases, and cumbersome processes for rearing and releasing male adult mosquitoes-present significant barriers for existing methods. As the host range of Aedes aegypti continues to advance into new geographies due to increasing globalisation and climate change, traditional chemical-based approaches are under mounting pressure from both more stringent regulatory processes and the ongoing development of insecticide resistance. It is no exaggeration to state that new tools, which are equal parts effective and scalable, are needed now more than ever. This paper describes the development and field evaluation of a new self-sexing strain of Aedes aegypti that has been designed to combine targeted vector suppression, operational simplicity, and cost-effectiveness for use in disease-prone regions. This conditional, self-limiting trait uses the sex-determination gene doublesex linked to the tetracycline-off genetic switch to cause complete female lethality in early larval development. With no female progeny survival, sex sorting is no longer required, eliminating the need for large-scale mosquito production facilities or physical sex-separation. In deployment operations, this translates to the ability to generate multiple generations of suppression for each mosquito released, while being entirely self-limiting. To evaluate these potential benefits, a field trial was carried out in densely-populated urban, dengue-prone neighbourhoods in Brazil, wherein the strain was able to suppress wild mosquito populations by up to 96%, demonstrating the utility of this self-sexing approach for biological vector control. In doing so, it has shown that such strains offer the critical components necessary to make these tools highly accessible, and thus they harbour the potential to transition mating-based approaches to effective and sustainable vector control tools that are within reach of governments and at-risk communities who may have only limited resources.

CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.

Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.

The Insect Pest Control Laboratory of the Joint FAO/IAEA Programme: Ten Years (2010-2020) of Research and Development, Achievements and Challenges in Support of the Sterile Insect Technique.

The Joint FAO/IAEA Centre (formerly called Division) of Nuclear Techniques in Food and Agriculture was established in 1964 and its accompanying laboratories in 1961. One of its subprograms deals with insect pest control, and has the mandate to develop and implement the sterile insect technique (SIT) for selected key insect pests, with the goal of reducing the use of insecticides, reducing animal and crop losses, protecting the environment, facilitating international trade in agricultural commodities and improving human health. Since its inception, the Insect Pest Control Laboratory (IPCL) (formerly named Entomology Unit) has been implementing research in relation to the development of the SIT package for insect pests of crops, livestock and human health. This paper provides a review of research carried out between 2010 and 2020 at the IPCL. Research on plant pests has focused on the development of genetic sexing strains, characterizing and assessing the performance of these strains (e.g., Ceratitis capitata), elucidation of the taxonomic status of several members of the Bactrocera dorsalis and Anastrepha fraterculus complexes, the use of microbiota as probiotics, genomics, supplements to improve the performance of the reared insects, and the development of the SIT package for fruit fly species such as Bactrocera oleae and Drosophila suzukii. Research on livestock pests has focused on colony maintenance and establishment, tsetse symbionts and pathogens, sex separation, morphology, sterile male quality, radiation biology, mating behavior and transportation and release systems. Research with human disease vectors has focused on the development of genetic sexing strains (Anopheles arabiensis, Aedes aegypti and Aedes albopictus), the development of a more cost-effective larvae and adult rearing system, assessing various aspects of radiation biology, characterizing symbionts and pathogens, studying mating behavior and the development of quality control procedures, and handling and release methods. During the review period, 13 coordinated research projects (CRPs) were completed and six are still being implemented. At the end of each CRP, the results were published in a special issue of a peer-reviewed journal. The review concludes with an overview of future challenges, such as the need to adhere to a phased conditional approach for the implementation of operational SIT programs, the need to make the SIT more cost effective, to respond with demand driven research to solve the problems faced by the operational SIT programs and the use of the SIT to address a multitude of exotic species that are being introduced, due to globalization, and established in areas where they could not survive before, due to climate change.

Transcribed sex-specific markers on the Y chromosome of the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: The Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise. RESULTS: Using Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis. CONCLUSIONS: Our random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

Irradiation induced inversions suppress recombination between the M locus and morphological markers in Aedes aegypti.

BACKGROUND: Aedes aegypti is the primary vector of arthropod-borne viruses and one of the most widespread and invasive mosquito species. Due to the lack of efficient specific drugs or vaccination strategies, vector population control methods, such as the sterile insect technique, are receiving renewed interest. However, availability of a reliable genetic sexing strategy is crucial, since there is almost zero tolerance for accidentally released females. Development of genetic sexing strains through classical genetics is hindered by genetic recombination that is not suppressed in males as is the case in many Diptera. Isolation of naturally-occurring or irradiation-induced inversions can enhance the genetic stability of genetic sexing strains developed through genetically linking desirable phenotypes with the male determining region. RESULTS: For the induction and isolation of inversions through irradiation, 200 male pupae of the 'BRA' wild type strain were irradiated at 30 Gy and 100 isomale lines were set up by crossing with homozygous 'red-eye' (re) mutant females. Recombination between re and the M locus and the white (w) gene (causing a recessive white eye phenotype when mutated) and the M locus was tested in 45 and 32 lines, respectively. One inversion (Inv35) reduced recombination between both re and the M locus, and wand the M locus, consistent with the presence of a rather extended inversion between the two morphological mutations, that includes the M locus. Another inversion (Inv5) reduced recombination only between w and the M locus. In search of naturally-occurring, recombination-suppressing inversions, homozygous females from the red eye and the white eye strains were crossed with seventeen and fourteen wild type strains collected worldwide, representing either recently colonized or long-established laboratory populations. Despite evidence of varying frequencies of recombination, no combination led to the elimination or substantial reduction of recombination. CONCLUSION: Inducing inversions through irradiation is a feasible strategy to isolate recombination suppressors either on the M or the m chromosome for Aedes aegypti. Such inversions can be incorporated in genetic sexing strains developed through classical genetics to enhance their genetic stability and support SIT or other approaches that aim to population suppression through male-delivered sterility.

Using Moderate Transgene Expression to Improve the Genetic Sexing System of the Australian Sheep Blow Fly Lucilia cuprina.

The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a "moderate strategy" could be considered for the TESS development in other pest species.

Evaluation of Rearing Parameters of a Self-Limiting Strain of the Mediterranean Fruit Fly, Ceratitis capitata (Diptera: Tephritidae).

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a significant pest of stone and pome fruit that causes considerable economic losses worldwide. Current control is primarily based on insecticides, which are often mixed with protein baits. Oxitec has developed a self-limiting medfly strain (OX3864A) that demonstrates conditional female-specific mortality in the early life stages. Sustained release of OX3864A males offers medfly control, which should lead to substantial economic benefits in area-wide programmes. In the current study, the optimum quantities of mature and immature stages of the strain are assessed under semi-mass production. Moreover, the rearing and quality control limitations related to the production of this strain are provided. The data here demonstrate that the egg hatch rate can reach >85% under optimum rearing conditions. However, this depends on the number of pupae loaded in a cage and their ages. The suggested pupal density ranges between 14,000 and 18,000 pupae per cage to provide optimum egg production. In parallel, the embryo densities of 1.25-1.5 mL/kg larval Tet+ diet are recommended for strain propagation, while embryo densities of 1.25-2.0 mL/kg larval Tet- diet are suggested for male-only pupal production.

Advances and Challenges of Using the Sterile Insect Technique for the Management of Pest Lepidoptera.

Over the past 30 years, the sterile insect technique (SIT) has become a regular component of area-wide integrated pest management (AW-IPM) programs against several major agricultural pests and vectors of severe diseases. The SIT-based programs have been especially successful against dipteran pests. However, the SIT applicability for controlling lepidopteran pests has been challenging, mainly due to their high resistance to the ionizing radiation that is used to induce sterility. Nevertheless, the results of extensive research and currently operating SIT programs show that most problems with the implementation of SIT against pest Lepidoptera have been successfully resolved. Here, we summarize the cytogenetic peculiarities of Lepidoptera that should be considered in the development and application of SIT for a particular pest species. We also discuss the high resistance of Lepidoptera to ionizing radiation, and present the principle of derived technology based on inherited sterility (IS). Furthermore, we present successful SIT/IS applications against five major lepidopteran pests, and summarize the results of research on the quality control of reared and released insects, which is of great importance for their field performance. In the light of new research findings, we also discuss options for the development of genetic sexing strains, which is a challenge to further improve the applicability of SIT/IS against selected lepidopteran pests.

Slow Larvae Mutant and Its Potential to Improve the Pupal Color-Based Genetic Sexing System in Mexican Fruit Fly, (Diptera: Tephritidae).

For many years, an area-wide fruit fly control campaign against the Mexican fruit fly, Anastrepha ludens (Loew) has been implemented in some regions of Mexico and Texas, using the sterile insect technique (SIT) as its principal component. To improve the efficiency of the SIT, a genetic sexing strain based on black pupae mutation (bp) was developed for A. ludens, namely, 'Tapachula-7' (Tap-7 genetic sexing strains [GSSs]). This strain was introduced into the AW-IPM program recently and allows male-only releases for SIT applications. Here, we report the genetic and biological characterization of a new mutation, slow larvae (sl), which was introduced to the original translocation of the Tap-7 GSS resulting in two new GSS (slow-7 and Tap/slow-7). In both GSSs, the translocated wild-type males emerge from brown pupae that develop faster than females. The females are homozygous for sl mutation in the slow-7 GSS and homozygous for sl and bp mutations in the Tap/slow-7 GSS, reaching larval maturity 2 d after most of the wild-type males, allowing the separation of most males during pupariation. The potential use of the slow-7 and Tap/slow-7 GSSs in mass rearing and large-scale population suppression programs is discussed.

A perspective on the need and current status of efficient sex separation methods for mosquito genetic control.

Major efforts are currently underway to develop novel, complementary methods to combat mosquito-borne diseases. Mosquito genetic control strategies (GCSs) have become an increasingly important area of research on account of their species-specificity, track record in targeting agricultural insect pests, and their environmentally non-polluting nature. A number of programs targeting Aedes and Anopheles mosquitoes, vectors of human arboviruses and malaria respectively, are currently being developed or deployed in many parts of the world. Operationally implementing these technologies on a large scale however, beyond proof-of-concept pilot programs, is hampered by the absence of adequate sex separation methods. Sex separation eliminates females in the laboratory from male mosquitoes prior to release. Despite the need for sex separation for the control of mosquitoes, there have been limited efforts in recent years in developing systems that are fit-for-purpose. In this special issue of Parasites and Vectors we report on the progress of the global Coordinated Research Program on "Exploring genetic, molecular, mechanical and behavioural methods for sex separation in mosquitoes" that is led by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture with the specific aim of building efficient sex separation systems for mosquito species. In an effort to overcome current barriers we briefly highlight what we believe are the three main reasons why progress has been so slow in developing appropriate sex separation systems: the availability of methods that are not scalable, the difficulty of building the ideal genetic systems and, finally, the lack of research efforts in this area.

Construction of a genetic sexing strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito.

BACKGROUND: Aedes albopictus is an invasive mosquito species of global medical concern as its distribution has recently expanded to Africa, the Americas and Europe. In the absence of prophylaxis protecting human populations from emerging arboviruses transmitted by this mosquito species, the most straightforward control measures rely on the suppression or manipulation of vector natural populations. A number of environmental-friendly methods using mass releases of sterilizing males are currently under development. However, these strategies are still lacking an efficient sexing method required for production of males at an industrial scale. RESULTS: We present the first Genetic Sexing Strain (GSS) in Ae. albopictus, hereafter referred as Tikok, obtained by sex linkage of the rdl gene conferring dieldrin resistance. Hatch rate, larval survival and sex ratio were followed during twelve generations. The use of dieldrin at the third larval stage allowed selecting 98 % of males on average. CONCLUSION: A good production rate of Tikok males makes this GSS suitable for any control method based on mass production of Ae. albopictus males. Despite limitations resulting from reduced egg hatch as well as the nature of the used insecticide, the construction of this GSS paves the way for industrial sex separation of Ae. albopictus.

Isolation and characterization of a temperature-sensitive lethal strain of Anopheles arabiensis for SIT-based application.

BACKGROUND: Malaria is still a global health problem and vector control is the cornerstone of disease control strategies using indoor residual insecticide spraying (IRS) and insecticide-treated nets. The situation is becoming acute with widespread resistance to the limited arsenal of available insecticide classes. Therefore, new and innovative tools to reduce Plasmodium transmission are in need and this situation raised considerable interest in using sterile insect technique (SIT) against human pest insects, particularly Anopheles malaria vectors. When considering a mosquito release programme, one of the first issues to be addressed is how to eliminate/separate the hematophagous vector females. In this paper, we report the development and evaluation of an Anopheles arabiensis temperature-sensitive lethal (tsl) strain towards its potential use for the development of a genetic sexing strain (GSS). Anopheles arabiensis male mosquitoes originated from North Cameroon were treated with 0.05% ethyl methanesulfonate (EMS). The mutagen was added to 10% sucrose solution and mosquitoes fed ad-libitum for 24h and 48h on a cotton wool soaked with sucrose-mutagen solution placed at the top of the cup. Treated males were then allowed to mate with wild virgin females and their progenies were screened for heat sensitivity from F3 to F8, until the isolation and establishment of a tsl strain which was further characterized by assessing its productivity (fecundity and fertility), larval development, adult longevity as well as nature and inheritance pattern of the tsl. RESULTS: Observations showed that the number of eggs laid and their hatch rate were similar between females that mated with mutagenized males and those from the control suggesting that mutagenesis didn't affect An. arabiensis male fecundity and fertility. At F3, fourteen isofemale families out of 88 tested, which showed mortalities ranged between 50 and 80 %, were selected as lines potentially containing a tsl mutation. From F4 to F8, a tsl strain was isolated and established by screening L1 larvae at 41°C for 3 hours. This strain showed similar life history traits compared to the wild type strain in terms of fertility, larval development time and adult's emergence. Crossing experiments to further assess the nature and inheritance pattern of the tsl phenotype showed that it is due to a recessive allele located on an autosome. CONCLUSION: The successful establishment of the An. arabiensis tsl strain is a valuable tool towards the development of a GSS for SIT applications against this species. This will be done by induction of a Y-autosome translocation to link the wild-type allele to the Y chromosome in such manner that males are wild-type (temperature resistant) and females are mutant (temperature sensitive).

A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis.

The feasibility of the sterile insect technique (SIT) as a malaria vector control strategy against Anopheles arabiensis has been under investigation over the past decade. One of the critical steps required for the application of this technique to mosquito control is the availability of an efficient and effective sex-separation system. Sex-separation systems eliminate female mosquitoes from the production line prior to irradiation and field release of sterile males. This is necessary because female mosquitoes can transmit pathogens such as malaria and, therefore, their release must be prevented. Sex separation also increases the efficiency of an SIT programme. Various sex-separation strategies have been explored including the exploitation of developmental and behavioural differences between male and female mosquitoes, and genetic approaches. Most of these are however species-specific and are not indicated for the major African malaria vectors such as An. arabiensis. As there is currently no reliable sex-separation method for An. arabiensis, various strategies were explored in an attempt to develop a robust system that can be applied on a mass-rearing scale. The progress and challenges faced during the development of a sexing system for future pilot and/or large-scale SIT release programmes against An. arabiensis are reviewed here. Three methods of sex separation were examined. The first is the use of pupal size for gender prediction. The second is the elimination of blood-feeding adult females through the addition of an endectocide to a blood meal source. The third is the establishment of a genetic sexing strain (GSS) carrying an insecticide resistance selectable marker (dieldrin-resistance rdl gene and/or other GABA receptor antagonists that can be used as alternative insecticides to dieldrin) or a temperature-sensitive lethal marker.

Effects of stable ectopic expression of the primary sex determination gene Yob in the mosquito Anopheles gambiae.

BACKGROUND: Mosquito-borne diseases, such as malaria, are controlled primarily by suppressing mosquito vector populations using insecticides. The current control programmes are seriously threatened by the emergence and rapid spread of resistance to approved insecticides. Genetic approaches proposed to complement the existing control efforts may be a more sustainable solution to mosquito control. All such approaches would rely on releases of modified male mosquitoes, because released females would contribute to biting and pathogen transmission. However, no sufficiently large-scale methods for sex separation in mosquitoes exist. RESULTS: Here we exploited the female embryo-killing property of the sex determining gene Yob from the African malaria mosquito, Anopheles gambiae, to evaluate the feasibility of creating transgenic An. gambiae sexing strains with a male-only phenotype. We generated An. gambiae lines with Yob expression, in both sexes, controlled by the vas2 promoter. Penetrance of the female-lethal phenotype was highly dependent on the location of the transgenic construct within the genome. A strong male bias was observed in one of the lines. All the females that survived to adulthood in that line possessed masculinized head appendages and terminal abdominal segments. They did not feed on blood, lacked host-seeking behavior, and thus were effectively sterile. Males, however, were not affected by Yob overexpression. CONCLUSIONS: Our study demonstrates that ectopic expression of Yob results in a recovery of viable, fertile males, and in death, or otherwise strongly deleterious effects, in females. This result shows potential for generation of transgenic sexing strains of Anopheles gambiae with a conditional male-only phenotype.

Development of a genetic sexing strain of Anopheles arabiensis for KwaZulu-Natal, South Africa.

An efficient sexing system is important for the release of sterile males for any control programme using the sterile insect technique. This study describes the development and characterization of a new genetic sexing strain from South Africa (GMK), needed for the planned implementation of such a programme in northern KwaZulu-Natal Province. The base colony used was a locally modified laboratory strain of Anopheles arabiensis containing a sex-linked gene conferring dieldrin resistance to male mosquitoes. Female A. arabiensis mosquitoes from northern KwaZulu-Natal were mated with these males and backcrossed to introduce the dieldrin resistance gene to the Y chromosome. The resulting strain therefore had an overall genotype representing the local population but with the Y chromosome containing the dieldrin resistance gene. Life-history characteristics, stability of the sex-linked resistance marker, and reduction in dieldrin waste were investigated. The strain showed semi-sterility exhibited by low egg hatch rates, faster development in the immature stages and longer adult survivorship compared with the parental strains. While the GMK strain carrying the dieldrin-resistant gene was successfully established, the stability of the gene is limited, requiring periodic purification. Dieldrin waste can be limited by treating many more eggs than currently recommended.