Life-history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background.

BACKGROUND: South Africa has set a mandate to eliminate local malaria transmission by 2023. In pursuit of this objective a Sterile Insect Technique programme targeting the main vector Anopheles arabiensis is currently under development. Significant progress has been made towards operationalizing the technology. However, one of the main limitations being faced is the absence of an efficient genetic sexing system. This study is an assessment of an An. arabiensis (AY-2) strain carrying the full Y chromosome from Anopheles gambiae, including a transgenic red fluorescent marker, being introgressed into a South African genetic background as a potential tool for a reliable sexing system. METHODS: Adult, virgin males from the An. arabiensis AY-2 strain were outcrossed to virgin females from the South African, Kwazulu-Natal An. arabiensis (KWAG strain) over three generations. Anopheles arabiensis AY-2 fluorescent males were sorted as first instar larvae (L1) using the Complex Object Parametric Analyzer and Sorter (COPAS) and later screened as pupae to verify the sex. Life history traits of the novel hybrid KWAG-AY2 strain were compared to the original fluorescent AY-2 strain, the South African wild-type KWAG strain and a standard laboratory An. arabiensis (Dongola reference strain). RESULTS: The genetic stability of the sex-linked fluorescent marker and the integrity and high level of sexing efficiency of the system were confirmed. No recombination events in respect to the fluorescent marker were detected over three rounds of introgression crosses. KWAG-AY2 had higher hatch rates and survival of L1 to pupae and L1 to adult than the founding strains. AY-2 showed faster development time of immature stages and larger adult body size, but lower larval survival rates. Adult KWAG males had significantly higher survival rates. There was no significant difference between the strains in fecundity and proportion of males. KWAG-AY2 males performed better than reference strains in flight ability tests. CONCLUSION: The life history traits of KWAG-AY2, its rearing efficiency under laboratory conditions, the preservation of the sex-linked fluorescence and perfect sexing efficiency after three rounds of introgression crosses, indicate that it has potential for mass rearing. The potential risks and benefits associated to the use of this strain within the Sterile Insect Technique programme in South Africa are discussed.

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.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

Developing methods for chilling, compacting, and sterilizing adult Aedes aegypti (Diptera: Culicidae) and comparing mating competitiveness between males sterilized as adults versus pupae for sterile male release.

The yellow fever mosquito, Aedes aegypti L., can transmit several pathogens responsible for human diseases. With insecticide resistance development becoming a concern, alternative control strategies are needed for Ae. aegypti. Sterile insect technique (SIT) is an increasingly popular option being explored. However, logistical issues in mass production and sterilization make it difficult to maintain a SIT program. Male mosquitoes are typically irradiated as pupae because this is the earliest developmental point at which females can be separated from males, but asynchrony in pupation and high variability in pupal responses to irradiation based on pupal age make it difficult to sterilize mass quantities of pupae on a regular schedule in a rearing facility. Young adult mosquitoes have wider windows for irradiation sterilization than pupae, which can allow facilities to have fixed schedules for irradiation. We produced a workflow for adult Ae. aegypti irradiation in a mosquito control district with an operational SIT program that currently irradiates pupae. The impacts of chilling, compaction, and radiation dose on survival were all assessed before combining them into a complete adult irradiation protocol. Males chilled up to 16 h prior to compaction and compacted to 100 males/cm3 during radiation resulted in low mortality. Males irradiated as adults had increased longevity and similar sterility compared to males irradiated as pupae. Additionally, males sterilized as adults were more sexually competitive than males sterilized as pupae. Thus, we have shown that irradiating adult males can be a viable option to increase the efficiency of this operational mosquito SIT program.

Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti.

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

A sterile insect technique pilot trial on Captiva Island: defining mosquito population parameters for sterile male releases using mark-release-recapture.

BACKGROUND: The sterile insect technique (SIT), which involves area-wide inundative releases of sterile insects to suppress the reproduction of a target species, has proven to be an effective pest control method. The technique demands the continuous release of sterilized insects in quantities that ensure a high sterile male:wild male ratio for the suppression of the wild population over succeeding generations. METHODS: For these releases, it is important to determine several ecological and biological population parameters, including the longevity of the released males in the field, the dispersal of the released males and the wild pest population size. The Lee County Mosquito Control District initiated a study in a 47-ha portion of Captiva Island (Florida, USA), an island with a total area of 230 ha, to define biological SIT parameters for Aedes aegypti (L.), an invasive disease-vectoring mosquito known to be difficult to control due to a combination of daytime biting activity, use of cryptic breeding habitats that are difficult to target with conventional night-time ultra-low volume methods, and emerging resistance to commonly used insecticides. Another goal was to assess patterns of dispersal and survival for laboratory-reared sterile Ae. aegypti males released over time in the pilot site. These parameters will be used to evaluate the efficacy of a SIT suppression program for Ae. aegypti on Captiva Island. RESULTS: Over the course of seven mark-release-recapture studies using single- and multiple-point releases, 190,504 sterile marked males were released, for which the recapture rate was 1.5% over a mean period of 12 days. The mean distance traveled by sterile males of the local strain of Ae. aegypti that has colonized Captiva Island was 201.7 m from the release point, with an observed maximum traveled distance of 404.5 m. The released sterile mosquitoes had a probability of daily survival of 0.67 and an average life expectancy of ~ 2.46 days. CONCLUSIONS: These data together with the population size estimate and sterile:wild ratio provide a solid basis for planning the SIT operational phase which is aimed at mosquito population suppression.

Genetic Stability and Fitness of Aedes aegypti Red-Eye Genetic Sexing Strains With Pakistani Genomic Background for Sterile Insect Technique Applications.

The mosquito species Aedes aegypti is the primary transmitter of viruses that cause endemic diseases like dengue in Pakistan. It is also a cause of other vector-borne diseases like yellow fever, Zika fever, and chikungunya, which significantly impact human health worldwide. In the absence of efficient vaccines (except for yellow fever) or drugs, vector control methods, such as the sterile insect technique (SIT), have been proposed as additional tools for the management of these diseases. Mosquito SIT programs are based on the release of sterile males and it is important female releases to be ideally zero or to be kept at a minimum, since females are the ones that bite, blood-feed and transmit pathogens. Recently, an Ae. aegypti genetic sexing strain (GSS), with and without a recombination-suppressing inversion (Inv35), was developed using the eye color as a selectable marker, with males having black eyes and females red eyes. In the present study, we introgressed the sexing features and the Inv35 of the Ae. aegypti red-eye GSS into the Pakistani genomic background aiming to their future use for SIT applications in the country. Both introgressed strains, the Red-eye GSS-PAK and the Red-eye GSS/Inv35-PAK, were evaluated in respect to their genetic stability and biological quality by assessing parameters like recombination rate, fecundity, fertility, pupal and adult recovery, time of development, pupal weight, survival, and flight ability in comparison with a wild Pakistani population (PAK). The results suggest that the sexing features and the recombination suppression properties of Inv35 were not affected after their introgression into the local genomic background; however, some biological traits of the two newly constructed strains were affected, positively or negatively, suggesting that a thorough quality control analysis should be performed after the introgression of a GSS into a new genomic background prior to its use in SIT field trials or applications. The importance of using GSS with local genomic background for SIT applications against Aedes aegypti is also discussed.

Introgression of the Aedes aegypti Red-Eye Genetic Sexing Strains Into Different Genomic Backgrounds for Sterile Insect Technique Applications.

Aedes aegypti is an invasive mosquito species and major vector of human arboviruses. A wide variety of control methods have been employed to combat mosquito populations. One of them is the sterile insect technique (SIT) that has recently attracted considerable research efforts due to its proven record of success and the absence of harmful environmental footprints. The efficiency and cost-effectiveness of SIT is significantly enhanced by male-only releases. For mosquito SIT, male-only releases are ideally needed since females bite, blood-feed and transmit the pathogens. Ae. aegypti genetic sexing strains (GSS) have recently become available and are based on eye colour mutations that were chosen as selectable markers. These genetic sexing strains were developed through classical genetics and it was shown to be subjected to genetic recombination, a phenomenon that is not suppressed in males as is the case in many Diptera. The genetic stability of these GSS was strengthened by the induction and isolation of radiation-induced inversions. In this study, we used the red eye mutation and the inversion Inv35 line of the Ae. aegypti red-eye GSS s and introgressed them in six different genomic backgrounds to develop GSS with the respective local genomic backgrounds. Our goal was to assess whether the recombination frequencies in the strains with and without the inversion are affected by the different genomic backgrounds. In all cases the recombination events were suppressed in all Inv35 GSS strains, thus indicating that the genomic background does not negatively affect the inversion result. Absence of any effect that could be ascribed to genetic differences, enables the introgression of the key elements of the GSS into the local genomic background prior to release to the target areas. Maintaining the local background increases the chances for successful matings between released males and wild females and addresses potential regulatory concerns regarding biosafety and biosecurity.

The Effect of an Irradiation-Induced Recombination Suppressing Inversion on the Genetic Stability and Biological Quality of a White Eye-Based Aedes aegypti Genetic Sexing Strain.

Aedes aegypti is the primary vector of diseases such as dengue, chikungunya, Zika fever, and yellow fever. The sterile insect technique (SIT) has been proposed as a species-specific and environment-friendly tool for the suppression of mosquito vector populations as a major component of integrated vector management strategies. As female mosquitoes are blood-feeders and may transmit pathogenic microorganisms, mosquito SIT depends on the release of sterile males. Genetic sexing strains (GSS) can be used for the efficient and robust separation of males from females. Two Ae. aegypti GSS were recently developed by exploiting eye colour mutations, resulting in the Red-eye GSS (RGSS) and the White-eye GSS (WGSS). In this study, we compared two WGSS, with and without the chromosomal inversion 35 (Inv35), and evaluated their biological quality, including genetic stability. Our results suggest that the WGSS/Inv35 presents a low recombination rate and long-term genetic stability when recombinants are removed from the colony (filtering) and a slow accumulation of recombinants when they are not removed from the colony (non-filtering). The two strains were similar with respect to fecundity, pupal and adult recovery rates, pupation curve, and pupal weight. However, differences were detected in fertility, survival rate of females, and flight ability of males. The WGSS/Inv35 presented lower fertility, higher survival rate of females, and better flight ability of males compared to the WGSS.

Exploring Conditions for Handling Packing and Shipping Aedes aegypti Males to Support an SIT Field Project in Brazil.

The sterile insect technique (SIT) application, as an alternative tool for conventional mosquito control methods, has recently gained prominence. Nevertheless, some SIT components require further development, such as protocols under large-scale conditions, focusing on packing and shipping mosquitoes, and considering transporting time. Immobilization of Aedes aegypti males was tested at temperatures 4, 7, 10, and 14 °C, and each temperature was assessed for 60, 90, and 120 min. The recovery after 24 h was also studied. Chilled and control-reared males had comparable survival rates for all conditions, although 4 °C for 120 min impacted male survival. The male escape rate was affected after 60 min of exposure at 4 °C; this difference was not significant, with 24 h of recovery. First, we defined the successful immobilization at 4 °C for 60 min, thus enabling the evaluation of two transportation intervals: 6 and 24 h, with the assessment of different compaction densities of 100 and 150 mosquitoes/cm3 at 10 °C to optimize the shipment. Compaction during simulated mosquito shipments reduced survival rates significantly after 6 and 24 h. In the mating propensity and insemination experiments, the sterile males managed to inseminate 40 to 66% for all treatments in laboratory conditions. The male insemination propensity was affected only by the highest compaction condition concerning the control. The analysis of the densities (100 and 150 males/cm3) showed that a higher density combined with an extended shipment period (24 h) negatively impacted the percentage of inseminated females. The results are very helpful in developing and improving the SIT packing and shipment protocols. Further studies are required to evaluate all combined parameters' synergetic effects that can combine irradiation to assess sexual competitiveness when sterile males are released into the field.

Population Dynamics of Aedes aegypti and Aedes albopictus in Two Rural Villages in Southern Mexico: Baseline Data for an Evaluation of the Sterile Insect Technique.

Indoor and outdoor ovitraps were placed in 15 randomly selected houses in two rural villages in Chiapas, southern Mexico. In addition, ovitraps were placed in five transects surrounding each village, with three traps per transect, one at the edge, one at 50 m, and another at 100 m from the edge of the village. All traps were inspected weekly. A transect with eight traps along a road between the two villages was also included. Population fluctuations of Aedes aegypti and Ae. albopictus were examined during 2016-2018 by counting egg numbers. A higher number of Aedes spp. eggs was recorded at Hidalgo village with 257,712 eggs (60.9%), of which 58.1% were present in outdoor ovitraps and 41.9% in indoor ovitraps, compared with 165,623 eggs (39.1%) collected in the village of Río Florido, 49.0% in outdoor and 51.0% in indoor ovitraps. A total of 84,047 eggs was collected from ovitraps placed along transects around Río Florido, compared to 67,542 eggs recorded from transects around Hidalgo. Fluctuations in egg counts were associated with annual variation in precipitation, with 2.3 to 3.2-fold more eggs collected from ovitraps placed in houses and 4.8 to 5.1-fold more eggs in ovitraps from the surrounding transects during the rainy season than in the dry season, respectively. Aedes aegypti was the dominant species during the dry season and at the start of the rainy season in both villages. Aedes albopictus populations were lower for most of the dry season, but increased during the rainy season and predominated at the end of the rainy season in both villages. Aedes albopictus was also the dominant species in the zones surrounding both villages. The numbers of eggs collected from intradomiciliary ovitraps were strongly correlated with the numbers of eggs in peridomiciliary ovitraps in both Río Florido (R2 adj = 0.92) and Hidalgo (R2 adj = 0.94), suggesting that peridomiciliary sampling could provide an accurate estimate of intradomiciliary oviposition by Aedes spp. in future studies in these villages. We conclude that the feasibility of sterile insect technique (SIT)-based program of vector control could be evaluated in the isolated Ae. aegypti populations in the rural villages of our baseline study.

Genetic sexing strains for the population suppression of the mosquito vector Aedes aegypti.

Aedes aegypti is the primary vector of arthropod-borne viruses including dengue, chikungunya and Zika. Vector population control methods are reviving to impede disease transmission. An efficient sex separation for male-only releases is crucial for area-wide mosquito population suppression strategies. Here, we report on the construction of two genetic sexing strains using red- and white-eye colour mutations as selectable markers. Quality control analysis showed that the Red-eye genetic sexing strains (GSS) is better and more genetically stable than the White-eye GSS. The introduction of an irradiation-induced inversion (Inv35) increases genetic stability and reduces the probability of female contamination of the male release batches. Bi-weekly releases of irradiated males of both the Red-eye GSS and the Red-eye GSS/Inv35 fully suppressed target laboratory cage populations within six and nine weeks, respectively. An image analysis algorithm allowing sex determination based on eye colour identification at the pupal stage was developed. The next step is to automate the Red-eye-based genetic sexing and validate it in pilot trials prior to its integration in large-scale population suppression programmes. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Sexual Competitiveness and Induced Egg Sterility by Aedes aegypti and Aedes albopictus Gamma-Irradiated Males: A Laboratory and Field Study in Mexico.

The sterile insect technique may prove useful for the suppression of mosquito vectors of medical importance in regions where arboviruses pose a serious public health threat. In the present study, we examined the effects of sterilizing irradiation doses across different ratios of fertile:irradiated males on the mating competitiveness of Ae. aegypti and Ae. albopictus under laboratory and field-cage conditions. For both species, the percentage of females inseminated and the number of eggs laid over two gonotrophic cycles varied significantly in mating treatments involving 1:1, 1:5, and 1:10 fertile:irradiated males compared to controls of entirely fertile or entirely irradiated males but was not generally affected by the irradiation dose. Egg hatching was negatively affected in females exposed to increasing proportions of irradiated males in both laboratory and field cages. Male competitiveness (Fried's index) values varied from 0.19 to 0.58 in the laboratory and were between 0.09 and 1.0 in field cages, depending on th species. Competitiveness values were negatively affected by th eirradiation dose in both species under field-cage conditions, whereas in the laboratory, Ae. albopictus was sensitive to the dose but Ae. aegypti was not. In general, male competitiveness was similar across all mating regimes. Most importantly, induced egg sterility was positively correlated with the proportion of irradiated males present in the mating treatments, reaching a maximum of 88% under field-cage conditions for both Ae. aegypti and Ae. albopictus males treated with 50 and 40 Gy irradiation, respectively. These results indicate that sterile males produced at our facility are suitable and competitive enough for field pilot SIT projects and provide guidance to decide the optimal sterile:fertile ratios.

The role of oxygen depletion and subsequent radioprotective effects during irradiation of mosquito pupae in water.

BACKGROUND: Radiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in water versus in air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions. METHODS: Pupae of Aedes aegypti, Ae. albopictus, and Anopheles arabiensis were submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae of Ae. aegypti (aged 40-44 h), Ae. albopictus (aged 40-44 h) and An. arabiensis (aged 20-24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with < 0.5% O2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility. RESULTS: All three species depleted the water of DO to levels under 0.5% within 30 minutes, with An. arabiensis consuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses (P < 0.0001), increasing at higher doses. This effect was most pronounced in An. arabiensis. CONCLUSIONS: The consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.

Optimization of irradiation dose to Aedes aegypti and Ae. albopictus in a sterile insect technique program.

The sterile insect technique (SIT) may offer a means to control the transmission of mosquito borne diseases. SIT involves the release of male insects that have been sterilized by exposure to ionizing radiation. We determined the effects of different doses of radiation on the survival and reproductive capacity of local strains of Aedes aegypti and Ae. albopictus in southern Mexico. The survival of irradiated pupae was invariably greater than 90% and did not differ significantly in either sex for either species. Irradiation had no significant adverse effects on the flight ability (capacity to fly out of a test device) of male mosquitoes, which consistently exceeded 91% in Ae. aegypti and 96% in Ae. albopictus. The average number of eggs laid per female was significantly reduced in Ae. aegypti at doses of 15 and 30 Gy and no eggs were laid by females that had been exposed to 50 Gy. Similarly, in Ae. albopictus, egg production was reduced at doses of 15 and 25 Gy and was eliminated at 35 Gy. In Ae. aegypti, fertility in males was eliminated at 70 Gy and was eliminated at 30 Gy in females, whereas in Ae. albopictus, the fertility of males that mated with untreated females was almost zero (0.1%) in the 50 Gy treatment and female fertility was eliminated at 35 Gy. Irradiation treatments resulted in reduced ovary length and fewer follicles in both species. The adult median survival time of both species was reduced by irradiation in a dose-dependent manner. However, sterilizing doses of 35 Gy and 50 Gy resulted in little reduction in survival times of males of Ae. albopictus and Ae. aegypti, respectively, indicating that these doses should be suitable for future evaluations of SIT-based control of these species. The results of the present study will be applied to studies of male sexual competitiveness and to stepwise evaluations of the sterile insect technique for population suppression of these vectors in Mexico.

Correction: Mosquito pornoscopy: Observation and interruption of Aedes aegypti copulation to determine female polyandric event and mixed progeny.

[This corrects the article DOI: 10.1371/journal.pone.0193164.].

Mosquito pornoscopy: Observation and interruption of Aedes aegypti copulation to determine female polyandric event and mixed progeny.

Ades aegypti is the most important arbovirus vector in the world, and new strategies are under evaluation. Biological studies mentioning the occurrence of a second mate in Aedes aegypti can interfere with vector control program planning, which involves male mosquito release technique. This study presents different experiments to show the occurrence of mixed progeny. Mixed male crosses (using a combination of different type of males in confinement with virgin females) showed no polyandric female. Individual crosses with male substitution in every gonotrophic cycle also did not show any polyandric female. Individual crosses with a 20 minutes interval, with subsequent male change, showed that only a few females presented mixed offspring. The copulation breach in three different moments, group A with full coitus length, group B the coitus was interrupted in 5-7 seconds after the start; and group C, which the copulation was interrupted 3 seconds after started. In summary, group A showed a majority of unique progeny from the first male; group B showed the higher frequency of mixed offspring and group C with the majority of the crosses belonging to the second male. To conclude, the occurrence of a viable second mate and mixed offspring is only possible when the copulation is interrupted; otherwise, the first mate is responsible for mixed progeny.

Dispersal of Engineered Male Aedes aegypti Mosquitoes.

BACKGROUND: Aedes aegypti, the principal vector of dengue fever, have been genetically engineered for use in a sterile insect control programme. To improve our understanding of the dispersal ecology of mosquitoes and to inform appropriate release strategies of 'genetically sterile' male Aedes aegypti detailed knowledge of the dispersal ability of the released insects is needed. METHODOLOGY/PRINCIPAL FINDINGS: The dispersal ability of released 'genetically sterile' male Aedes aegypti at a field site in Brazil has been estimated. Dispersal kernels embedded within a generalized linear model framework were used to analyse data collected from three large scale mark release recapture studies. The methodology has been applied to previously published dispersal data to compare the dispersal ability of 'genetically sterile' male Aedes aegypti in contrasting environments. We parameterised dispersal kernels and estimated the mean distance travelled for insects in Brazil: 52.8 m (95% CI: 49.9 m, 56.8 m) and Malaysia: 58.0 m (95% CI: 51.1 m, 71.0 m). CONCLUSIONS/SIGNIFICANCE: Our results provide specific, detailed estimates of the dispersal characteristics of released 'genetically sterile' male Aedes aegypti in the field. The comparative analysis indicates that despite differing environments and recapture rates, key features of the insects' dispersal kernels are conserved across the two studies. The results can be used to inform both risk assessments and release programmes using 'genetically sterile' male Aedes aegypti.

Aedes aegypti Control Strategies in Brazil: Incorporation of New Technologies to Overcome the Persistence of Dengue Epidemics.

Dengue is considered to be the most important mosquito-borne viral disease in the world. The Aedes aegypti mosquito, its vector, is highly anthropophilic and is very well adapted to urban environments. Although several vaccine candidates are in advanced stages of development no licensed dengue vaccine is yet available. As a result, controlling the spread of dengue still requires that mosquitoes be targeted directly. We review the current methods of dengue vector control focusing on recent technical advances. We first examine the history of Brazil's National Dengue Control Plan in effect since 2002, and we describe its establishment and operation. With the persistent recurrence of dengue epidemics, current strategies should be reassessed to bring to the forefront a discussion of the possible implementation of new technologies in Brazil's mosquito control program.

Suppression of a Field Population of Aedes aegypti in Brazil by Sustained Release of Transgenic Male Mosquitoes.

The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males (0.031; 95% CI: 0.025-0.036) was similar to that estimated in the Cayman trials (0.059; 95% CI: 0.011-0.210), indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.