Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of female Aedes mosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Could Sterile Aedes albopictus Male Releases Interfere with Aedes aegypti Population in Reunion Island?

In Reunion Island, the feasibility of an Aedes albopictus control program using the Sterile Insect Technique (SIT) is studied. Because, in some regions, Ae. albopictus is living in sympatry with Aedes aegypti, the impact of releasing millions of sterile male Ae. albopictus on female Ae. aegypti reproduction needs to be assessed. Thus, to study the potential heterospecific matings, a marking technique using rhodamine B has been used. Rhodamine is given in solution to male mosquitoes to be incorporated into the male body and seminal fluid and transferred during mating into the bursa inseminalis and spermathecae of females. The presence of rhodamine in females occurred in 15% of cases when Ae. aegypti females were offered non-irradiated Ae. albopictus males, 5% when offered irradiated Ae. albopictus males and 18% of cases in the inverse heterospecific matings. Moreover, our results also showed that these matings gave few eggs but were not viable. Finally, the results showed that whatever the type of mating crosses, females in cages previously crossed with males of another species can re-mate with males of their species and produce an equivalent amount of egg compared to females only mated with conspecific males. Despite the promiscuity of the males and females in small cages for three days, heterospecific mating between sterile male Ae. albopictus and female Ae aegypti, 95% of the females have not been inseminated suggesting that in the field the frequency satyrization would be very low.

Comparing sterile male releases and other methods for integrated control of the tiger mosquito in temperate and tropical climates.

The expansion of mosquito species worldwide is creating a powerful network for the spread of arboviruses. In addition to the destruction of breeding sites (prevention) and mass trapping, methods based on the sterile insect technique (SIT), the autodissemination of pyriproxyfen (ADT), and a fusion of elements from both of these known as boosted SIT (BSIT), are being developed to meet the urgent need for effective vector control. However, the comparative potential of these methods has yet to be explored in different environments. This is needed to propose and integrate informed guidelines into sustainable mosquito management plans. We extended a weather-dependent model of Aedes albopictus population dynamics to assess the effectiveness of these different vector control methods, alone or in combination, in a tropical (Reunion island, southwest Indian Ocean) and a temperate (Montpellier area, southern France) climate. Our results confirm the potential efficiency of SIT in temperate climates when performed early in the year (mid-March for northern hemisphere). In such a climate, the timing of the vector control action was the key factor in its success. In tropical climates, the potential of the combination of methods becomes more relevant. BSIT and the combination of ADT with SIT were twice as effective compared to the use of SIT alone.

Estimates of the population size and dispersal range of Anopheles arabiensis in Northern KwaZulu-Natal, South Africa: implications for a planned pilot programme to release sterile male mosquitoes.

BACKGROUND: Anopheles arabiensis is a major malaria vector, recently implicated as contributing to ongoing residual malaria transmission in South Africa, which feeds and rests both indoors and outdoors. This species is, therefore, not effectively targeted using core malaria vector control interventions alone. Additionally, increasing resistance to available insecticides necessitates investigations into complementary non-insecticide-based vector control methods for outdoor-resting mosquitoes. The feasibility of the sterile insect technique (SIT) as a complementary vector control intervention is being investigated in South Africa. Successful implementation of an SIT programme largely depends on inundating a target insect population with sterilized laboratory-bred males. Therefore, knowledge of the native population size and dispersal ability of released sterile laboratory-reared males is critical. In this study, we estimated the male An. arabiensis population size and the dispersal of released males in an area targeted for a pilot sterile male release programme. METHODS: Three separate releases were performed within a 2-year period. Approximately 5000-15,000 laboratory-reared male An. arabiensis (KWAG) were produced and marked for mark-release-recapture experiments. To recapture released mosquitoes, cloth tubes were deployed in widening concentric circles. The average dispersal distance of released males was calculated and the wild male An. arabiensis population size was estimated using two Lincoln index formulae. The natural population was sampled concurrently and Anopheles species diversity examined. RESULTS: The Anopheles gambiae complex and An. funestus group species made up the majority of wild collections along with other anophelines. The An. arabiensis population size was estimated to be between 550 and 9500 males per hectare depending on time of year, weather conditions and method used. Average dispersal distance of marked males ranged from 58 to 86 m. Marked males were found in swarms with wild males, indicating that laboratory-reared males are able to locate and participate in mating swarms. CONCLUSIONS: It was logistically feasible to conduct mark-release-recapture studies at the current scale. The population size estimates obtained may provide a guideline for the initial number of males to use for a pending SIT pilot trial. It is promising for future SIT trials that laboratory-reared marked males participated in natural swarms, appearing at the right place at the right time.

Strategic Approach, Advances, and Challenges in the Development and Application of the SIT for Area-Wide Control of Aedes albopictus Mosquitoes in Reunion Island.

The global expansion of Aedes albopictus, together with the absence of specific treatment and vaccines for most of the arboviruses it transmits, has stimulated the development of more sustainable and ecologically acceptable methods for control of disease transmission through the suppression of natural vector populations. The sterile insect technique (SIT) is rapidly evolving as an additional tool for mosquito control, offering an efficient and more environment-friendly alternative to the use of insecticides. Following the devastating chikungunya outbreak, which affected 38% of the population on Reunion Island (a French overseas territory in the southwest of the Indian Ocean), there has been strong interest and political will to develop effective alternatives to the existing vector control strategies. Over the past 10 years, the French Research and Development Institute (IRD) has established an SIT feasibility program against Ae. albopictus on Reunion Island in collaboration with national and international partners. This program aimed to determine whether the SIT based on the release of radiation-sterilized males is scientifically and technically feasible, and socially acceptable as part of a control strategy targeting the local Ae. albopictus population. This paper provides a review of a multi-year and a particularly broad scoping process of establishing the scientific and technological feasibility of the SIT against Ae. albopictus on Reunion Island. It also draws attention to some prerequisites of the decision-making process, through awareness campaigns to enhance public understanding and support, social adoption, and regulatory validation of the SIT pilot tests.

Complementarity of empirical and process-based approaches to modelling mosquito population dynamics with Aedes albopictus as an example-Application to the development of an operational mapping tool of vector populations.

Mosquitoes are responsible for the transmission of major pathogens worldwide. Modelling their population dynamics and mapping their distribution can contribute effectively to disease surveillance and control systems. Two main approaches are classically used to understand and predict mosquito abundance in space and time, namely empirical (or statistical) and process-based models. In this work, we used both approaches to model the population dynamics in Reunion Island of the 'Tiger mosquito', Aedes albopictus, a vector of dengue and chikungunya viruses, using rainfall and temperature data. We aimed to i) evaluate and compare the two types of models, and ii) develop an operational tool that could be used by public health authorities and vector control services. Our results showed that Ae. albopictus dynamics in Reunion Island are driven by both rainfall and temperature with a non-linear relationship. The predictions of the two approaches were consistent with the observed abundances of Ae. albopictus aquatic stages. An operational tool with a user-friendly interface was developed, allowing the creation of maps of Ae. albopictus densities over the whole territory using meteorological data collected from a network of weather stations. It is now routinely used by the services in charge of vector control in Reunion Island.

Aedes albopictus Adult Medium Mass Rearing for SIT Program Development.

For the production of several hundred thousands of Aedes albopictus sterile males for the implementation of a Sterile Insect Technique (SIT) program, no costly mass-rearing equipment is needed during the initial phases, as optimized rearing at laboratory scale can be sufficient for the first steps. The aim of this study was to maximize the egg production by optimizing adult rearing methods for Ae. albopictus. The effect of parameters such as male/female ratio, density of adults, membrane type for blood feeding, quantity of blood delivered, continuous or discontinuous blood feeding, and surface of substrates for egg laying on overall egg production was tested to find optimized conditions. Based on the number of eggs produced per cage in response to the parameters tested, the optimum cage set-up was seen to be 1500 adults in a 30 × 30 × 30 cm cage with a male/female sex ratio of 1:3, fed by fresh bovine blood for periods of 30 min using a cellulose membrane covering a 10 cm stainless steel plate heated by a Hemotek device, and the provision of five oviposition cups to collect eggs. With this set-up, production per cage can reach a maximum of 35,000 eggs per week.

Field evaluation of seasonal trends in relative population sizes and dispersal pattern of Aedes albopictus males in support of the design of a sterile male release strategy.

BACKGROUND: To develop an efficient sterile insect technique (SIT) programme, the number of sterile males to release, along with the spatial and temporal pattern of their release, has to be determined. Such parameters could be estimated from a reliable estimation of the wild population density (and its temporal variation) in the area to treat. Here, a series of mark-release-recapture experiments using laboratory-reared and field-derived Aedes albopictus males were carried out in Duparc, a selected pilot site for the future application of SIT in the north of La Reunion Island. METHODS: The dispersal, longevity of marked males and seasonal fluctuations in the population size of native mosquitoes were determined from the ratio of marked to unmarked males caught in mice-baited BG-Sentinel traps. The study was conducted during periods of declining population abundance (April), lowest abundance (September) and highest abundance (December). RESULTS: According to data collected in the first 4 days post-release, the Lincoln index estimated population size as quite variable, ranging from 5817 in April, to 639 in September and 5915 in December. Calculations of daily survival probability to 4 days after release for field and laboratory males were 0.91 and 0.98 in April, respectively, and 0.88 and 0.84 in September, respectively. The mean distance travelled (MDT) of released field males were 46 m, 67 m and 37 m for December, April and September experiments, respectively. For released laboratory males, the MDT was 65 m and 42 m in April and September, respectively. CONCLUSIONS: Theoretically, the most efficient release programme should be started in July/August when the mosquito population size is the lowest (c.600 wild males/ha relative to 5000 wild males estimated for December and April), with a weekly release of 6000 males/ha. The limited dispersal of Ae. albopictus males highlights the nessecity for the widespread release of sterile males over multiple sites and in a field setting to avoid topographical barriers and anthropogenic features that may block the migration of the released sterile male mosquitoes.

Assessment of the developmental success of Anopheles coluzzii larvae under different nutrient regimes: effects of diet quality, food amount and larval density.

BACKGROUND: In a context of increasing resistance of both vectors toward main classes of insecticides used in public health and parasites toward anti-malarial drugs, development of new and complementary molecules or control approaches is fundamental to achieve the objective of controlling or even eliminating malaria. Concerning vector control, the sterile insect technique and other genetic control approaches are among promising complementary tools in an integrated management strategy for malaria control. These approaches rely not only on a good understanding of vector biology (especially during larval stages), but also on the availability of adequate supplies and protocols for efficient mosquito rearing. The aim of this study was to assess the factors impacting the life history of Anopheles coluzzii mosquitoes at the larval stage, in the context of genetic and sterile insect approaches to control malaria vectors. METHODS: The effect of different larval diets and larval rearing densities on the development of An. coluzzii were evaluated in the laboratory. Emergence rate (ER), pre-imaginal developmental time (DT) and adult wing length (WL) were measured under different food regimes. Four diets were tested among which three were provided by the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint division. RESULTS: Data showed significant differences in the quality of the different diets and suggested a negative density dependence in all three life history parameters measured under tested rearing conditions. ER and WL increased with food availability, but decreased with increasing larval density. Conversely DT was shortened with increasing food availability but increased with larval density. These data demonstrates intraspecific larval competition modulated by food amount and space availability. Of the four diets tested, the one made of a mix of tuna meal, bovine liver powder, brewer's yeast, squid liver powder and vitamin mix (diet 2) yielded the best results as it produced a good balance between ER, DT and WL. Food availability for optimal development (highest survival at shortest time) was in the range of 180-400 µg/larvae/day for the three diets provided by the IPCL. CONCLUSION: There is an interaction between diet type, diet concentration and larval density. Best results in terms of optimal larvae development parameters happen when moderately high values of those three variables are observed.

Laboratory evaluation of the effects of sterilizing doses of γ-rays from Caesium-137 source on the daily flight activity and flight performance of Aedes albopictus males.

The control of Aedes albopictus through Sterile Male Releases requires that the most competitive males be mass-reared and sterilized usually with gamma- or X-ray radiation prior to release. Developing an understanding of the impact of irradiation treatment on flight performance in sterile males is very important because any fitness cost may reduce the efficacy of SIT intervention in the field. Here, we examined the role of irradiation exposure and sugar-feeding on daily flight activity and performance of Ae. albopictus males sterilized during pupal stage with gamma-radiation at 35Gray from a Caesium 137 source. We used a previously developed automated video tracking system to monitor the flight activity of different groups of sterile and control non-sterile males over 24 hours in a flight arena. This monitoring took place under controlled laboratory conditions and we wished to quantify the daily flight activity and to highlight any changes due to radiation treatment and nutritional conditions (starved versus sugar fed). Our experimental evidence demonstrated a characteristic diurnal flight activity with a bimodal pattern regardless of the treatment. Precisely, both irradiated and non-irradiated males exhibited two distinct peaks in flight activity in the morning (6-8 a.m.) and late afternoon (4-6 p.m.). Under changing physiological conditions, irradiated males were generally more active over time and flew longer overall distances than control male populations. These results suggest some internal circadian control of the phase relation to the light-dark cycle, with evidence for modification of flight performance by nutritional status. The fact that daily activity patterns are alike in irradiated and control Ae. albopictus males, and that sterile males could display higher flight performance, is in contrast with the hypothesis that irradiation treatment appears to reduce the fitness of male mosquitoes. We discuss the implications of the present study in sterile-male release programs against Ae. albopictus.

Comparison of Efficiency of BG-Sentinel Traps Baited with Mice, Mouse-Litter, and CO2 Lures for Field Sampling of Male and Female Aedes albopictus Mosquitoes.

Determining the abundance and distribution of male mosquitoes in the wild and establishing species seasonality in candidate pilot sites is of particular interest with respect to the use of the sterile-male technique. With the knowledge that using mice as bait in BG-Sentinel traps effectively enhances Aedes albopictus male and female trapping success, the present study was designed to determine whether attractants derived from mouse odour blend could be a viable substitute for live mice to lure Ae. albopictus mosquitoes into traps. The effects of baiting BG-Sentinel traps with mice, carbon dioxide (CO2), and attractants derived from litter mouse odours (mouse litter (ML)) and a mouse odour blend (MOB) on the efficiency of trapping Ae. albopictus males and females were tested using a Latin square design. The BG-Sentinel trap baited with CO2 + ML caught a significantly larger number of mosquitoes compared to traps baited with mice only. The BG-Sentinel traps containing only CO2 or CO2 + MOB, however, did not catch significantly more mosquitoes compared to the other traps. The proportions of males caught in the BG-Sentinel traps did not differ significantly between the respective attractants. The results from this study confirm that CO2 bait is efficient to provide a reliable estimation method for Ae. albopictus adult male abundance in the wild, and suggest that mouse litter baits in combination with CO2 could be used to enhance Aedes trapping success in BG-Sentinel traps.

Cross-Mating Compatibility and Competitiveness among Aedes albopictus Strains from Distinct Geographic Origins - Implications for Future Application of SIT Programs in the South West Indian Ocean Islands.

The production of large numbers of males needed for a sustainable sterile insect technique (SIT) control program requires significant developmental and operational costs. This may constitute a significant economic barrier to the installation of large scale rearing facilities in countries that are undergoing a transition from being largely dependent on insecticide use to be in a position to integrate the SIT against Aedes albopictus. Alternative options available for those countries could be to rely on outsourcing of sterile males from a foreign supplier, or for one centralised facility to produce mosquitoes for several countries, thus increasing the efficiency of the mass-rearing effort. However, demonstration of strain compatibility is a prerequisite for the export of mosquitoes for transborder SIT applications. Here, we compared mating compatibility among Ae. albopictus populations originating from three islands of the South Western Indian Ocean, and assessed both insemination rates and egg fertility in all possible cross-mating combinations. Furthermore, competitiveness between irradiated and non-irradiated males from the three studied strains, and the subsequent effect on female fertility were also examined. Although morphometric analysis of wing shapes suggested phenoptypic differences between Ae. albopictus strains, perfect reproductive compatibility between them was observed. Furthermore, irradiated males from the different islands demonstrated similar levels of competitiveness and induced sterility when confronted with fertile males from any of the other island populations tested. In conclusion, despite the evidence of inter-strain differences based on male wing morphology, collectively, our results provide a new set of expectations for the use of a single candidate strain of mass-reared sterile males for area-wide scale application of SIT against Ae. albopictus populations in different islands across the South Western Indian Ocean. Cross-mating competitiveness tests such as those applied here are necessary to assess the quality of mass reared strains for the trans-border application of sterile male release programs.

Darwin's finches treat their feathers with a natural repellent.

Darwin's finches are highly innovative. Recently we recorded for the first time a behavioural innovation in Darwin's finches outside the foraging context: individuals of four species rubbed leaves of the endemic tree Psidium galapageium on their feathers. We hypothesised that this behaviour serves to repel ectoparasites and tested the repellency of P. galapageium leaf extracts against parasites that negatively affect the fitness of Darwin's finches, namely mosquitoes and the invasive hematophagous fly Philornis downsi. Mosquitoes transmit pathogens which have recently been introduced by humans and the larvae of the fly suck blood from nestlings and incubating females. Our experimental evidence demonstrates that P. galapageium leaf extracts repel both mosquitoes and adult P. downsi and also inhibit the growth of P. downsi larvae. It is therefore possible that finches use this plant to repel ectopoarasites.

Enhancement of the BG-sentinel trap with varying number of mice for field sampling of male and female Aedes albopictus mosquitoes.

BACKGROUND: Trapping male mosquitoes in the field is essential for the development of area-wide vector control programs with a sterile insect technique (SIT) component. To determine the optimal temporal and spatial release strategy, an estimation of the wild population density and its temporal dynamics is essential. Among the traps available for such data collection, the BG-Sentinel trap developed by the Biogents company uses a combination of visual cues, convection currents and olfactory signals. Although in numerous cases, this trap has shown high efficiency in sampling Aedes albopictus, in some cases low capture rates of Ae. albopictus males were recorded for the BG-sentinel mosquito trap baited with synthetic attractants. METHODS: The effects of modifying the BG-sentinel trap (by adding one mouse, two or three live mice to the trap) on the efficiency of trapping Ae. albopictus males and females was tested. The experiment was carried out in three distinct areas on La Réunion that have been selected for pilot field testing of the release of sterile male Ae. albopictus mosquitoes. The effect of four types of attractant (including the generic BG-Lure, one mouse or two to three mice) in baited BGS traps was tested with a Latin square design in order to control for the variability of different sampling positions and dates. RESULTS: At the three studied sites, the number of Ae. albopictus adults caught and the proportion of males per trap consistently increased with the number of mice present in the trap. CONCLUSION: The results from this study suggest that some new attractants derived from, or similar to, mouse odors could be developed and tested in combination with other existing attractive components, such as CO2 and heat, in order to provide a reliable estimation method for Ae. albopictus adult male abundance in the wild.

Large-scale Anopheles arabiensis egg quantification methods for mass-rearing operations.

BACKGROUND: The success of the sterile insect technique relies, among other things, on the continuous release of over flooding numbers of sexually competitive sterile males into the target area. To produce sufficiently large quantities of sterile males, rearing protocols need to be optimized including the development and validation of a standardized egg quantification method. METHODS: Batches of 1000 freshly laid eggs collected from standard rearing cages were counted, gently dried under laboratory conditions (27 ± 1 °C, 75 ± 5 % RH) and combined so that 1000-8000 eggs were weighed, to calculate the correlation between weight and number. The actual counted egg number and the egg number estimated by weighing were further compared for samples of 1000, 3000 and 4000 eggs collected from both standard and mass-rearing cages. The effect of drying, brushing and weighing on egg hatch rate was evaluated in three samples each of 1000 fresh and 1000 dried eggs, and in batches of 1000, 3000 and 4000 dried eggs. Pupal production and adult life history traits were assessed for dried eggs hatched and reared in mass-rearing trays. Expected egg numbers and actual observed mean egg numbers were compared after gentle drying, and after applying a rapid drying method exposure to wind speed of 1.8 m/s for 30 min. RESULTS: A significant positive relationship between the number of dried eggs and egg weight was observed and the equation 'Weight (mg) = (0.00399 × Number of counted eggs) + 0.536 was derived. The actual counted mean egg number and the egg number estimated by weighing were similar for samples from small rearing cages but significantly lower for samples of 3000 and 4000 egg samples collected from mass-rearing cages. No negative effect of the drying, brushing and weighing process on egg hatch rate was observed. No significant difference was observed in any life history trait between adults reared from dried or from fresh eggs up to twenty-one days post emergence. The mean number of eggs counted from a given replicate's weight was significantly higher for egg batches fast dried with a suction device compared to those dried with a gentle drying method (fast: 1075 ± 9, gentle: 1024 ± 7). CONCLUSION: An equation has been derived to allow accurate quantification of dried Anopheles arabiensis eggs based on weight, enabling more accurate quantification of eggs for consistent larval rearing density to be achieved. Eggs can be dried for weighing in a manner which does not impair the quality of resulting adults.

Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) - II - Egg storage and hatching.

BACKGROUND: Management of large quantities of eggs will be a crucial aspect of the efficient and sustainable mass production of mosquitoes for programmes with a Sterile Insect Technique component. The efficiency of different hatching media and effectiveness of long term storage methods are presented here. METHODS: The effect on hatch rate of storage duration and three hatching media was analysed: deionized water, boiled deionized water and a bacterial broth, using Two-way ANOVA and Post hoc Tukey tests, and the Pearson correlation coefficient was used to find the effect on the proportion of collapsed eggs. Two long term storage methods were also tested: conventional storage (egg paper strips stored in zip lock bags within a sealed plastic box), and water storage (egg papers in a covered plastic cup with deionized water). Regression analyses were used to find the effect of water storage and storage duration on hatch rate. RESULTS: Both species hatched most efficiently in bacterial broth. Few eggs hatched in deionized water, and pre-boiling the water increased the hatch rate of Ae. aegypti, but not Ae. albopictus. A hatch rate greater than 80% was obtained after 10 weeks of conventional storage in Ae. aegypti and 11 weeks in Ae. albopictus. After this period, hatching decreased dramatically; no eggs hatched after 24 weeks. Storing eggs in water produced an 85% hatch rate after 5 months in both species. A small but significant proportion of eggs hatched in the water, probably due to combined effects of natural deoxygenation of the water over time and the natural instalment hatching typical of the species. CONCLUSIONS: The demonstrated efficiency of the bacterial broth hatching medium for both Ae. albopictus and Ae. aegypti facilitates mass production of these two important vector species in the same facility, with use of a common hatching medium reducing cost and operational complexity. Similarly the increased hatch rate of eggs stored in water would allow greater flexibility of egg management in a large programme over the medium term, particularly if oxygenation of the water by bubbling oxygen through the storage tray could be applied to prevent hatching during storage.

Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) - I - egg quantification.

BACKGROUND: Quantification of eggs prior to rearing the immature stages of mosquitoes is an essential step in establishing a standardized mass rearing system. To develop a simple and accurate method of egg quantification for Aedes aegypti and Aedes albopictus, the relationship between egg number and weight, as well as egg number and volume, were studied. METHODS: Known quantities of eggs (1,000, 3,000, 6,000, 12,000, 15,000, 18,000, 21,000 and 27,000) were counted and subsequently their weight and volume were measured. Best-fit curves and regression equations were used to describe relationships between Aedes egg number and both weight and volume. RESULTS: Eighteen thousand Ae. aegypti eggs weighed 159.8 mg and had a volume of 277.4 µl, compared to measurements of 131.5 mg and 230.3 µl for Ae. albopictus. The eggs of Ae. aegypti were thus larger and heavier than those of Ae. albopictus. The use of weight and volume to quantify egg number was validated by counting volumes and weights of eggs expected to correspond to 3,000 and 18,000 eggs of each species; significant correlations were found in all cases except in the case of 3,000 Ae. albopictus eggs measured by volume. CONCLUSION: Methods for egg quantification were validated and shown to be a consistent and practical means to achieve uniform distribution of Aedes larvae between rearing trays, important for optimal mass rearing of the immature stages of Aedes mosquitoes.

Participation of irradiated Anopheles arabiensis males in swarms following field release in Sudan.

BACKGROUND: The success of the sterile insect technique (SIT) depends the release of large numbers of sterile males, which are able to compete for mates with the wild male population within the target area. Unfortunately, the processes of colonisation, mass production and irradiation may reduce the competitiveness of sterile males through genetic selection, loss of natural traits and somatic damage. In this context, the capacity of released sterile Anopheles arabiensis males to survive, disperse and participate in swarms at occurring at varying distances from the release site was studied using mark-release-recapture (MRR) techniques. METHODS: In order to assess their participation in swarms, irradiated and marked laboratory-reared male mosquitoes were released 50, 100 or 200 m from the known site of a large swarm on three consecutive nights. Males were collected from this large swarm on subsequent nights. Over the three days a total of 8,100 males were released. Mean distance travelled (MDT), daily probability of survival and estimated population size were calculated from the recapture data. An effect of male age at the time of release on these parameters was observed. RESULTS: Five per cent of the males released over three days were recaptured. In two-, three- and four-day-old males, MDT was 118, 178 and 170 m, and the daily survival probability 0.95, 0.90 and 0.75, respectively. From the recapture data on the first day following each release, the Lincoln index gives an estimation of 32,546 males in the natural population. DISCUSSION: Sterile An. arabiensis males released into the field were able to find and participate in existing swarms, and possibly even initiate swarms. The survival probability decreased with the age of male on release but the swarm participation and the distance travelled by older males seemed higher than for younger males. The inclusion of a pre-release period may thus be beneficial to male competitiveness and increase the attractiveness of adult sexing techniques, such as blood spiking.

Mating competitiveness of sterile male Anopheles coluzzii in large cages.

BACKGROUND: Understanding the factors that account for male mating competitiveness is critical to the development of the sterile insect technique (SIT). Here, the effects of partial sterilization with 90 Gy of radiation on sexual competitiveness of Anopheles coluzzii allowed to mate in different ratios of sterile to untreated males have been assessed. Moreover, competitiveness was compared between males allowed one versus two days of contact with females. METHODS: Sterile and untreated males four to six days of age were released in large cages (~1.75 sq m) with females of similar age at the following ratios of sterile males: untreated males: untreated virgin females: 100:100:100, 300:100:100, 500:100:100 (three replicates of each) and left for two days. Competitiveness was determined by assessing the egg hatch rate and the insemination rate, determined by dissecting recaptured females. An additional experiment was conducted with a ratio of 500:100:100 and a mating period of either one or two days. Two controls of 0:100:100 (untreated control) and 100:0:100 (sterile control) were used in each experiment. RESULTS: When males and females consort for two days with different ratios, a significant difference in insemination rate was observed between ratio treatments. The competitiveness index (C) of sterile males compared to controls was 0.53. The number of days of exposure to mates significantly increased the insemination rate, as did the increased number of males present in the untreated: sterile male ratio treatments, but the number of days of exposure did not have any effect on the hatch rate. DISCUSSION: The comparability of the hatch rates between experiments suggest that An. coluzzii mating competitiveness experiments in large cages could be run for one instead of two days, shortening the required length of the experiment. Sterilized males were half as competitive as untreated males, but an effective release ratio of at least five sterile for one untreated male has the potential to impact the fertility of a wild female population. However, further trials in field conditions with wild males and females should be undertaken to estimate the ratio of sterile males to wild males required to produce an effect on wild populations.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.