Successful introgression of wMel Wolbachia into Aedes aegypti populations in Fiji, Vanuatu and Kiribati.

Pacific Island countries have experienced periodic dengue, chikungunya and Zika outbreaks for decades. The prevention and control of these mosquito-borne diseases rely heavily on control of Aedes aegypti mosquitoes, which in most settings are the primary vector. Introgression of the intracellular bacterium Wolbachia pipientis (wMel strain) into Ae. aegypti populations reduces their vector competence and consequently lowers dengue incidence in the human population. Here we describe successful area-wide deployments of wMel-infected Ae. aegypti in Suva, Lautoka, Nadi (Fiji), Port Vila (Vanuatu) and South Tarawa (Kiribati). With community support, weekly releases of wMel-infected Ae. aegypti mosquitoes for between 2 to 5 months resulted in wMel introgression in nearly all locations. Long term monitoring confirmed a high, self-sustaining prevalence of wMel infecting mosquitoes in almost all deployment areas. Measurement of public health outcomes were disrupted by the Covid19 pandemic but are expected to emerge in the coming years.

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

BACKGROUND: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. CONCLUSION: These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.

A standardised method of marking male mosquitoes with fluorescent dust.

BACKGROUND: Prior to a major release campaign of sterile insects, including the sterile insect technique, male mosquitoes must be marked and released (small scale) to determine key parameters including wild population abundance, dispersal and survival. Marking insects has been routinely carried out for over 100 years; however, there is no gold standard regarding the marking of specific disease-transmitting mosquitoes including Anopheles arabiensis, Aedes aegypti and Aedes albopictus. The research presented offers a novel dusting technique and optimal dust colour and quantities, suitable for small-scale releases, such as mark-release-recapture studies. METHODS: We sought to establish a suitable dust colour and quantity for batches of 100 male An. arabiensis, that was visible both by eye and under UV light, long-lasting and did not negatively impact longevity. A set of lower dust weights were selected to conduct longevity experiments with both Ae. aegypti and Ae. albopictus to underpin the optimal dust weight. A further study assessed the potential of marked male An. arabiensis to transfer their mark to undusted males and females. RESULTS: The longevity of male An. arabiensis marked with various dust colours was not significantly reduced when compared to unmarked controls. Furthermore, the chosen dust quantity (5 mg) did not negatively impact longevity (P = 0.717) and provided a long-lasting mark. Dust transfer was found to occur from marked An. arabiensis males to unmarked males and females when left in close proximity. However, this was only noticeable when examining individuals under a stereomicroscope and thus deemed negligible. Overall, male Ae. aegypti and Ae. albopictus displayed a greater sensitivity to dusting. Only the lowest dust weight (0.5 mg) did not significantly reduce longevity (P = 0.888) in Ae. aegypti, whilst the lowest two dust weights (0.5 and 0.75 mg) had no significant impact on longevity (P = 0.951 and 0.166, respectively) in Ae. albopictus. CONCLUSION: We have devised a fast, inexpensive and simple marking method and provided recommended dust quantities for several major species of disease-causing mosquitoes. The novel technique provides an evenly distributed, long-lasting mark which is non-detrimental. Our results will be useful for future MRR studies, prior to a major release campaign.

Demonstration of resistance to satyrization behavior in Aedes aegypti from La Réunion island.

Aedes aegypti and Aedes albopictus are competent vectors of arboviruses such as dengue and chikungunya viruses which co-exist in some areas, including La Réunion island, Indian Ocean. A type of reproductive interference called satyrization has been described in sympatric species where dominant species mating fails to produce hybrids and thus reduces the fitness and tends to control the spread of the other species. Here, we investigated satyrization in laboratory experiments to provide insights on the potential impact on Ae. aegypti of a control campaign including a sterile insect technique component against Ae. albopictus. Different mating crosses were used to test sympatric, conspecific-interspecific and allopatric effects of irradiated and non-irradiated male Ae. albopictus on female Ae. aegypti, including in a situation of skewed male ratio. Our results suggest that there was only a low level of satyrization between sympatric populations of Ae. aegypti and Ae. albopictus colonized from La Réunion island. A male Ae. albopictus to female Ae. aegypti ratio of 3:1 did not increase the level of satyrization. Female Ae. aegypti previously mated to male Ae. albopictus were not prevented from being inseminated by conspecific males. A satyrization effect was not seen between allopatric Ae. albopictus and Ae. aegypti strains from La Réunion Island either. The tested Ae. aegypti strain from La Réunion island has therefore developed full resistance to satyrization and so releasing sterile male Ae. albopictus may not suppress Ae. aegypti populations if an overflooding of irradiated male Ae. albopictus leads to similar results. The management strategy of two competent species in a sympatric area is discussed.

TITLE: Démonstration de la résistance au comportement de satyrisation chez Aedes aegypti de l'île de La Réunion. ABSTRACT: Aedes aegypti et Aedes albopictus sont des vecteurs compétents d'arbovirus, tels que les virus de la dengue et du chikungunya, qui coexistent dans certaines régions, y compris à La Réunion, dans l'Océan Indien. Une interférence sur la reproduction appelée satyrisation a été décrite chez les espèces sympatriques où l'accouplement d'une espèce dominante ne produit pas d'hybrides et donc réduit le succès reproducteur et tend à contrôler la propagation des autres espèces. Ici, nous avons étudié la satyrisation dans des expériences de laboratoire pour fournir des informations sur l'impact potentiel sur Ae. aegypti d'une campagne de lutte contre Ae. albopictus basée sur la technique de l'insecte stérile. Différents croisements d'accouplements ont été utilisés pour tester les effets sympatriques, conspécifiques-interspécifiques et allopatriques de mâles Ae. albopictus irradiés et non irradiés sur les femelles Ae. aegypti, y compris dans une situation de ratio masculin asymétrique. Nos résultats suggèrent qu'il n'y avait qu'un faible niveau de satyrisation entre les populations sympatriques d'Ae. aegypti et Ae. albopictus colonisées à La Réunion. Un rapport mâles Ae. albopictus sur femelles Ae. aegypti de 3 contre 1 n'a pas augmenté le niveau de satyrisation. Les femelles Ae. aegypti déjà accouplées à un mâle Ae. albopictus n'ont pas été empêchées d'être inséminées par des mâles conspécifiques. Aucun effet de satyrisation n'a été observé entre les souches allopatriques d'Ae. albopictus et d'Ae. aegypti de La Réunion. La souche testée d'Ae. aegypti de La Réunion a donc développé une résistance totale à la satyrisation et en conséquence la libération de mâles stériles d'Ae. albopictus peut ne pas supprimer les populations d'Ae. aegypti si des lâchers massifs de mâles irradiés d'Ae. albopictus conduisent à des résultats similaires. La stratégie de gestion de deux espèces compétentes dans une zone sympatrique est discutée.

Insects to feed insects - feeding Aedes mosquitoes with flies for laboratory rearing.

The black soldier fly, yellow mealworm and house fly are known for their wide distribution, ease of breeding, and environmental and nutritional attributes. Diets based on these fly proteins for the rearing of mosquito larvae are more accessible and affordable when compared to the reference IAEA diet which consists largely of costly livestock products such as bovine liver powder. Following a step-by-step assessment, we developed diet mixtures based on insect meal for the optimal mass production of Aedes albopictus and Ae. aegypti. Based on the assessed parameters including mosquito egg hatch, body size, flight ability, longevity and diet cost reduction, two mixtures are recommended: 1/2 tuna meal (TM) + 7/20 black soldier fly (BSF) + 3/20 brewer's yeast and 1/2 TM + 1/2 BSF. These findings, which could be adapted to other mosquito species, provide alternative protein sources for mass rearing insects for genetic control strategies.

Investigating the impact of chilling temperature on male Aedes aegypti and Aedes albopictus survival.

In genetic control programmes, including the sterile insect technique (SIT), it is crucial to release insects of the highest quality with maximum survival. It is likely that male mosquitoes will follow the trend of other insects in SIT programmes and be stored, transported and eventually released under chilled conditions. The aim of our study was to investigate the impact of different chilling temperatures on male Aedes aegypti and Ae. albopictus survival by exposing them to a range of temperatures for different durations. Ae. aegypti were found to be less sensitive to the impact of chilling, with only 6°C causing a marginal decrease in survival in comparison to non-chilled controls. Conversely, Ae. albopictus displayed a significantly reduced survival at all chilling temperatures even when exposed for a short time. In both species, longer exposure to low temperatures reduced survival. Our results uncovered that Ae. albopictus are more sensitive to chilling, regardless of the temperature, when compared to Ae. aegypti. Such results indicate differences in thermal tolerances between species and the necessity of conducting experiments on a species by species basis when determining temperature limits for any insect destined for release as part of a genetic control programme.

Incompatible and sterile insect techniques combined eliminate mosquitoes.

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

Optimization of Mass-Rearing Methods for Anopheles arabiensis Larval Stages: Effects of Rearing Water Temperature and Larval Density on Mosquito Life-History Traits.

Insect mass-rearing is an essential requirement for the sterile insect technique. Production at a large scale requires the development of standardized rearing procedures to produce good quality males able to compete with wild males to mate with wild females. Three sets of experiments (using trays placed on the table, the whole tray-rack system, and climate-controlled chambers) have been conducted aiming to determine the optimal water temperature and number of eggs to aliquot into each larval rearing tray to achieve the highest production of pupae. No difference was found in time to pupation, sex ratio, or male body size as a result of altering larval density. However, higher larval densities resulted in decreased emergence rate and female body size. A constant water temperature of 22°C delayed hatching and did not allow Anopheles arabiensis to complete larval development. Hatching eggs in water at 22°C and then increasing water temperature to 27°C resulted in decreased pupae production compared to eggs hatched and larvae maintained at a water temperature of 27°C throughout. Water temperature and larval density affected the production parameters of An. arabiensis mosquitoes, which has implications for mass release programs. We conclude that 4,000 eggs per 4 liter and a water temperature of 27°C were the optimal conditions for mass-rearing this mosquito species which yielded 105,000 pupae/larval rearing unit. These results are valuable information in the development of standard operation procedures for the efficient large-scale rearing of An. arabiensis mosquitoes.

Establishment of a medium-scale mosquito facility: tests on mass production cages for Aedes albopictus (Diptera: Culicidae).

BACKGROUND: Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency. METHODS: Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm2/mosquito, 0.675 cm2/mosquito and 0.54 cm2/mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m2 space. RESULTS: Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm2/mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days. CONCLUSIONS: Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory.

Establishment of a medium-scale mosquito facility: optimization of the larval mass-rearing unit for Aedes albopictus (Diptera: Culicidae).

BACKGROUND: Standardized larval rearing units for mosquito production are essential for the establishment of a mass-rearing facility. Two larval rearing units, developed respectively by the Guangzhou Wolbaki Biotech Co. Ltd. (Wolbaki) and Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (FAO/IAEA-IPCL), are tested to assess their potential uses to mass-rear the larval stages of Aedes albopictus in support of the establishment of a medium-scale mosquito facility for the application of mosquito genetic control strategies. METHODS: The triple Wolbachia-infected Ae. albopictus strain (HC strain) was used in this study. The effects of larval densities of two larval rearing trays (corresponding to 2.4, 3.0 and 3.6 larvae/cm2) and tray size/position (top, middle and bottom layers) on the pupae production and larval survival were assessed when trays were stacked within the larval rearing units. The male pupae production, female pupae contamination after sex separation, and male mating competitiveness were also studied by using both larval rearing units in their entirety. RESULTS: The optimal larval rearing density for Wolbaki-tray (Wol-tray) was 6,600 larvae (equal to 3.0 larvae/cm2) and 18,000 larvae (3.6 larvae/cm2) for the FAO/IAEA-IPCL tray (IAEA-tray). No significant difference in pupae production was observed when trays were stacked within top, middle or bottom layers for both units. At thirty-four hours after the first pupation, the average male pupae production was (0.89 × 105) for the Wol-unit and (3.16 × 105) for the IAEA-unit. No significant difference was observed in female pupae contamination between these two units. The HC males showed equal male mating competitiveness to wild type males for mating with wild type females in large cages, regardless of whether they were reared in the Wol-unit or IAEA-unit. CONCLUSIONS: The current study has indicated that both the Wol-unit and IAEA-unit are suitable for larvae mass-rearing for Ae. albopictus. However, the IAEA-unit, with higher male production and less space required compared to the Wol-unit, is recommended to be used in support of the establishment of a medium-sized mosquito facility.

Does mosquito mass-rearing produce an inferior mosquito?

BACKGROUND: The success of the sterile insect technique depends, among other things, on continuous releases of sexually competitive sterile males within the target area. Several factors (including high rearing density and physical manipulation, such as larvae and pupae separation) can influence the quality of males produced in mass-rearing facilities. The different steps in mass production in the laboratory may modify the behaviour of mosquitoes, directly or through loss of natural characters as a result of adaptation to lab rearing, and lead to the competitiveness of sterile male being reduced. In the present study, the objective was to evaluate the effect of mass-rearing conditions on sterile male sexual competitiveness in semi-field cages compared to routine small scale laboratory rearing methods. METHODS: Anopheles arabiensis immature stages were reared both on a large scale using a rack and tray system developed by the FAO/IAEA (MRS), and on a small scale using standard laboratory rearing trays (SRS). Mosquito life history traits such as pupation rate, emergence rate, adult size as well as the effect of irradiation on adult longevity were evaluated. Moreover, 5-6 day old mosquitoes were released into field cages and left for two nights to mate and the mating competitiveness between sterile mass-reared males and fertile males reared on a small scale when competing for small scale reared virgin females was investigated. Resulting fertility in a treatment ratio of 1:1:1 (100 irradiated males: 100 non-irradiated males: 100 virgin females) was compared to control cages with 0:100:100 (non-irradiated control) and 100:0:100 (irradiated control). RESULTS: No significant differences in life history parameters were observed between rearing methods. The competitiveness index of mass reared males (0.58) was similar to males reared on a small scale (0.59). A residual fertility rate of 20% was observed in the irradiated control (100:0:100), measured as the percentage of eggs collected from the cages which developed to adulthood. No significant difference was observed (t = 0.2896, df = 4, P = 0.7865) between the rearing treatments (MRS and SRS) in the fertility rate, a measure of mating competitiveness. CONCLUSIONS: The results showed that the FAO/IAEA mass-rearing process did not affect mosquito life history parameters or the mating competitiveness of males.

Reverse osmosis and ultrafiltration for recovery and reuse of larval rearing water in Anopheles arabiensis mass production: Effect of water quality on larval development and fitness of emerging adults.

BACKGROUND: Countries around the world are showing increased interest in applying the sterile insect technique against mosquito disease vectors. Many countries in which mosquitoes are endemic, and so where vector control using the sterile insect technique may be considered, are located in arid zones where water provision can be costly or unreliable. Water reuse provides an alternate form of water supply. In order to reduce the cost of mass rearing of Anopheles arabiensis mosquitoes, the possibility of recycling and reusing larval rearing water was explored. METHODS: The used rearing water ('dirty water') was collected after the tilting of rearing trays for collection of larvae/pupae, and larvae/pupae separation events and underwent treatment processes consisting of ultrafiltration and reverse osmosis. First-instar An. arabiensis larvae were randomly assigned to different water-type treatments, 500 larvae per laboratory rearing tray: 'clean' dechlorinated water, routinely used in rearing; dirty water; and 'recycled' dirty water treated using reverse osmosis and ultrafiltration. Several parameters of insect quality were then compared: larval development, pupation rate, adult emergence, body size and longevity. Water quality of the samples was analyzed in terms of ammonia, nitrite, nitrate, sulphate, dissolved oxygen, chloride, and phosphate concentrations after the larvae had all pupated or died. Surface water temperatures were also recorded continuously during larval development. RESULTS: Pupation rates and adult emergence were similar in all water treatments. Adult body sizes of larvae reared in recycled water were similar to those reared in clean water, but larger than those reared in the dirty larval water treatment, whereas the adult longevity of larvae reared in recycled water was significantly increased relative to both 'clean' and 'dirty' water. Dirty larval water contained significantly higher concentrations of ammonium, sulfate, phosphate and chloride and lower levels of dissolved oxygen than clean water. These parameters significantly varied during the period of larval development. After dirty water was recycled by ultrafiltration and reverse osmosis, all the parameters measured were the same as those in clean water. CONCLUSION: This study demonstrated the potential for using recycled larval rearing water to supplement clean dechlorinated water supplies. Recycling used water improved its quality and of the reared mosquitoes. As water demands and environmental pressures grow, recycling of larval rearing water will improve the sustainability and affordability of mosquito mass-rearing.

Optimization of mosquito egg production under mass rearing setting: effects of cage volume, blood meal source and adult population density for the malaria vector, Anopheles arabiensis.

BACKGROUND: Anopheles arabiensis is one of the major malaria vectors that put millions of people in endemic countries at risk. Mass-rearing of this mosquito is crucial for strategies that use sterile insect technique to suppress vector populations. The sterile insect technique (SIT) package for this mosquito species is being developed by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. To improve mass-rearing outcomes for An. arabiensis, the question of whether the egg production by females would be affected by the size of the adult holding cages, the source of the blood meal and the total number of pupae that could be loaded into the cages was addressed and finally the impact of adding additional pupae to the cage daily to maintain adult numbers on egg productivity assessed. METHODS: Mass production cages of two different volumes, two different sources of blood meal (bovine and porcine) and two different population densities (cages originally loaded with either 15,000 or 20,000 pupae) were tested and evaluated on the basis of eggs produced/cage or per female. Males and females pupae with a ratio of 1:1 were added to the cages at day 1 and 2 of pupation. The emerging adults had constant access to 5% sugar solution and blood fed via the Hemotek membrane feeding system. Eggs were collected either twice a week or daily. A generalized linear model was used to identify factors which gave significantly higher egg production. RESULTS: Neither cage volume nor blood meal source affected egg production per cage or per female. However, increasing population density to 20,000 pupae had a negative effect on eggs produced per cage and per female. Although high density negatively impacted egg production, adding 1000 daily additional pupae compensating for daily mortality resulted in a substantial increase in egg production. Moreover, in all tests the first and the third egg batches collected were significantly higher than others eggs batches. With the equipment and protocols described here and routinely used at the Insect Pest Control Laboratory (IPCL), it was possible to produce up to 120,000 eggs/cage/day. CONCLUSION: These results demonstrated that 15,000 is the optimal number of pupae to be loaded into the Anopheles Mass production cages. Under this condition, an average of 40 eggs per female was obtained for five gonotrophic cycles. However, an improvement in egg production can be achieved by daily addition, to the original 15,000 pupae, of one thousand pupae a day. Interestingly, feeding females with bovine or porcine blood using both large and small versions of the mass production cage did not affect egg productivity.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

BACKGROUND: Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. METHODS: The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. RESULTS: Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the highest sterility in the representative wild-type population, with potential to effectively suppress reproduction. CONCLUSION: Laboratory-reared and sterilised GAMA male An. arabiensis at a release ratio of 3:1 (3 sterile males to 1 wild, fertile male) can successfully compete for insemination of wild-type females. These results will be used to inform subsequent small-scale pilot field releases in South Africa.

Combining the Sterile Insect Technique with the Incompatible Insect Technique: III-Robust Mating Competitiveness of Irradiated Triple Wolbachia-Infected Aedes albopictus Males under Semi-Field Conditions.

Combination of the sterile insect technique with the incompatible insect technique is considered to be a safe approach to control Aedes albopictus populations in the absence of an accurate and scalable sex separation system or genetic sexing strain. Our previous study has shown that the triple Wolbachia-infected Ae. albopictus strain (wAlbA, wAlbB and wPip) was suitable for mass rearing and females could be completely sterilized as pupae with a radiation dose of at least 28 Gy. However, whether this radiation dose can influence the mating competitiveness of the triple infected males was still unknown. In this study we aimed to evaluate the effects of irradiation on the male mating competitiveness of the triple infected strain under laboratory and semi-field conditions. The results herein indicate that irradiation with a lower, female-sterilizing dose has no negative impact on the longevity of triple infected males while a reduced lifespan was observed in the wild type males (wAlbA and wAlbB) irradiated with a higher male-sterilizing dose, in small cages. At different sterile: fertile release ratios in small cages, triple-infected males induced 39.8, 81.6 and 87.8% sterility in a wild type female population at 1:1, 5:1 and 10:1 release ratios, respectively, relative to a fertile control population. Similarly, irradiated triple infected males induced 31.3, 70.5 and 89.3% sterility at 1:1, 5:1 and 10:1 release ratios, respectively, again relative to the fertile control. Under semi-field conditions at a 5:1 release ratio, relative to wild type males, the mean male mating competitiveness index of 28 Gy irradiated triple-infected males was significantly higher than 35 Gy irradiated wild type males, while triple infected males showed no difference in mean mating competitiveness to either irradiated triple-infected or irradiated wild type males. An unexpected difference was also observed in the relative male mating competitiveness of the triple infected strain after irradiation at 28 Gy dose in small vs large cages, with a higher male mating competitiveness index calculated from results of experiments in the large cages. Based on these results, we consider that the male mating performance of the triple infected strain after irradiation at 28 Gy, a dose required for complete female sterility and the avoidance of population replacement, is approximately equal to that of the wild type males under semi-field conditions. Though field evaluation is required, this suggests that the triple infected strain is suitable for irradiation and release as part of a combined SIT-IIT approach to Ae. albopictus control.

Reusing larval rearing water and its effect on development and quality of Anopheles arabiensis mosquitoes.

BACKGROUND: There is growing interest in applying the sterile insect technique (SIT) against mosquitoes. Mass production of mosquitoes for large-scale releases demands a huge amount of water. Yet, many arid and/or seasonally arid countries face the difficulties of acute water shortage, deterioration of water quality and environmental constraints. The re-use of water to rear successive generations of larvae is attractive as a way to reduce water usage and running costs, and help to make this control method viable. METHODS: To determine whether dirty larval water was a suitable rearing medium for Anopheles arabiensis, in place of the 'clean' dechlorinated water routinely used, a series of three experiments was carried out to evaluate the effect of dirty water or mixed clean and dirty water on several parameters of insect quality. Batches of 100 fresh eggs were distributed in dirty water or added to clean water to test the effect of dirty water on egg hatching, whereas first-instar larvae were used to determine the effect on immature development time, pupation, adult emergence, body size, and longevity. Moreover, to assess the effect of dirty water on larval mortality, pupation rate, adult emergence, and longevity, L4 larvae collected after the tilting or larvae/pupae separation events were returned either to the dirty water or added to clean water. RESULTS: Results indicated that reusing dirty water or using a 50:50 mix of clean and dirty water did not affect egg hatching. Moreover, no difference was found in time to pupation, larval mortality or sex ratio when first-instar larvae were added to clean water, dirty water, or a 75:25, 50:50 or 25:75 mix of clean and dirty water and reared until emergence. When late-instar larvae were put back into their own rearing water, there was no effect on pupation rate, emergence rate or female longevity, though male longevity was reduced. When reared from first-instar larvae, however, dirty water decreased pupation rate, emergence rate, body size, and adult longevity. CONCLUSIONS: Re-used larval-rearing water has no impact on egg hatching, development time or mortality of the immature stages of An. arabiensis. However, dirty water is not suitable for the production of high quality adult mosquitoes. Recycling processes to improve water quality and increase insect quality will be investigated, since it may have important implications for the implementation of the SIT in areas where clean water is a scarce or costly resource.

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.

Combining the Sterile Insect Technique with Wolbachia-Based Approaches: II--A Safer Approach to Aedes albopictus Population Suppression Programmes, Designed to Minimize the Consequences of Inadvertent Female Release.

Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple-infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F1 and F2 progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population, released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.

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.

Combining the sterile insect technique with the incompatible insect technique: I-impact of wolbachia infection on the fitness of triple- and double-infected strains of Aedes albopictus.

The mosquito species Aedes albopictus is a major vector of the human diseases dengue and chikungunya. Due to the lack of efficient and sustainable methods to control this mosquito species, there is an increasing interest in developing and applying the sterile insect technique (SIT) and the incompatible insect technique (IIT), separately or in combination, as population suppression approaches. Ae. albopictus is naturally double-infected with two Wolbachia strains, wAlbA and wAlbB. A new triple Wolbachia-infected strain (i.e., a strain infected with wAlbA, wAlbB, and wPip), known as HC and expressing strong cytoplasmic incompatibility (CI) in appropriate matings, was recently developed. In the present study, we compared several fitness traits of three Ae. albopictus strains (triple-infected, double-infected and uninfected), all of which were of the same genetic background ("Guangzhou City, China") and were reared under the same conditions. Investigation of egg-hatching rate, survival of pupae and adults, sex ratio, duration of larval stages (development time from L1 to pupation), time to emergence (development time from L1 to adult emergence), wing length, female fecundity and adult longevity indicated that the presence of Wolbachia had only a minimal effect on host fitness. Based on this evidence, the HC strain is currently under consideration for mass rearing and application in a combined SIT-IIT strategy to control natural populations of Ae. albopictus in mainland China.