Hanano Yamada

X-Ray-Induced Sterility in Aedes albopictus (Diptera: Culicidae) and Male Longevity Following Irradiation

ABSTRACT The mosquito Aedes albopictus (Skuse, 1895) is a potent vector of several arboviral diseases, most notably chikungunya and dengue fever. In the context of the sterile insect technique (SIT), the sterilization of the male mosquitoes before their release can be achieved by gamma-ray irradiation. As gamma-ray irradiators are becoming increasingly problematic to purchase and transport, the suitability of an X-ray irradiator as an alternative for the sterilization of Ae. albopictus males was studied. The sterilization of up to 200,000 pupae at one time can be achieved with relative ease, and the sterility results obtained were comparable with those achieved by gamma irradiation, where 99% sterility is induced with a dose of 40 Gy. A significant reduction of longevity was observed in the latter stages of the males’ life after irradiation treatments, especially at doses >40 Gy, which is consistent with the negative effects on longevity induced by similar radiation doses using gamma rays. Females irradiated at 40 Gy were not only 100% sterile, but also failed to oviposit entirely, i.e., all of the females laid 0 eggs. Overall, it was found that the X-ray irradiator is generally suitable for the sterilization process for sterile insect technique programs, as it showed a high processing capacity, practicality, high effectiveness, and reproducibility.

Genetic sex separation of the malaria vector, Anopheles arabiensis, by exposing eggs to dieldrin

BackgroundThe sterile insect technique (SIT) has been used with success for suppressing or eliminating important insect pests of agricultural or veterinary importance. In order to develop SIT for mosquitoes, female elimination prior to release is essential as they are the disease-transmitting sex. A genetic sexing strain (GSS) of Anopheles arabiensis was created based on resistance to dieldrin, and methods of sex separation at the egg stage were developed. The use of this strain for SIT will require sexually sterile males: useful radiation doses for this purpose were determined for pupae and adults.MethodsFor the creation of the sexing strain, dieldrin-resistant males were irradiated with 40 Gy using a 60Co source and were subsequently crossed to homozygous susceptible virgin females. Individual families were screened for semi-sterility and for male resistance to dieldrin. For sex separation, eggs of a resulting GSS, ANO IPCL1, were exposed to varying concentrations of dieldrin for different durations. Percent hatch, larval survival, and male and female emergence were recorded. Radiation induced sterility was determined following adult and pupa exposure to gamma rays at 0–105 Gy. Mortality induced by dieldrin treatment, and levels of sterility post radiation were investigated.ResultsANO IPCL1 contains a complex chromosome aberration that pseudo-links the male-determining Y chromosome and dieldrin resistance, conferring high natural semi-sterility. Exposure of eggs to 2, 3, and 4 ppm dieldrin solutions resulted in complete female elimination without a significant decrease of male emergence compared to the controls. A dose of 75 Gy reduced the fertility to 3.8 and 6.9% when males were irradiated as pupae or adults respectively, but the proportions of progeny of these males reaching adulthood were 0.6 and 1.5% respectivelyConclusionThe GSS ANO IPCL1 was shown to be a suitable strain for further testing for SIT though high semi-sterility is a disadvantage for mass rearing.

Anopheles arabiensis egg treatment with dieldrin for sex separation leaves residues in male adult mosquitoes that can bioaccumulate in goldfish (Carassius auratus auratus)

The sterile insect technique (SIT) is a biological control tactic that is used as a component of area-wide integrated pest management (AW-IPM) programs. The SIT can only be applied against disease-transmitting mosquitoes when only sterile male mosquitoes are released, and the blood-sucking and potentially disease-transmitting females are eliminated from the production line. For Anopheles arabiensis, a potent vector of malaria, a genetic sexing strain was developed whereby females can be eliminated by treating the eggs or larvae with the insecticide dieldrin. To evaluate the presence of dieldrin residues in male mosquitoes designated for SIT releases, a simple, sensitive, and accurate gas chromatography–electron capture detector (GC–ECD) method was developed. In addition, bioaccumulation and food chain transfer of these residues to fish after feeding with treated mosquitoes was demonstrated. The overall recovery from method validation studies was 77.3 ± 2.2% (mean ± relative standard deviation [RSD]) for the mosquitoes, and 99.1 ± 4.4% (mean ± RSD) for the fish. The average dieldrin concentration found in adult male An. arabiensis was 28.1 ± 2.9 µg/kg (mean ± standard deviation [SD]). A range of 23.9 ± 1.1 µg/kg to 73.9 ± 5.2 µg/kg (mean ± SD) of dieldrin was found in the fish samples. These findings indicate the need to reassess the environmental and health implications of control operations with a SIT component against An. arabiensis that involves using persistent organochlorines in the sexing process.

The Anopheles arabiensis genetic sexing strain ANO IPCL1 and its application potential for the sterile insect technique in integrated vector management programmes.

The Anopheles arabiensis genetic sexing strain ANO IPCL1 was developed based on a dieldrin resistant mutation. The strain has been shown to be practical and reliable in terms of female elimination by dieldrin treatments at larval stages, but has provided some difficulties when treatments were applied at the egg stage. The high natural sterility of this strain has advantages and disadvantages in both mass rearing and the sterilization process. In addition, its recombination rate, although relatively low, poses a threat of strain deterioration if left unchecked in a mass-rearing setting. The males of the ANO IPCL1 have been shown to be equally competitive as lab-reared males of the wild-type Dongola strain, but competitiveness decreased by half when irradiated with 75 Gy—a dose conferring >98% sterility. More controversial issues surround the use of dieldrin—a highly persistent organochlorine that is known to bioaccumulate in the food chain. The prospective use of large volumes of dieldrin in a mass-rearing facility and the retention of its residues by the male mosquitoes makes the use of the strain in the context of the sterile insect technique against this vector highly questionable, and therefore its implementation at a large scale cannot be recommended.

Enhancements to the mass-rearing cage for the malaria vector, Anopheles arabiensis for improved adult longevity and egg production

Innovations in mosquito mass‐rearing techniques are essential in the quest to develop SIT (sterile insect technique) methods to fight mosquito vectors of disease. This study reports modifications to the Food and Agriculture Organisation/International Atomic Energy Agency (FAO/IAEA) mass‐rearing cage (MRC) for mosquitoes to support the behaviour of adult Anopheles arabiensis Patton (Diptera: Culicidae) and to maximize egg production. The effects of an improved sugar‐feeding device, and the addition of resting sites and a black cloth shroud to create an artificial horizon (visual contrast of light vs. dark at the edge) were assessed for their effect on adult longevity and egg production. Egg production of adults resulting from larvae reared in individual free‐standing trays vs. those reared in the same trays in the FAO/IAEA larval rearing rack was also compared. Finally, the effect of blood feeding and frequency of egg collection from the MRC on average egg production per batch was investigated. Overall, the modifications to the MRC enhanced adult longevity, and the improved cage prototype allowed the collection of more eggs overall from a cohort of adults than was possible using the original and previous cage prototypes. These stepwise improvements are important for the development of economical and logistically efficient mass‐rearing systems for the malaria vector An. arabiensis.

Cost-effective larval diet mixtures for mass rearing of Anopheles arabiensis Patton (Diptera: Culicidae)

BackgroundLarval nutrition, particularly diet quality, is a key driver in providing sufficient numbers of high quality mosquitoes for biological control strategies such as the sterile insect technique. The diet currently available to mass rear Anopheles arabiensis, referred here to as the “IAEA diet”, is facing high costs and difficulties concerning the availability of the bovine liver powder component. To promote more affordable and sustainable mosquito production, the present study aimed to find alternative diet mixtures. Eight cheaper diet mixtures comprised of varying proportions of tuna meal (TM), bovine liver powder (BLP), brewer’s yeast (BY), and chickpea (CP) were developed and evaluated through a step by step assessment on An. arabiensis larvae and adult life history traits, in comparison to the IAEA diet which served as a basis and standard.ResultsFour mixtures were found to be effective regarding larval survival to pupation and to emergence, egg productivity, adult body size and longevity. These results suggest that these different diet mixtures have a similar nutritional value that support the optimal development of An. arabiensis larvae and enhance adult biological quality and production efficiency, and thus could be used for mass rearing.ConclusionsOur study demonstrated that four different diet mixtures, 40 to 92% cheaper than the IAEA diet, can result in a positive assessment of the mosquitoes’ life history traits, indicating that this mosquito species can be effectively mass reared with a significant reduction in costs. The mixture comprised of TM + BY + CP is the preferred choice as it does not include BLP and thus reduces the cost by 92% compared to the IAEA diet.

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

BackgroundStandardized 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.

Evaluation of radiation sensitivity and mating performance of Glossina brevipalpis males

Background Area-wide integrated pest management strategies that include a sterile insect technique component have been successfully used to eradicate tsetse fly populations in the past. To ensure the success of the sterile insect technique, the released males must be adequately sterile and be able to compete with their native counterparts in the wild. Methodology/Principal findings In the present study the radiation sensitivity of colonised Glossina brevipalpis Newstead (Diptera; Glossinidae) males, treated either as adults or pupae, was assessed. The mating performance of the irradiated G. brevipalpis males was assessed in walk-in field cages. Glossina brevipalpis adults and pupae were highly sensitive to irradiation, and a dose of 40 Gy and 80 Gy induced 93% and 99% sterility respectively in untreated females that mated with males irradiated as adults. When 37 to 41 day old pupae were exposed to a dose of 40 Gy, more than 97% sterility was induced in untreated females that mated with males derived from irradiated pupae. Males treated as adults with a dose up to 80 Gy were able to compete successfully with untreated fertile males for untreated females in walk-in field cages. Conclusions/Significance The data emanating from this field cage study indicates that, sterile male flies derived from the colony of G. brevipalpis maintained at the Agricultural Research Council-Onderstepoort Veterinary Institute in South Africa are potential good candidates for a campaign that includes a sterile insect technique component. This would need to be confirmed by open field studies.

A rapid quality control test to foster the development of genetic control in mosquitoes

Vector-borne diseases are responsible for more than one million deaths per year. Alternative methods of mosquito control to insecticides such as genetic control techniques are thus urgently needed. In genetic techniques involving the release of sterile insects, it is critical to release insects of high quality. Sterile males must be able to disperse, survive and compete with wild males in order to inseminate wild females. There is currently no standardized, fast-processing method to assess mosquito male quality. Since male competitiveness is linked to their ability to fly, we developed a flight test device that aimed to measure the quality of sterile male mosquitoes via their capacity to escape a series of flight tubes within two hours and compared it to two other reference methods (survival rate and mating propensity). This comparison was achieved in three different stress treatment settings usually encountered when applying the sterile insect technique, i.e. irradiation, chilling and compaction. In all treatments, survival and insemination rates could be predicted by the results of a flight test, with over 80% of the inertia predicted. This novel tool could become a standardised quality control method to evaluate cumulative stress throughout the processes related to genetic control of mosquitoes.

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

BackgroundMass 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.MethodsThree 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.ResultsFemale 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.ConclusionsOur 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.