Effects of drying eggs and egg storage on hatchability and development of Anopheles arabiensis.

BACKGROUND: The mass rearing of insects requires a large colony from which individuals can be harvested for sterilization and release. Attention is given to larval food requirements and to handling and rearing conditions to ensure predictability and synchrony of development. Maximizing production requires optimized adult holding to ensure mating success, blood feeding and oviposition. Appropriate egg storage and harvesting is necessary to compensate any unpredicted reduction in egg production. METHODS: Anopheles arabiensis eggs were collected on wet filter paper in eggs cups. The eggs were cleaned and then dried over a suction device with adjustable speed and time. The effects of drying, storage time and storage condition (wet, dry and bulk with relative humidity 75 ± 5% and storage temperatures of 10, 15 and 20°C) on hatch rate, duration of larval stages (L1 to pupal stage), duration of L1 to adult emergence, survival of L1 to pupal stage and the survival of L1 to adult emergence were investigated. Post drying and post storage hatch rates were determined by counting hatched and unhatched eggs and were confirmed by counting the viable larvae in the rearing medium. RESULTS: The hatch rate of eggs dried at wind speeds of 1.0 or 1.8 m/s was not significantly different from the control, but eggs dried at 3.0 m/s resulted in very low (64%) hatchability as compared to the control (82%). Eggs stored at 20°C and 75 ± 5% RH in bulk in an aerated vial showed better survival than eggs stored in wet or dry conditions at 10 or 15°C. No significant changes in larval duration and survival were recorded after six days of bulk storage. CONCLUSION: Anopheles arabiensis eggs can be stored in bulk at 20°C and 75 ± 5% RH for six days without any decrease in hatch rate, and up to 9 days with no impact on larval development.

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

BACKGROUND: The 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. METHODS: For 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. RESULTS: ANO 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% respectively CONCLUSION: The 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.

Mosquito mass rearing technology: a cold-water vortex device for continuous unattended separation of Anopheles arabiensis pupae from larvae.

In mass rearing of anopheline mosquitoes, pupae are usually separated from larvae on a daily basis to prevent unwanted adult emergence from trays. Depending on the device and species, 2 physical characteristics have most often been used for separation: buoyant density and size. In this report, we describe a system for continuous separation of Anopheles arabiensis larvae from pupae based on the natural difference in buoyant density and behavior between the 2 stages. We determined that temperatures 4-15 degrees C caused neither mortality nor reduction in likelihood of pupation or emergence. Separation improved as temperatures decreased down to 4 degrees C. We devised and demonstrated a 15 degrees C water vortex separator that we anticipate can process approximately 1 million larvae and pupae per hour with a < 0.3% pupal contamination rate and which operates unattended.

Ovipositional behavior in the context of mass rearing of Anopheles arabiensis.

Large-scale production of mosquitoes is a key factor for a successful sterile insect technique program. A manageable mass-production cage must contain appropriate features for adult resting, mating, feeding, and ovipositional activities. In order to maximize egg collections, tests were conducted to determine the physical characteristics of ovipositional sites for caged Anopheles arabiensis. Effects of texture, shade, height, and shape of the ovipositional container on female behavior were investigated. Results indicate a strong preference for oviposition on humid substrates over free-standing water. The shade and texture of the cup's walls also influenced site choice, with black rough inner vertical walls of the cup resulting in the largest number of eggs. Ovipositional sites with square shape were preferred rather than circular cups, and in the square cups, >60% of the eggs were laid in the corners. Height also affected oviposition, as An. arabiensis significantly favored the lowest sites even though some oviposition occurred at higher sites. Based on this study and from available literature, we determined the characteristics of an artificial ovipositional site that includes all these characteristics, which will yield large numbers of eggs required for mass production of this species.

Eliminating female Anopheles arabiensis by spiking blood meals with toxicants as a sex separation method in the context of the sterile insect technique.

BACKGROUND: Ivermectin has longevity reducing effects in several insect species, including disease transmitting mosquitoes after feeding on hosts that have received ivermectin treatment. This has important implications in mosquito population control and thus the reduction of disease transmission. In addition, ivermectin could play an enormous role in mosquito control operations by its use in the female elimination process during mass-rearing, enabling the release of only sterile males in the context of the sterile insect technique (SIT). METHODS: Blood meals were spiked with various toxicants and were then offered to adult Anopheles arabiensis and killing effects were observed. Varying concentrations of the most effective substance were then tested in subsequent trials to obtain an optimal dose for quick and total female elimination. The remaining males were mated with untreated virgin females to assess whether their mating efficiency had been compromised. The most promising substance at the optimal concentration was further tested on a larger number of adults, after they had been irradiated and partially sterilised as pupae with 70 Gy to evaluate the feasibility of the method in a mass-rearing, and SIT context. The males resulting from the latter trial were also checked for mating efficiency post treatments. RESULTS: Ivermectin (Virbamec®) at a concentration of 7.5 ppm was chosen from the toxicants tested as sufficiently effective in eliminating all female An. arabiensis in 4 days, the shortest time required for female elimination of all chemicals tested. Mating efficiency of the non-blood feeding male mosquitoes was not compromised significantly compared to controls even when they were kept for a total of 4 days (from emergence) before theoretical release. The irradiation treatment did not affect overall female feeding behaviour in this setting, nor were the sterile males less competitive for mating with virgin females after the treatments than virgin sterile males that had not been in the ivermectin treatment environment. CONCLUSIONS: Spiking bloodmeals with ivermectin has shown potential as a viable treatment to eliminate female An. arabiensis from laboratory colonies although its practical use in a mass-rearing facility still needs to be tested.