Fabrizio Balestrino

Pilot Field Trials with Aedes albopictus Irradiated Sterile Males in Italian Urban Areas

ABSTRACT The pilot field studies here presented are part of a long-term research program aimed to develop a cost-effective sterile insect technique (SIT) methodology to suppress Aedes albopictus (Skuse) populations. Aedes albopictus is a mosquito species mainly developing in man-made containers and with an island-like urban and suburban distribution. These two features make the application of the sterile insect technique a possible control strategy. Five trials have been performed in three small towns from 2005 to 2009 (Emilia-Romagna region, northern Italy). Reared male pupae, sexed by a sieving technique allowing the recovery of ≈26–29% of males, were exposed to gamma rays and immediately released in the field. Adult population density was estimated based on a weekly monitoring of egg density in the ovitraps, whereas induced sterility was estimated by measuring the hatching percentage of weekly collected eggs in SIT and control areas. Results showed that sterile males released at the rate of 896–1,590 males/ha/wk induced a significant sterility level in the local population. In addition, when the sterility level achieved values in the range of 70–80%, a similar reduction also was found for the egg density in the ovitraps. We could estimate that the minimum egg sterility value of 81% should be maintained to obtain suppression of the local population. Immigration of mated females was not a main issue in the small villages where trials have been run.

γ Ray Dosimetry and Mating Capacity Studies in the Laboratory on Aedes albopictus Males

ABSTRACT In Italy, Aedes albopictus Skuse is currently recognized as the most dangerous mosquito, and as currently applied conventional control methods gave unsatisfactory results, we are developing alternative strategies such as the sterile insect technique. To find the optimal sterilizing dose, male pupae were exposed to different doses of &ggr; rays in the range 20–80 Gy, generated by a Cesium-137 source. The effects of male pupal age at irradiation and &ggr; ray dose on adult male emergence, sterility level, longevity, and mating capacity were evaluated, and dose-response curves of residual fertility were calculated. Radiation tests were also performed on female pupae to observe their reproductive capacity in case of accidental release. Results confirmed that the age at which the male pupa is irradiated is an important factor that affects the longevity of the adult, whereas the effect of age on the induced sterility level is less pronounced. When male pupae older than 30 h were irradiated, the longevity of the adults was not affected by doses up to 40 Gy. The 40-Gy dose appeared sufficient to induce high level of sterility (>99%) at any male pupal age for all the strains tested. The duration of coupling and the number of mated females per male appeared to be affected by the radiation received by male pupae only at doses higher than 40 Gy. The female pupae were more sensitive to radiation than male pupae, with strong reduction in fecundity and fertility at 20 Gy and complete suppression of oviposition at higher doses.

Dispersal and survival of Aedes albopictus (Diptera: Culicidae) males in Italian urban areas and significance for sterile insect technique application.

ABSTRACT The dispersal and survival of laboratory-reared Aedes albopictus Skuse males were investigated during the summer of 2007 in three Northern Italy urban localities by mark-release-recapture techniques. Two marking methods were compared: one group of males was dusted with fluorescent pigments on the body (FP), and the other group was obtained from a strain whose natural infection of Wolbachia had been removed (WB0). FP- and WB0-marked males were released as adults and pupae, respectively, in one fixed station at each locality. Recaptures were performed by skilled technicians, within a radius of 350 m from the release site, on days 4, 5, and 7 after the release, and the males were collected while flying around the technicians body or in swarms. Recapture rates ranged from 0.63 to 4.72% for FP males and from 2.39 to 11.05% for WB0 males. The mean distance traveled for WBO males was significantly higher than for FP males; no difference was observed between the dispersal distance measured for the males recaptured on human host versus males recaptured while swarming. No further increase of the dispersal occurred during the postrelease period investigated (from day 4 to day 7 after release). The mean survival rate at the release was 0.51 for FP-marked males and 0.81 for WBO males. The data obtained are discussed for their significance in planning sterile insect technique programs against Ae. albopictus.

Efficiency of three diets for larval development in mass rearing Aedes albopictus (Diptera: Culicidae).

ABSTRACT A fundamental step in establishing a mass production system is the development of a larval diet that promotes high adult performance at a reasonable cost. To identify a suitable larval diet for Aedes albopictus (Skuse), three diets were compared: a standard laboratory diet used at the Centro Agricoltura Ambiente, Italy (CAA) and two diets developed specifically for mosquito mass rearing at the FAO/IAEA Laboratory, Austria. The two IAEA diets, without affecting survival to the pupal stage, resulted in a shorter time to pupation and to emergence when compared with the CAA diet. At 24 h from pupation onset, 50 and 90% of the male pupae produced on the CAA and IAEA diets, respectively, had formed and could be collected. The diet received during the larval stage affected the longevity of adult males with access to water only, with best results observed when using the CAA larval diet. However, similar longevity among diet treatments was observed when males were supplied with sucrose solution. No differences were observed in the effects of larval diet on adult male size or female fecundity and fertility. Considering these results, along with the relative costs of the three diets, the IAEA 2 diet is found to be the preferred choice for mass rearing of Aedes albopictus, particularly if a sugar meal can be given to adult males before release, to ensure their teneral reserves are sufficient for survival, dispersal, and mating in the field.

A new larval tray and rack system for improved mosquito mass rearing.

ABSTRACT The requirement for efficient mosquito mass rearing technology has been one of the major obstacles preventing the large scale application of the Sterile Insect Technique against mosquitoes. At the Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) Insect Pest Control Laboratories we developed a larval rearing unit based on the use of a stainless steel rack that operates 50 thermoformed ABS plastic trays and is expected to be able to successfully rear 140,000–175,000 Anopheles arabiensis (Patton) adult mosquitoes per rack. The mechanized rearing unit is simple to handle, maintains minimal water temperature variation and negligible water evaporation and allows normal larval development. The mosquito mass-rearing tray was designed to provide a large surface area of shallow water that would closely mimic natural breeding sites. The trays stack into a dedicated rack structure and filling and draining were easily performed. The close stacking of the trays in the rack and the possibility to tightly line up several racks makes this rearing unit a valid solution for maximal use of the space thus reducing construction, heating, and cooling costs. The low amount of labor required to operate the system also reduces labor costs that represent one of the main expenditures in any mass rearing facility operation. Preliminary experiments performed on Aedes albopictus (Skuse) also confirm the possibility of successfully extending the use of this technology to other mosquito species. Our larval rearing unit could enhance any mosquito control strategy in which large-scale releases of mosquitoes are needed to suppress or replace natural populations.

Validation of a New Larval Rearing Unit for Aedes albopictus (Diptera: Culicidae) Mass Rearing

The mosquito larval rearing unit developed at the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint Division was evaluated for its potential use for Aedes albopictus (Skuse, 1895) mass rearing in support of the development of a sterile insect technique (SIT) package for this species. The use of the mass rearing trays and rack did not adversely affect larval development, pupation and survival rates and allowed the management of large larval rearing colonies with reduced space requirements in comparison with classical individual trays. The effects of larval density, water temperature and diet composition on pupal production and size differentiation for sex separation efficacy were analyzed for individual mass rearing trays as well as multiple trays stacked within the dedicated rack unit. Best results were obtained using eighteen thousand larvae per tray at a density of 3 larvae per ml of deionized water at a temperature of 28°C on a diet consisting of 50% tuna meal, 36% bovine liver powder, 14% brewers yeast and, as an additive, 0.2 gr of Vitamin Mix per 100 ml of diet solution. Pupae were harvested on the sixth day from larval introduction at L1 stage and males were separated out by the use of a 1400 µm sieve with 99.0% accuracy with a recovery rate of ca. 25% of the total available males. With the use of this larval rearing unit, an average production of 100,000 male pupae per week can be achieved in just 2 square meter of laboratory space. Compared to previous laboratory rearing method, the same pupal production and sex separation efficacy could only be achieved by use of ca. 200 plastic trays which required the space of two 5 square meter climatic-controlled rooms.

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.

Mass Production Cage for Aedes albopictus (Diptera: Culicidae)

ABSTRACT Mass production is an important component of any pest or vector control program that requires the release of large number of insects. As part of efforts to develop an area-wide program involving the sterile insect technique (SIT) for the control of mosquitoes, the Insect Pest Control Laboratory of the Food and Agriculture Organization—International Atomic Energy Agency (FAO—IAEA Joint Division) has developed a mass production cage (Aedes MPC) for brood stock colonies in a mass production system for Aedes albopictus (Skuse, 1895). A preliminary experiment using Plexiglas cages was carried out to estimate the impact of cage volume on egg productivity. Transparent Plexiglas cages of different dimensions but loaded with the same adult density were tested. Egg productivity (number of eggs laid per adult female) and adult survival were recorded and analyzed. According to the results, the optimal volume of 100 liters has been chosen to develop the Aedes MPC. The numbers of adults introduced into the Aedes MPC did not affect the egg production and adult survival in comparison with the Plexiglas cage experiment data, confirming the possible use of Aedes MPC for mass-rearing procedures. Finally, the modification of Aedes MPC and creation of a new prototype model of MPC (Anopheles MPC) to effectively contain Anopheles arabiensis (Patton, 1905) adults is discussed with major changes pioneered to oviposition devices and systems for automatic collection of the eggs.

Sugar administration to newly emerged Aedes albopictus males increases their survival probability and mating performance

Aedes albopictus male survival in laboratory cages is no more than 4-5 days when kept without any access to sugar indicating their need to feed on a sugar source soon after emergence. We therefore developed a device to administer energetic substances to newly emerged males when released as pupae as part of a sterile insect technique (SIT) programme, made with a polyurethane sponge 4 cm thick and perforated with holes 2 cm in diameter. The sponge was imbibed with the required sugar solution and due to its high retention capacity the sugar solution was available for males to feed for at least 48 h. When evaluated in lab cages, comparing adults emerged from the device with sugar solution vs the device with water only (as negative control), about half of the males tested positive for fructose using the Van Handel anthrone test, compared to none of males in the control cage. We then tested the tool in semi-field and in field conditions with different sugar concentrations (10%, 15%, and 20%) and compared results to the controls fed with water only. Males were recaptured by a battery operated manual aspirator at 24 and 48 h after pupae release. Rather high share 10-25% of captured males tested positive for fructose in recollections in the vicinity of the control stations, while in the vicinity of the sugar stations around 40-55% of males were positive, though variability between replicates was large. The sugar positive males in the control test may have been released males that had access to natural sugar sources found close to the release station and/or wild males present in the environment. Only a slight increase in the proportion of positive males was obtained by increasing the sugar concentration in the feeding device from 10% to 20%. Surprisingly, modification of the device to add a black plastic inverted funnel above the container reduced rather than increased the proportion of fructose positive males collected around the station. No evidence of difference in the capacity of sterile (irradiated with 30 Gy) males to take a sugar meal relative to fertile males was observed in field comparison. A clear effect of temperature and relative humidity (RH) on the rate of sugar positive males was observed, with an increase of temperature and a decrease in RH strongly increasing the % of sugar positive males. In large enclosures we tested the effect of our sugar supplying tool on the mating competitiveness of sterile vs fertile males, which produced an evident favorable effect both on sterile and fertile males.

Mosquito Mass Rearing Technology: A Cold-Water Vortex Device for Continuous Unattended Separation of Anopheles arabiensis Pupae from Larvae

Abstract 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°C caused neither mortality nor reduction in likelihood of pupation or emergence. Separation improved as temperatures decreased down to 4°C. We devised and demonstrated a 15°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.