Arianna Puggioli

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.

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.

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.

Wolbachia Density and Cytoplasmic Incompatibility in Aedes albopictus: Concerns with Using Artificial Wolbachia Infection as a Vector Suppression Tool.

The mosquito Aedes albopictusi is a competent vector of harmful human pathogens, including viruses causing dengue and chikungunya. Cytoplasmic incompatibility (CI) induced by endosymbiotic Wolbachia can be used to produce functionally sterile males that can be released in the field as a suppression tool against this mosquito. Because the available sexing methods are not efficient enough to avoid unintentional release of a few transinfected females, we assessed the CI pattern in crosses between wPip Wolbachia-transinfected (ARwP) females and wild-type males of Ae. albopictus in this study. Quantitative polymerase chain reaction was used to monitor the titer of the Wolbachia strains that naturally infect Ae. albopictus, that is, wAlbA and wAlbB, in age-controlled males and females. Data were coupled with incompatibility level detected when the above-mentioned males were crossed with ARwP females. Wolbachia infection titer was also monitored in samples of wild caught males. Incompatibility level was positively correlated only with wAlbA density. Crosses between wild-type males having very low wAlbA density (<0.001 wAlbA/actin copy numbers) and ARwP females were partially fertile (CIcorr = 68.06 ± 6.20). Individuals with low wAlbA titer were frequently found among sampled wild males (30%–50% depending on the site and period). ARwP males can be as considered as a very promising tool for suppressing Ae. albopictus. However, crosses between wild males having low wAlbA density and ARwP females may be partially fertile. In the case of local establishment of the transinfected mosquito line, this occurrence may favor the replacement of the wild-type mosquitoes with the ARwP line, thus reducing the long-term efficacy of incompatible insect technique. Various alternative strategies have been discussed to prevent this risk and to exploit Wolbachia as a tool to control Ae. albopictus.

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.

Striking diflubenzuron resistance in Culex pipiens , the prime vector of West Nile Virus

Culex pipiens mosquitoes cause severe nuisance and transmit human diseases including West Nile. Vector control by insecticides is the main tool to prevent these diseases and diflubenzuron is one of the most effective mosquito larvicides used in many places. Here, high levels of resistance were identified in Cx. pipiens from Italy, with a Resistance Ratio of 128 fold. The phenotype was associated with mutations at amino acid I1043 (I1043M and I1043L) of the Chitin synthase gene, which showed significantly higher frequency in bioassay survivors. Both mutations have been introduced in the Drosophila melanogaster chitin synthase gene using the genome editing method CRISPR/Cas9 and validated to confer significant levels of resistance, although at different levels. The I→M mutation results in a Resistance Ratio >2,900 fold and the I→L mutation >20 fold. Two PCR based diagnostics were developed for monitoring of the resistant mutations in field populations. The findings are of major concern for public health given the importance of diflubenzuron in mosquito control in many places, the intensity of the resistance phenotype and the limited availability of alternative larvicides.

Establishment of a satellite rearing facility to support the release of sterile Aedes albopictus males. I. Optimization of mass rearing parameters.

The vector species Aedes albopictus (Skuse, 1894) was recorded in Turkey for the first time, near the Greek border, in 2011 and a high risk of expansion towards Aegean and Mediterranean coasts of Turkey was estimated. A preliminary study was planned to evaluate the possibility of creating a satellite mass rearing facility for this species and manage a larval rearing procedure by using the new mass-rearing technology proposed by the International Atomic Energy Agency (IAEA). For this purpose, the effects of different larval densities (1, 2, 3 and 4 larvae per ml) on the preimaginal development were evaluated by observing pupal, adult and male productivity using life cycle trials. Geometric morphometric analyses were also performed to define all phenotypic differences that occurred on the wing size and shape morphology of adult stage at the four different rearing conditions tested. A high pupation productivity was obtained with a larval density of 2 larvae/ml while adult emergence ratio was not affected by the densities tested. No significant difference was observed in shape of the wings among different densities in males and females. Nevertheless, a significant difference in females centroid sizes was observed between the treatment groups 1-2 and 3-4 larvae/ml and in males centroid size reared at 1 larvae/ml versus the other densities.

Comparative study on the effectiveness of different mosquito traps in arbovirus surveillance with a focus on WNV detection.

The selection of the ideal trap for arbovirus surveillance is an issue of primary importance to increase the sensitivity of virus detection and the cost-effectiveness of the entomological surveillance. During the summer 2011, the effectiveness of five types of mosquito traps (CDC gravid trap, CO2(-)baited trap, BG-Sentinel™ and two experimental prototypes) to attract females potentially infected with West Nile virus were assessed. The study was carried out in three natural wetland sites located in the Emilia-Romagna Region (Northern Italy), using a Latin square scheme. Single night collections of adult females were performed and determination of species and physiological state (gravid, nulliparous or parous) was made upon return to the laboratory. The species most frequently collected in the gravid trap was Culex pipiens sl. L., being gravid females the large majority of the individuals. Species diversity was much higher in CO2(-)baited traps, which may therefore enable a more comprehensive description of the vector species composition and their role in arboviruses circulation. Our findings indicate that gravid traps can be a valid tool and should be integrated in the West Nile virus surveillance system in the Emilia-Romagna region, mainly based on collections made with CO2-baited traps.

Quality control methods for Aedes albopictus sterile male production

The capacity of the released sterile males to survive, disperse, compete with wild males and inseminate wild females is an essential prerequisite to be evaluated in any area-wide integrated pest management (AW-IPM) programs including a sterile insect release method. Adequate quality control tests supported by standardized procedures need to be developed to measure these parameters and to identify and correct potential inappropriate rearing or handling methods affecting the overall male quality. In this study, we report results on the creation and validation of the first quality control devices designed to infer the survival and mating capacity of radio-sterilized Aedes albopictus males through the observation of their flight capacity under restricted conditions (flight organ device) and after stress treatment (aspirator device). Results obtained consistently indicate comparable flight capacity and quality parameters between untreated and 35 Gy irradiated males while a negative impact was observed with higher radiation doses at all observation time performed. The male flight capacity registered with the proposed quality control devices can be successfully employed, with different predictive capacities and response time, to infer the adult male quality. These simple and cost-effective tools provide a valuable method to detect and amend potentially sub-standard procedures in the sterile male production line and hence contribute to maintaining optimal quality and field performance of the mosquitoes being released.

Chikungunya and Dengue Risk Assessment in Greece

Objective: The aims of the study were: (1) To assess the mutated CHIKV E1-A226V and DENV II infection and dissemination rates of an Ae. albopictus population established in Athens (Greece) (2) To assess the risk of outbreaks in four Greek localities based on Ae. albopictus population density whose estimate was based on the number of eggs laid in ovitraps. Methods: Under laboratory conditions females were offered blood meal infected with the CHIKV titer of 1X106 TCID 50/mL and DENV II titer of 1.76 X106 TCID 50/mL; at day 11 after oral infection, females were sacrificed, legs were removed and processed for PCR analysis to assess the presence of viral replicates. In order to evaluate the risk of outbreak of CHIKV and DENV II, a pilot monitoring program was started in three Greek localities and in Chania (Crete), to estimate the adult female population density on the base of the number of eggs in the ovitraps. Results: We proved the vector competence of the Greek Ae. albopictus strain for E1-A226V mutated CHIKV and DENV II. Combining the data on the vector competence with those on the female population density, based on the egg density data, the estimated risk of outbreak was relatively low but not negligible. Conclusion: As the vector competence estimated under laboratory conditions was obtained by offering females moderately low initial virus titers, it can be expected a higher vector competence in the field. This consideration, together with a possible increase of the mosquito population due to the global warming effects, make the quantitative ovitrap-based monitoring a necessary and useful tool to estimate the risk of outbreaks.