Mass rearing of Aedes albopictus (Skuse) in the Sterile Insect Technique (SIT)

Dal 1999 presso il laboratorio del Centro Agricoltura Ambiente “G. Nicoli” a Crevalcore (BO) è in corso una sperimentazione finalizzata a verificare la possibilità di attuare la tecnica del maschio sterile (SIT) in Italia contro Aedes albopictus. Alcuni aspetti per migliorare l’efficienza di questa struttura pilota, oggetto della presente ricerca, sono stati: 1) studio degli effetti di determinati costituenti della dieta larvale a) sullo sviluppo larvale stesso, per individuare intervalli limite di densità larvale e di concentrazione di cibo in cui è possibile lo sviluppo di tale specie, e b) sulla qualità dei maschi adulti ottenuti; 2) la valutazione di attrezzatura per l’allevamento massale e 3) la possibilità di migliorare la dieta larvale mediante integrazione di carboidrati. Dalle prove di valutazione della dieta larvale si è potuto osservare che, per quanto riguarda i parametri larvali, le due diete denominate “IAEA” (1 e 2) sono risultate più efficaci rispetto alla dieta standard “CAA”. Tali diete sono perciò da preferirsi nel loro possibile impiego in biofabbriche per l’allevamento massale. Le prove condotte sugli adulti allevati con le diverse diete hanno suggerito la necessità di valutare una possibile integrazione di componenti per migliorarne la longevità. Risulta altresì opportuno continuare la ricerca per ottimizzare la dieta larvale così da ottenere maschi di elevata qualità. Grazie ai risultati ottenuti dalle prove per valutare l’impiego di attrezzatura massale (vassoi di grandi dimensioni e carrello) si è potuto definire un modello per l’allevamento di Ae. albopictus con parametri standardizzati di densità larvale, dose di dieta, temperatura dell’acqua di allevamento, percentuale di maschi passati al setacciamento e rendimento di allevamento. Prove future saranno necessarie per testare altri componenti della dieta ricchi in carboidrati, quali saccarosio, da aggiungere alla dieta larvale per migliorare le qualità degli adulti ottenuti senza provocare effetti negativi sui parametri dello sviluppo larvale.Since 1999 the Centro Agricoltura Ambiente “G. Nicoli” in Crevalcore (BO) has been conducting research to evaluate the application of the sterile insect technique (SIT) together with other existing control methods to suppress Ae. albopictus. Some aspects to improve the efficiency of this pilot facility, object of the present research, were: 1) study of the effects of certain constituents of the larval diet a) on larval development, in order to determine the boundary conditions of larval density and concentration of food in which the development of such species is possible, and b) on the quality of adult males obtained; 2) the evaluation of equipment for the mass rearing and 3) the possibility of improving the larval diet by integration of carbohydrates. From the experiments on the larval diet it has been observed that, as regards larval development, the two "IAEA" diets were more effective than the "CAA" standard diet. Such diets are therefore to be preferred in their possible use for the mass rearing of Ae. albopictus. The tests carried out on the adults reared on the different diets have suggested the need to evaluate a possible integration with components to improve longevity. It is also appropriate to continue the research for the optimization of larval diet as a key factor for obtaining high-quality males. From the results achieved by testing the use of mass rearing equipment (large trays and rack) it was possible to define a model for the breeding of Ae. albopictus with standardized parameters of larval density, amount of diet, water temperature, percentage of males passed to the sieving and productivity. Future trials will be necessary to test other components rich in carbohydrates, such as sucrose, to be added to the larval diet to improve the quality of the adults without causing negative effects on larval development

Exploring protandry and pupal size selection for Aedes albopictus sex separation

Background: We explored the possibility to improve male/female separation (sexing) in Aedes albopictus by selection of two strains, one toward increasing sex dimorphism and another toward increasing protandry. In the laboratory we selected and crossed small males with large females to exploit dimorphism, and early pupating males with late pupating females to exploit protandry. Results: While selection for enhanced dimorphism was not a profitable character, the selection for enhanced protandry up to F10 produced significant improvement in the time interval between male and female pupation. By collecting the pupae at 24 h from the beginning of pupation, without any sieving operation, we obtained about 28.50% of pupae (calculated in relation to the estimated initial number of first instar larvae used), vs 26.49% we had in the control strain, and, more interestingly, when checking the sex ratio of these pupae we observed a presence of females of 0.92% vs 23.02% in the control strain. We also modified our egg hatching protocol from the previous standard procedure that required keeping the eggs in the glass hatching container overnight (for about 14-16 h) to a new protocol where eggs are kept in the hatching container for 4 h in order to obtain more synchronized larvae. This was possible without any reduction in the egg hatching rate. Conclusions: In Aedes albopictus it is possible to develop hyper-protandric strains useful to produce male pupae without applying other sexing systems. This represents a considerable achievement assisting the Sterile Insect Technique application, allowing improvement of the current sexing method based on mechanical separation. More investigations are under way in order to further enhance the male productivity capacity of the strain and to determine whether the selection has any impact on the male fitness parameters

Cannibalism in temporary waters. Simulations and laboratory experiments revealed the role of spatial shape in the mosquito Aedes albopictus

Cannibalism is a commonly observed phenomenon in arthropod species having relevant consequences for population dynamics and individual fitness. It is a context-dependent behaviour and an understanding of the factors affecting cannibalism rate is crucial to highlight its ecological relevance. In mosquitoes, cannibalism between larval stages has been widely documented, and the role of density, food availability and length of contact between individuals also ascertained. However, although mosquitoes can develop in temporary water habitats with very heterogeneous topologies, the role of the site shape where cannibals and victims co-occur has been instead overlooked. In this paper, we investigated this issue by using a simulation approach and laboratory cannibalism experiments between old (third- and fourth-instars) and young (first-instar) larvae of the tiger mosquito Aedes albopictus. Three virtual spaces with different shapes were simulated and the number of larval encounters was estimated in each one to assess whether the spatial shape affected the number of encounters between cannibal and victims. Then, experimental trials in containers with similar shapes to those used in the simulations were performed, and the cannibalism rate was estimated at 24 and 48h. Our results showed that the spatial shape plays a role on cannibalism interactions, affecting the number of encounters between individuals. Indeed, in the experimental trials performed, we observed the highest cannibalism rate in the container with the highest number of encounters predicted by the simulations. Interestingly, we found also that spatial shape can affect cannibalism not only by affecting the number of encounters, but also the number of encounters ªfavorableº for cannibalistic events. Temporary waters are inhabited by several species other than mosquitoes. Our results, showing an influence of the spatial shape on cannibalism in Ae. albopictus larvae, add a new critical factor to those affecting ecological interactions in these habitats

Identification and Detection of a Novel Point Mutation in the Chitin Synthase Gene of Culex Pipiens Associated With Diflubenzuron Resistance

Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide

Efficiency of Three Diets for Larval Development in Mass Rearing Aedes albopictus (Diptera: Culicidae)

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

From Bio-Prospecting to Field Assessment: The Case of Carvacrol Rich Essential Oil as a Potent Mosquito Larvicidal and Repellent Agent

Contemporary legislation tends to increase limitation on the use of all synthetic pesticides, promoting bio-pesticides as a safer alternative. Bio-prospecting efforts for bio-pesticides provide results, which rarely reach the industry. Present essay elaborates on our efforts to chart the path from the laboratory bench to field assessment. Eight Mediterranean wild gathered foods provided the essential oils that were assessed as mosquito control agents against the Asian tiger mosquito (Aedes albopictus). Three Lamiaceae essential oils, derived from Satureja thymbra, Origanum onites, and Thymbra spicata presented carvacrol as principal component. All exhibited DEET-like repellent performance and total larvae mortality defining the carvacrol rich essential oil (CREO) as a promising mosquito control agent. A commercial variety of Origanum vulgare ssp. hirtum, was selected as CREO source and subjected to dose-response and eco-toxicity studies. We have found significant larvicidal (LC90 of 58,747 mg/L), and repellent (0.2 μL/cm2) properties, but also severe toxicity (LC90 of 12,806 mg/L) against Macrocyclops albidus. This last figure was the limit for the larvicidal field assessment; while for the repellent evaluation was used double the minimum indication (0.4 μL/cm2). CREO was tested per se as larvicidal agent, and emulsified for both repellent and larvicidal field activity. The emulsified CREO's spatial repellent assessment showed maximum efficacy of 86% in day 1 that gradually declined in the following 2 days (81%, 69%). Both emulsified and crude CREO proved to be efficient larvicidal agents, with crude CREO (3 weeks) overrunning slightly the emulsified (2 weeks) in terms of endurance. Conclusively, CREO in its emulsified form may be considered as a promising mosquito larvicidal and repellent agent, applicable in both precautionary and emergency response measures

wPip Wolbachia contribution to Aedes albopictus SIT performance: Advantages under intensive rearing

As a part of a project aiming at the suppression of the mosquito vector Aedes albopictus, a specific Ae. albopictus line producing sterile males, ARwP, was tested for its suitability to intense rearing conditions compatible with mass production and field release. This line was developed by the Italian National Agency for New Technologies, Energy and Sustainable Economic Development thanks to the artificial infection with a heterologous Wolbachia strain, resulting in a bidirectional incompatibility pattern with wild-type Ae. albopictus. ARwP was reared under Standard Operating Procedures at the Centro Agricoltura Ambiente and compared with a wild-type strain in terms of time of pupation onset, production of male pupae in the following 24 h and mechanical sexing efficacy. Mating competitiveness of ARwP males was also evaluated in comparison with irradiated wild-type males in large field enclosures. ARwP males demonstrated a significantly shorter time of pupation onset, a higher rate of production of male pupae in the following 24 h and a lower percentage of residual contaminant females when applying mechanical sexing procedures. In addition, ARwP males were more efficient than wild-types in competing for wild-type females in large enclosures, thus inducing a level of sterility significantly higher than that expected for an equal mating competitiveness. These results encourage the use of this Ae. albopictus strain as suppression tool against Ae. albopictus based on considerations thoroughly discussed in the manuscript

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% brewer's 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

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

Field Competitiveness of Aedes albopictus (Diptera: Culicidae) Irradiated Males in Pilot Sterile Insect Technique Trials in Northern Italy

Vector-borne diseases account for 17% of infectious diseases, leading to more than one million deaths each year. Mosquitoes are responsible for 90% of the casualties and alternative control methods to insecticides are urgently needed, especially against Aedes vectors. Aedes albopictus is a particularly important species, causing major public health problems because it is a vector of several arboviruses and has a strong invasive behavior. Various genetic control methods have been proposed to be integrated into the management strat-egies of Aedes species, among which the sterile insect technique (SIT), which proved efficient against various insect pests and vectors. However, the ability of released irradiated sterile male mosquitoes to compete with their wild counterparts and induce sterility in wild females, which is critical to the success of this strategy, re-mained poorly defined. Here, we assessed the field competitiveness of Ae. albopictus irradiated male using data from eight release trials implemented in Northern Italy for 3 yr. Sterile males were capable of inducing a good level of sterility in the wild female population, however, with high variability in time and space. The field competitiveness of the released males was strongly negatively correlated with the ratio of sterile to wild males. This should be taken into consideration when designing future programs to suppress field populations of Aedes mosquitoes