Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions.

The global expansion of Aedes albopictus has stimulated the development of environmentally friendly methods aiming to control disease transmission through the suppression of natural vector populations. Sterile male release programmes are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation which can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic Wolbachia-free Ae. albopictus line. We then measured the penetrance of cytoplasmic incompatibility and life-history traits of both transinfected lines, selected the wPip-IV line and combined it with the genetic sexing strain. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Optimization of Dieldrin Selection for the Genetic Sexing of Aedes albopictus.

The mass production of mosquitoes at an industrial scale requires efficient sex separation, which can be achieved through mechanical, genetic or artificial intelligence means. Compared with other methods, the genetic sexing approach offers the advantage of limiting costs and space by removing females at the larval stage. We recently developed a Genetic Sexing Strain (GSS) in Aedes albopictus based on the sex linkage of the rdlR allele, conferring resistance to dieldrin, to the male (M) locus. It has been previously reported that dieldrin ingested by larvae can be detected in adults and bioaccumulated in predators, raising the question of its use at a large scale. In this context, we performed several experiments aiming at optimizing dieldrin selection by decreasing both dieldrin concentration and exposure time while maintaining a stable percentage of contaminating females averaging 1%. We showed that the previously used dieldrin exposure induced an important toxicity as it killed 60% of resistant males at the larval stage. We lowered this toxicity by reducing the dose and/or the exposure time to recover nearly all resistant males. We then quantified the residues of dieldrin in resistant male adults and showed that dieldrin toxicity in larvae was positively correlated with dieldrin concentrations detected in adults. Interestingly, we showed that the use of reduced dieldrin exposure led to a dieldrin quantification in adult males that was below the quantity threshold of the Gas Chromatography-Mass Spectrometry detection method. Presented data show that dieldrin exposure can be adjusted to suppress toxicity in males while achieving efficient sexing and lowering the levels of dieldrin residues in adults to barely quantifiable levels.

Development of an automated mosquito pupae counter.

The production of mosquitoes for control programs or basic research is facilitated by the standardization of rearing conditions allowing the daily manipulation of thousands of individuals. It is crucial to develop mechanical or electronic systems for controlling the density of mosquitoes at each development stage with the aim of reducing costs, time, and human errors. We present herein an automatic mosquito counter using a recirculating water system allowing rapid and reliable counting of pupae without detectable increased mortality. Using Aedes albopictus pupae, we determined the density of pupae and the time of counting for which the device is most accurate, and evaluated the time saved using this device. Lastly, we discuss how this mosquito pupae counter can be useful in small-scale or mass-rearing contexts enabling a number of applications for research purposes as well as operational mosquito control programs.

Could Sterile Aedes albopictus Male Releases Interfere with Aedes aegypti Population in Reunion Island?

In Reunion Island, the feasibility of an Aedes albopictus control program using the Sterile Insect Technique (SIT) is studied. Because, in some regions, Ae. albopictus is living in sympatry with Aedes aegypti, the impact of releasing millions of sterile male Ae. albopictus on female Ae. aegypti reproduction needs to be assessed. Thus, to study the potential heterospecific matings, a marking technique using rhodamine B has been used. Rhodamine is given in solution to male mosquitoes to be incorporated into the male body and seminal fluid and transferred during mating into the bursa inseminalis and spermathecae of females. The presence of rhodamine in females occurred in 15% of cases when Ae. aegypti females were offered non-irradiated Ae. albopictus males, 5% when offered irradiated Ae. albopictus males and 18% of cases in the inverse heterospecific matings. Moreover, our results also showed that these matings gave few eggs but were not viable. Finally, the results showed that whatever the type of mating crosses, females in cages previously crossed with males of another species can re-mate with males of their species and produce an equivalent amount of egg compared to females only mated with conspecific males. Despite the promiscuity of the males and females in small cages for three days, heterospecific mating between sterile male Ae. albopictus and female Ae aegypti, 95% of the females have not been inseminated suggesting that in the field the frequency satyrization would be very low.

Aedes albopictus Adult Medium Mass Rearing for SIT Program Development.

For the production of several hundred thousands of Aedes albopictus sterile males for the implementation of a Sterile Insect Technique (SIT) program, no costly mass-rearing equipment is needed during the initial phases, as optimized rearing at laboratory scale can be sufficient for the first steps. The aim of this study was to maximize the egg production by optimizing adult rearing methods for Ae. albopictus. The effect of parameters such as male/female ratio, density of adults, membrane type for blood feeding, quantity of blood delivered, continuous or discontinuous blood feeding, and surface of substrates for egg laying on overall egg production was tested to find optimized conditions. Based on the number of eggs produced per cage in response to the parameters tested, the optimum cage set-up was seen to be 1500 adults in a 30 × 30 × 30 cm cage with a male/female sex ratio of 1:3, fed by fresh bovine blood for periods of 30 min using a cellulose membrane covering a 10 cm stainless steel plate heated by a Hemotek device, and the provision of five oviposition cups to collect eggs. With this set-up, production per cage can reach a maximum of 35,000 eggs per week.