The results showed that through temperature manipulation it is possible to delay emergence of mosquitoes by up to 3 days; the approximate length of the gonotrophic cycle of Anopheline females. These finding are important for ecological studies that require small punctual releases and for interventions requiring mass releases focussing on Anopheline vector species. Currently the logistics and planning for Anopheline production revolve around the assumption that achieved mosquito numbers, at a particular time point, directly depend on the quantity of eggs produced by a single gonotrophic cycle. The findings of this study offer the potential to effectively double the progeny produced from one female cohort, thereby bringing much needed flexibility to Anopheline rearing practices.
The 3-day delay was achieved by subjecting first instar larvae to a 5-day cooling period at 19oC. The alteration in temperature had no effect on pupation rates although there was a difference in the rate of pupation between An. coluzzii and An. gambiae. It was also found that cooling had a minimal effect on emergence rates, that were > 85%, but affected the two species conversely. In An. coluzzii, it resulted in an increase in emergence rate, but in An. gambiae it resulted in small decrease in emergence rates. Overall, pupation and emergence rates were high and in line with reports elsewhere for laboratory-reared Anopheles [41, 42].
There was no effect of temperature reduction on sex ratio, which was equivalent to a 1:1 male to female ratio in both species. Any evidence of female bias would have important consequences for male-focused mass release programmes. Imbalances have been reported following temperature and diet alterations for Aedes mosquitoes [43, 44]. However, for Anopheles mosquitoes no such differences have been found [45, 46].
Adult phenotypic quality and mating competitiveness are crucial to the success of release programmes [39, 47, 48]. Several studies have reported negative carry-over effects on the phenotypic quality of adult mosquitoes following experimental manipulations of larval conditions, such as temperature, density and food availability [31, 38, 49]. This study found that male and female adults reared at 19oC were smaller than those reared at 27oC, but the 0.05 mm (1.5%) reduction in size observed was unlikely to be biologically important. Indeed, the negligible size differences found did not translate to a negative impact on insemination rates. In the natural setting, An. gambiae s.l. mate in swarms that are typically composed of males and females visit to choose a mate and leave in copula [17, 50]. Smaller males have reduced spermatogenesis and are less competitive in terms of mating than medium-to-large sized mosquitoes, making them poor candidates for release programmes [51, 52]. Compared to the size distribution from those reports (2.48-3.12), males produced in this study at either temperature, were relatively large (2.98-3.08 mm) and consistent with the optimal size of 3mm for mating found in field studies [17].
Smaller females have reduced fecundity, have been shown to require multiple blood feeds before completion of a gonotophic cycle and may be less attractive to males [34, 39, 53, 54]. Although the current study found no difference in overall insemination rates in relation to larval cooling, inseminated An. coluzzii and An. gambiae females were 0.08 mm (2.7%) and 0.09 mm (2.9%) larger than non-inseminated ones, respectively. Although, this is again a very small size difference, the finding that larger females were more likely to mate is consistent with results from insectary and field swarm studies that suggest that males might prefer to mate with larger females [17].
The current study opted to slow down larval development rate by lowering the temperature rather than speed it up by increasing the temperature. Studies elsewhere have shown that at temperatures >34oC there are negative, irreversible carry-over effects on surviving adult mosquitoes and overall survival is lower [32, 34, 55]. Indeed, although adults develop quicker, they are smaller [31, 34, 56] possibly because food consumption cannot sustain the rate of metabolism [57]. Therefore, the current study corroborated previous reports which found that cooling temperatures serve as a reversible inhibitor to mosquito development with negligible impacts on mosquito phenotypic quality, provided they are not maintained throughout their entire development [32, 58]. A relatively short 5-day cooling period of 1st instars was employed, which allowed rearing at 10-fold higher density and ad libitum feeding. In preliminary studies, attempts to also maintain 1st instar larvae at comparable densities at 27oC, found that larval competition negatively affected development rates and success. Hence, keeping 1st instars at high densities was only possible for larvae kept at a cooler temperature which reduced their metabolism and food consumption [57, 59]. The optimized protocol presented here, therefore, exploits the relationship between development rate, temperature, density and food availability to adjust emergence time by ~3 days. As an incubator/fridge will be required for the cooled temperature condition, the 10-fold higher density culture at 19oC make the method both practical and scalable whilst minimizing pressure in terms of insectary space.