Background
Malaria is a persistent public health issue. Despite over 50 years of sustained efforts to control the disease through the use of anti-malarial drugs and vector control, transmission has been interrupted in only a limited number of countries. The World Health Organization reported 228 million cases and 405,000 deaths in 2018 [
1].
Most of these deaths occurred in children below five years of age living in sub-Saharan Africa. In recent years, the introduction of insecticide-treated bed nets (ITNs), long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) combined with artemisinin-based combination therapy have resulted in a decline in malaria incidence, thus providing renewed hope for elimination goals [
2,
3]. However, such gains are beginning to diminish, once again threatened by the development and spread of resistance to all anti-malarials and insecticides introduced [
4,
5]. Furthermore, behavioural changes in mosquito vectors, such as biting at dawn or early evening rather than at night when people are under bed net protection, diminishes the effectiveness of current intra-domiciliary control measures [
6‐
9]. Therefore, if reductions in malaria burden are to be at least sustained, alternative complementary approaches are necessary [
10].
Following recent advances in genetic engineering, genetic vector control strategies for malaria mosquitos are now at the forefront of research and development goals [
11‐
13]. These include a range of different approaches that are either self-limiting or self-sustaining. Self-limiting strategies involve the use of genetically modified sterile males or mosquitoes modified with a gene drive mechanism that is spatially or temporally self-limited [
14,
15]. These methods bear similarities with the traditional sterile insect technique (SIT). Their impact depends on effective mating between released mosquitoes and the target population, and require repeated, inundative mass release of mosquitoes [
16,
17]. Self-sustaining strategies employ a gene drive mechanism which means that a desirable trait such as male biased sex ratio [
18,
19], reduced female fertility [
20] or an antiparasitic effector gene [
21] is inherited at a higher rate than mendelian inheritance. The spread of such self-propagating genes can lead to population suppression, reducing the number of biting females or population replacement with mosquitoes that are refractory to the malaria parasite [
22,
23]. The self-sustaining strategies are a longer-term goal that would ideally require relatively smaller initial releases of mosquitoes, thereby making them more cost efficient [
13,
15,
16]. However, the deployment of such genetic tools on a broader scale will still necessitate the production and release of much larger numbers of mosquitoes [
13,
15,
16]. In addition to mosquito release interventions per se, ecological studies that focus on mosquito survival, dispersal or estimation of population sizes, such as mark-release-recapture studies, also rely on the punctual release of mosquitoes reared at a much smaller scale [
24].
One major challenge in rearing
Anopheline mosquitoes for release studies and interventions is that their eggs hatch shortly after being laid and can only survive for a limited number of days without water, hence, egg-to-adult rearing needs to be continuous [
25]. This imposes constraints on rearing protocols and infrastructures and means that the release cohort largely depends on the number of adults in the preceding generation. There have been efforts towards the optimization of
Anopheline egg storage. Through elaborate drying and cooling methods, it is now possible to increase egg storage times by up to 4–6 days, however beyond that point, hatch rate and larval development are negatively impacted [
26‐
28]. Therefore, other avenues to bulk-up
Anopheles mosquitoes for mass release, without affecting their phenotypic quality, should be explored.
The development rate of insects is mainly temperature dependent and offers the potential opportunity to slow or accelerate development [
29]. In
Anopheles gambiae, the relationship between mosquito aquatic stage development and temperature has been well studied [
30‐
34]. Within a minimum and maximum threshold, development rate increases linearly with an increase in temperature. Indeed, Barreaux et al. [
31] reported a 1.4-day difference in time to pupation between larvae maintained at 21
oC and 29
oC. Similarly, Christiansen-Jucht and colleagues reported a linear increase in development rate from 23 to 31
oC, but at 35
oC all larvae died before emergence [
34]. Bayoh and Lindsay [
32] showed that development rate increased linearly with temperatures from 22
oC to 28
oC resulting in an approximate 10-day shift in egg to adult development time. No adults emerged at temperatures below 18
oC or above 34
oC.
This study, aimed to exploit this relationship to mimic synchronization of successive egg batches obtained from repeated blood-feeding of a single female cohort, without impacting negatively on mosquito survival. The rearing temperature of 1st instar larvae of
An. gambiae sensu stricto (
s.s.) and
Anopheles coluzzii laboratory strains was reduced with the aim of slowing down development by approximately 3 days, the time required for one gonotrophic cycle by females at 27
oC [
35‐
37]. The impact of the temperature alteration on the pupation and emergence rates, developmental times, adult phenotypic quality and mating success was evaluated. The ability to slow down a larval cohort by 3 days, hence to synchronize the emergence of adult progeny resulting from multiple blood feeds and successive egg batches from the same pool of females, has important implications for the optimization of mass production and release methods for
Anopheles sensu lato (
s.l.).
Discussion
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 19
oC. 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 rate. 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 19
oC were smaller than those reared at 27
oC, 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 which visit to choose a mate and then 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, are more likely 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 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 > 34
oC 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 27
oC, 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 appromimately 3 days. As an incubator/fridge will be required for the cooled temperature condition, the 10-fold higher density culture at 19
oC make the method both practical and scalable whilst minimizing pressure in terms of insectary space.
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