Background
Malaria infects 300–500 million people each year [
1]. More than 90% of malarial cases occur in Sub-Saharan Africa, where
Anopheles gambiae is the principal vector. Despite the high vectorial capacity of this mosquito, critical aspects of its biology bearing on population dynamics and ultimately malaria transmission remain to be elucidated. For example, little is known about how abiotic factors like temperature influence egg survival. Eggs of
An. gambiae are more frequently found on mud (soil) around puddle habitats of the larvae than on the water surface itself [
2]. Larvae developing and hatching on wet substrates like mud [
3] can crawl to puddles [
2,
4] or perhaps be washed there by rains [
2]. It is also not uncommon for puddles and the mud around them to desiccate. Thus, eggs can experience temperature regimes more wide-ranging and lethal than temperatures experienced by larvae or pupae in puddles.
Some research has been conducted on tolerance of
An. gambiae eggs to desiccation. Unlike some mosquitoes, e.g.,
Aedes and
Ochlerotatus, eggs of
An. gambiae cannot tolerate prolonged desiccation [
5]. Survivorship of
An. gambiae eggs in drying soils held in the laboratory was found to be inversely related to time after deposition; very few eggs in drying soils hatched after 12 to 15 days upon re-flooded [
6]. It has been suggested that the egg stage of
An. gambiae might contribute to the short-term survival of this vector during dry periods [
6]. However, these studies did not consider temperatures likely to be encountered when soils dry under natural, out-door conditions likely to be sunny.
The effects of temperature on embryonic development and egg hatching of
An. gambiae have received little attention. In contrast, considerable data are available for other mosquitoes and insects generally [
7‐
11]. Upper tolerable temperatures for eggs in these studies ranged from 33 to 48°C.
Growth, development, and survival of
An. gambiae as influenced by constant temperatures between 10 and 40°C have been analyzed under laboratory conditions [
12]. The optimal temperature for larval growth was 28°C, while maximal fitness of adults occurred between 28 and 32°C. Growth and development of instars 1–4 and pupae ceased at 40, 38, 36, 34, and 34°C, respectively, under constant temperature regimes [
12]. However, the ability of
An. gambiae eggs to withstand temperatures of 40°C and greater was not reported, nor was the effect of fluctuating temperatures evaluated.
The objectives of the current study were to: 1) establish lethal temperatures for An. gambiae eggs briefly exposed to elevated temperatures, and 2) determine whether and for how long eggs located in and around typical An. gambiae larval habitats would be exposed to damaging temperatures during the long rainy season in Kisumu, Kenya.
Discussion
It is well known that insect embryogenesis and egg hatching are influenced by temperature [
10]. There are definite thresholds below or above which no eggs hatch. For example, all the embryos of
Culex theileri died after eggs were incubated at a constant temperature between 39 to 42°C [
11]. Exposure of eggs of
Culex quinquefasciatus for 24 h at 39°C completely inhibited egg hatch [
10]. All eggs of
Aedes structys exposed to a constant 33°C failed to hatch [
9]. No larvae emerged after
Anopheles sergentii eggs were incubated at 34°C [
18]. The upper tolerable temperatures for egg development and hatching of other insects were: 46 to 48°C for the tephritid fruit fly,
Bactrocera latifrons [
19]; 42°C for the Queensland fruit fly,
Bactrocera tryoni [
8]; 39°C for the common cattle grub,
Hypoderma lineatum [
20]; 37°C for the reindeer warble fly,
Hypoderma tarandi [
21]; and 32°C for the Diaprepes root weevil,
Diaprepes abbreviatus [
7].
Survival of
An. gambiae eggs was strongly influenced both by temperature and exposure times (Figure
3; Table
1). The upper tolerable temperature for these eggs was 40°C. Above this threshold, the rate of egg kill was approximately linear over time for a given temperature (Figure
3). Estimated LT
50 values for the following temperatures were: 41°C – 147 min; 42°C – 66 min; 43°C – 30 min; 44°C – 14 min; 45°C – 6 min; and 46°C – 2.8 min (based on the equation from Figure
4). Thus, for each successive degree temperature rose beyond 41°C, the time required for 50 % egg kill was approximately halved. Stated conversely, the velocity of some time-dependent process killing eggs approximately doubled with each rise of one degree C.
Protein denaturation [
22] is a likely mechanism explaining this pattern of lethality. Nguyen et al. [
24] quantified the time-course for denaturation of a firefly luciferase and an
Escherichia coli β-galactosidase transfected into
Drosophila and mouse cell lines. Denaturation was detectable at 37°C, but with a half-life of more than three h. Incubation of these cells at 42°C yielded approximately linear protein degradation profiles with half-lives ranging from 5–40 min depending upon experimental conditions. Mortality profiles in the current study using
An. gambiae eggs strongly resemble these carefully quantified protein-denaturation profiles from
in vivo and
in vitro preparations using cell lines and proteins from organisms not known to be selected for high thermal tolerance. Such similarities suggest
An. gambiae is not uniquely adapted to tolerate temperatures above those lethal to animal cells generally.
Ability to tolerate high temperatures can vary with life stage. Results from the current study combined with the findings of Bayoh and Lindsay [
12] reveal that
An. gambiae eggs are the most (40°C) and pupae and the 4
th instars the least (34°C) heat-tolerant life stages, respectively. Notably, the temperatures within our puddles sometimes exceeded the tolerance limits for larvae and pupae as measured under a constant-temperature rearing regimes [
12]. Munga et al. [
23] reported that the mean maximum daily water temperature in puddle habitats in open farmland was 38.8 ± 0.3°C. In the current study, that value was 31.3 ± 0.8°C (mean ± SEM). Although daily temperature profiles in and around these larval habitats were measured only for 7 days, data collected at a weather station [
24] indicated that the mean air temperature was very stable throughout all of 2005. Further research is justified to document to what extent
Anopheles gambiae adult production is limited by maximal temperatures within puddles.
Eggs from the laboratory strain were slightly more sensitive to heat stress than eggs from house-collected mosquitoes. Perhaps rearing for more than five years under no thermal stress caused the laboratory strain to lose a bit of thermal tolerance. Enhanced thermal tolerance of individual insects following non-lethal heat shock has been reported in some insects [
8,
25,
26], including anopheline mosquito [
26]. The effect of inducing heat-shock proteins in larvae of
Anopheles albimanus was to increase the upper tolerable temperature by only 1.5°C. Thermal conditioning in
An. gambiae eggs and interaction of temperature with humidity are worthy of additional investigation.
An. gambiae eggs appear to be thermally adapted only to residing on water or moist mud. Rapid evaporation of water from mud apparently has a pronounced cooling effect, making mud not much different thermally from water (Figure
5). Perhaps this is why
An. gambiae readily oviposit on moist or wet soil [
2]. However, sun-exposed dry soil is inhospitable to these eggs because it readily exceeds lethal temperatures. Eggs on the surface of dry soils baked for several hours under full sun will not survive and thus cannot contribute to
An. gambiae populations and malaria transmission.
Acknowledgements
The authors thank Joseph Nduati (Kenya Medial Research Institute (KEMRI), Kisumu, Kenya) for PCR species identification. The authors greatly acknowledge assistance with daily temperature measurement by Charles Ochieng and Geraldine Mwende (Kisian, Kenya). The authors also thank Piera Siegert, Bill Morgan, and Blair Bullard (Michigan State University) for technical assistance. This study was funded by National Institute of Health grant AI50703.
Authors' contributions
JH and JRM designed and carried out the experiments, analysed and interpreted data, as well as drafted and revised the manuscript. EDW was P. I. of the grant supporting this work; he participated in study design, data interpretation, and revision of the manuscript. JV provided institutional support for this study. All authors read and approved the final manuscript.