Heat shock proteins contribute to mosquito dehydration tolerance

Abstract

This study examines the responses of heat shock protein transcripts, Hsp70 and Hsp90, to dehydration stress in three mosquito species, Aedes aegypti, Anopheles gambiae and Culex pipiens. We first defined the water balance attributes of adult females of each species, monitored expression of the hsp transcripts in response to dehydration, and then knocked down expression of the transcripts using RNA interference (RNAi) to evaluate potential functions of the Hsps in maintenance of water balance. Fully hydrated females of all three species contained nearly the same amount of water (66-68%), but water loss rates differed among the species, with A. aegypti having the lowest water loss rate (2.6%/h), followed by C. pipiens (3.3%/h), and A. gambiae (5.1%/h). In all three species water could be replaced only by drinking water (or blood). Both A. aegypti and C. pipiens tolerated a loss of 36% of their body water, but A. gambiae was more vulnerable to water loss, tolerating a loss of only 29% of its body water. Dehydration elicited expression of hsp70 in all three species, but only C. pipiens continued to express this transcript during rehydration. Hsp90 was constitutively expressed and expression levels remained fairly constant during dehydration and rehydration, except expression was not noted during rehydration of C. pipiens. Injection of dsRNA to knock down expression of hsp70 (83% reduction) and hsp90 (46% reduction) in A. aegypti did not alter water content or water loss rates, but the dehydration tolerance was lower. Instead of surviving a 36% water loss, females were able to survive only a 28% water loss in response to RNAi directed against hsp70 and a 26% water loss when RNAi was directed against hsp90. These results indicate a critical function for these Hsps in mosquito dehydration tolerance.

Fig. 1

Net water loss rates for females of Aedes aegypti, Anopheles gambiae, and Culex pipiens. The slope represents the water loss rate in % h⁻¹ . m_t represents the water mass at anytime t and m₀ is the initial water mass. Values represent the mean ± SE of 45 mosquitoes.

Fig. 2

Percent survival of adult females of Aedes aegypti, Anopheles gambiae, and Culex pipiens when held at 0% RH. Each point represents the mean ± SE of 6 groups of 10 mosquitoes.

Fig. 3

Water loss rates at relative humidities ranging from complete dryness (0% RH) to water saturation (100% RH) for adult females of Aedes aegypti, Anopheles gambiae, and Culex pipiens. Rates were determined in the same manner as Fig. 1.

Fig. 4

Northern blot hybridizations displaying mRNA expression of hsp70 and hsp90 during dehydration in three mosquitoes (Aedes aegypti, Anopheles gambiae, and Culex pipiens). 28s was used as a control gene. Control, fully-hydrated mosquitoes; 5, 15, and 25% represent percent reduction in water content; rehy, mosquitoes that lost 15% of their water content and were then rehydration at 100% RH in the presence of liquid water. Each blot was replicated three times.

Fig. 5

Northern blot hybridizations displaying knockdown of hsp70 and hsp90 transcripts after injection of dsHsp70, dsHsp90, and dsβ-galactosidase. A sham injection was conducted as an additional control. Percent expression changes are in comparison to the sham control. Each knockdown experiment was conducted three times.

Fig. 6

Physiological changes in water balance features in adult females of Aedes aegypti following injection of dsHsp70, dsHsp90, and dsβ-galactosidase. A sham injection was also performed to control for possible cuticle damage that may have altered water vapor exchange. (A) Time to 50% mortality for mosquitoes held at 0% RH (B) water loss rates (% h⁻¹) determined according to Fig. 1, and (C) dehydration tolerance displayed as percentage of water loss tolerated (mean of three groups of 20 mosquitoes each ). *, denotes significance difference from sham control.