Sub-lethal metal stress response of larvae of Aedes aegypti

Abstract

Aedes aegypti (Diptera: Culicidae) has adapted to urban environments; the urbanisation process provides suitable habitats for this disease vector subsequently increasing the probability of the transmission of pathogens in high-density environments. Urban environments provide metal stressed larval habitats. However, little is known about the physiological cost of metal stress or how this might affect the performance of this mosquito species. This study aims to characterise the sub-lethal physiological consequences of metal stress in Aedes aegypti. Various parameters of mosquito physiology under larval metal stress are assessed including larval metallothionein expression and the effects of larval metal stress on adult performance and their progeny. Results show that environmentally relevant larval metal stress compromises larval and adult development and performance, and results in larval metal tolerance along with an increase in lipid consumption. These performance costs are coupled to a dramatic increase in metallothionein expression in the midgut. Metal stress results in lowered adult body mass and neutral storage lipids at emergence, starvation tolerance, fecundity and starvation tolerance of offspring compared to non-metal stressed individuals. Ironically, larval metal stress results in increased adult longevity. Together, these findings indicate that even low levels of environmentally relevant larval metal stress have considerable physiological consequences for this important disease vector.

Keywords: Aedes aegypti; metal stress; metallothionein; performance; storage lipids.

Fig. 1

Low sub-lethal metal stress compromised larval and adult phenotypes: Larvae reared in in clean water (A) are easily distinguishable after 24 h from those reared 1 ppm Cu²⁺ (B). Ovaries (ov) and Malpighian tubules (mt) dissected from 3 d old adults were also compromised as a result of low sub-lethal larval metal exposure (C). Metal stressed larvae and adult tissues were less robust, pale and translucent in appearance (all images-40× magnification).

Fig. 2

Dose response curve for copper: A. aegypti 3^rd instars responded to Cu²⁺ in a dose dependent manner in a 24h acute toxicity assay (LOEC = 1.6, LC₅₀ = 2.06 ppm), (n = 25, assayed in triplicate) (Sigmoidal nonlinear curve fit, r² =0.95). Brackets indicate concentrations at which mosquito larvae were reared for subsequent experiments throughout this work.

Fig. 3

A. aegypti 3^rd instars exhibited tolerance: Larvae reared in 0.75 ppm Cu²⁺ to the 3^rd instar stage exhibited significantly longer survivorship (median LT₅₀, 44 vs. 14 h) in an acute toxicity assay (2.0 ppm Cu²⁺) than larvae reared in clean water (n=20 performed in duplicate, log rank test, Chi square = 4.65, P = 0.03).

Fig. 4

The nutritional cost of metal stress: Fourth instar larvae held in 1.25 ppm Cu²⁺ for 5 d resulted in a reduction in neutral lipid reserves (NSL) compared to larvae held in clean water (P = 0.018, students t test, one tailed) (n=3 assayed in triplicate). Representative image of fourth instar larvae stained with Oil Red O; ovals indicate area where fat body is located (inset).

Fig. 5

AaMtn was expressed predominately in the midgut of larvae: (A) AaMtn expression in midguts (mdgt) of 4th instars held in clean water and exposed to 1.25 ppm Cu²⁺ for 6 h was higher than AaMtn expression in the rest of the body. (B) Midguts of larvae exposed to metal stress expressed ~2 the AaMtn than midguts of larvae held in clean water. AaMtn expression in body tissues of metal exposed and clean exposed were the same (t test, one way, P < 0.01, df = 8) (Three independent biological replicates of five tissues each) (mdgt = midgut; body = rest of the body minus midgut; cln = clean reared; mtl = metal stress).

Fig. 6

AaMtn transcription is highly dynamic: Newly emerged 1^st instars were exposed to 1.25 ppm Cu²⁺ for 12 h or held in clean water. For the rescue effect, larvae that were metal exposed to Cu²⁺ for 6 h were transferred back to clean water and held for 6h. AaMtn mRNA is expressed as copy number of AaMtn mRNA/ 10,000 copies of rpL32 mRNA. Each q-PCR data point is a mean ± SEM of three independent biological replicates of ~50 larvae (one way ANOVA with a Tukey’s multiple comparison test, F=7.02, total df = 19, P = 0.0022). Letter designations indicate significant differences for multiple comparison tests (P < 0.05).