The effect of metal pollution on the life history and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae)

Abstract

Metal exposure is one of the commonest anthropogenic pollutants mosquito larvae are exposed to, both in agricultural and urban settings. As members of the Anopheles gambiae complex, which contains several major malaria vector species including An. arabiensis, are increasingly adapting to polluted environments, this study examined the effects of larval metal exposure on various life history traits of epidemiological importance. Two laboratory strains of An. arabiensis, SENN (insecticide susceptible) and SENN DDT (insecticide resistant), were reared in maximum acceptable toxicity concentrations, (MATC-the highest legally accepted concentration) of cadmium chloride, lead nitrate and copper nitrate. Following these exposures, time to pupation, adult size and longevity were determined. Larvae reared in double the MATC were assessed for changes in malathion and deltamethrin tolerance, measured by lethal time bottle bioassay, as well as changes in detoxification enzyme activity. As defence against oxidative stress has previously been demonstrated to affect the expression of insecticide resistance, catalase, glutathione peroxidase and superoxide dismutase activity was assessed. The relative metal toxicity to metal naïve larvae was also assessed. SENN DDT larvae were more tolerant of metal pollution than SENN larvae. Pupation in SENN larvae was significantly reduced by metal exposure, while adult longevity was not affected. SENN DDT showed decreased adult size after larval metal exposure. Adult insecticide tolerance was increased after larval metal exposure, and this effect appeared to be mediated by increased β-esterase, cytochrome P450 and superoxide dismutase activity. These data suggest an enzyme-mediated positive link between tolerance to metal pollutants and insecticide resistance in adult mosquitoes. Furthermore, exposure of larvae to metal pollutants may have operational consequences under an insecticide-based vector control scenario by increasing the expression of insecticide resistance in adults.

Fig 1. Relative toxicity of heavy metals on insecticide resistant and susceptible Anopheles arabiensis laboratory strains.

The insecticide resistant An. arabiensis strain SENN DDT showed a significantly higher tolerance for metal exposure than the insecticide susceptible SENN strain, based on LD50s. Copper nitrate was the most toxic metal to both strains, while both strains showed a high tolerance to lead nitrate. Significant differences (p<0.05) are indicated by an asterisk (*).

Fig 2. The effect of metal pollution on larval development in insecticide susceptible and resistant Anopheles arabiensis.

SENN and SENN DDT larvae were reared in the maximum acceptable toxicity concentration (MATC) of cadmium chloride, copper nitrate and lead nitrate. Both strains had the highest number of pupae emerging on day 9, regardless of whether they were reared in metal polluted or untreated control water. There was variation in the total number that pupated on day 9. Larval cadmium chloride and copper nitrate treatment significantly reduced the percentage that pupated on day 9 in the insecticide susceptible SENN strain, but not the resistant SENN DDT strain. Lead nitrate treatment did not affect pupation in either strain.

Fig 3. The effect of larval metal exposure on adult longevity in insecticide susceptible and resistant Anopheles arabiensis.

Larval metal exposure did not affect adult longevity in insecticide susceptible SENN males or females, and did not affect the longevity of insecticide resistant SENN DDT females. SENN DDT males, however, did show an increase in longevity after larval metal treatment (A). That change was not due to copper nitrate (B) or lead nitrate (C) treatment, but was due to larval cadmium treatment (D).

Fig 4. The effect of larval metal exposure on adult size in insecticide susceptible and resistant Anopheles arabiensis.

Although larval metal treatment had no significant effect on adult size in the insecticide susceptible SENN strain, it did result in significant changes in the size of adults of the resistant SENN DDT strain. SENN DDT males that emerged from larvae exposed to lead nitrate were significantly smaller than those reared in untreated water. Similarly, SENN DDT females that eclosed from larvae that were exposed to copper nitrate and cadmium chloride were significantly smaller than adult females reared in untreated water. Significant differences from the control (p<0.05) are indicated by an asterisk (*).

Fig 5. The effect of larval metal exposure on the insecticide tolerance of insecticide susceptible and resistant Anopheles arabiensis females.

Larval metal exposure resulted in increased tolerance of malathion and deltamethrin in the insecticide resistant SENN DDT strain. All larval metal treatments resulted in a significantly increased lethal time (LT50s) in adult females (A). Larval treatment with cadmium chloride and lead nitrate resulted in a significant increase in malathion and deltamethrin tolerance of adult females of the insecticide susceptible SENN strain. Copper nitrate did not result in a change in tolerance to either insecticide in the SENN strain. Significant changes from the control (p<0.05) are indicated by asterisk (*).

Fig 6. The effect of larval metal exposure on the detoxification enzyme activity of insecticide susceptible and resistant Anopheles arabiensis adults.

Glutathione S-transferase (GST) activity was significantly decreased in SENN DDT females after all metal treatments, but no other treatments resulted in changes in GST activity (A). Cytochrome P450 activity, however, was significantly affected by certain treatments. SENN females showed a significant increase in activity after larval cadmium exposure. SENN DDT adult males showed significantly increased P450 activity after larval copper nitrate and lead nitrate exposure, while SENN DDT female enzyme activity was significantly increased after cadmium chloride and lead nitrate exposure (B). Neither α-esterase activity (C) nor β-esterase activity (D) was significantly altered in adults after larval metal exposure. Significant changes from control treatment (p<0.05) are indicated by asterisk (*).

Fig 7. The effect of larval metal exposure on the oxidative stress enzyme activity of insecticide susceptible and resistant Anopheles arabiensis adults.

Larval metal exposure had a highly variable effect on the three classes of oxidative stress defence enzymes. Catalase was least affected by larval metal exposure, with only SENN DDT females showing increased activity after larval copper nitrate and cadmium chloride exposure (A). Glutathione peroxidase activity showed the greatest variability after larval metal treatment. In the insecticide susceptible SENN strain, all three larval metal treatments significantly decreased peroxidase activity in adult males. In SENN females, although copper nitrate treatment significantly decreased peroxidase activity, cadmium treatment significantly increased adult peroxidase activity. In contrast, larval metal treatment had no effect on adult peroxidase activity in males of the resistant SENN DDT strain, while lead nitrate treatment resulted in a significant increase in activity in females (B). Superoxide dismutase activity was the most uniformly affected, with only lead treatment failing to elicit a significant increase in superoxide dismutase activity (C). Significant changes from the control (p<0.05) are indicated by an asterisk (*).