Respones of mosquitoes to heavy metals
Odhiambo, Mireji Paul
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Investigations were conducted to determine the influence of heavy metals on urban mosquito populations spatial distribution and composition and Anopheles gambiae sensu stricto fitness. Differential induction of proteins as well as expression of metal lothionein, alpha tubulin gene expressions in third instar An. gambiae s.s larvae in response to heavy metals selection was also investigated. Heavy metal concentrations and mosquito larvae species composition in larval habitats of urban Kisumu and Malindi, Kenya were determined by atomic absorption spectroscopy (AAS) and taxonomic keys respectively. A susceptible strain of An. gambiae s.s third intar larvae was separately placed under selection pressure with cadmium, copper and lead at LC30 and control through five generations. Egg hatchability, fecundity and survivorship of the larvae and adults were monitored in the sixth generation in the absence of heavy metal selecting agent. First, third and fifth generation selection survivors were screened for expression of the metallothionein and alpha tubulin genes by semi quantitative RT PCR and for differentially expression proteins by 2D gel electrophoresis. Manganese and iron were the most prevalent heavy metals in larval habitats in urban Kisumu and Malindi respectively. Concentrations of most were above WHO acceptable limits for drinking water. Copper and lead concentrations significantly influenced presence of Ae. Aegypti, while presence of An. gambiae was affected by lead concentrations. Resistance to cadmium, copper and lead selection increased 13, 11 and 78 folds respectively between first and fifth generation. There was significant reduction in egg viability, larvae survivorship, pupation, magnitude of adult emergence, fecundity, and delay in pupation in the heavy metals selected populations relative to control. The innate rate of increase and mean generation time were significantly lower in heavy metal selected populations than control. Instantaneous birth rate was also significantly lower in all but lead selected populations, than control. Population doubling time was significantly higher in the heavy metals selected than in the non-selected control populations. Expression of metallothionein was significantly higher in cadmium and copper selected than control populations in third and fifth generations respectively. Expression of alpha tubulin was significantly higher in cadmium selected than in control populations in fifth generation. Most differentially expressed protein spots were acidic and of low molecular weight among all metals and generations. Type of heavy metals and generation (selection pressure) were main indicators of differential expression magnitude. Mosquitos that traditionally exclusively proliferate in clean water- like An. gambiae seems capable of expanding their niche into polluted habitats and other hostile environments, but at a considerable biological costs. Semi quantitative RT PCR was sufficiently sensitive to detect heavy metals resistance/ adaptation in An. gambiae s.s larvae in a metal, gene and selection pressure specific manner. Both genes display both qualitative and quantitative potential application in monitoring both An. gambiae s.s adaptation status to heavy metals selection as well as biomonitoring heavy metals pollution through larval habitat. 2D gel electrophoresis also appears sufficiently sensitive to detect heavy-metals and generation specific responsive genes in An. gambiae s.s larvae and can also be used to biomonitor heavy metals environmental anopheles adaptation to pollution. This process would greatly be facilitated by characterization of the spots by mass spectrometry and molecular functional analysis of the spots against An. gambiae protein database.