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Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.

The immune response of Anopheles mosquitoes to Plasmodium invasion has been extensively studied and shown to be mediated mainly by the nitric oxide synthase (NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity. Here, we studied the correlation between a heat shock insult, transcription of immune response genes, and subsequent susceptibility to Plasmodium berghei infection in Anopheles albimanus. We found that transcript levels of many immune genes were drastically affected by the thermal stress, either positively or negatively. Furthermore, the transcription of genes associated with modifications of nucleic acid methylation was affected, suggesting an increment in both DNA and RNA methylation. The heat shock increased PO and NOS activity in the hemolymph, as well as the transcription of several immune genes. As consequence, we observed that heat shock increased the resistance of mosquitoes to Plasmodium invasion. The data provided here could help the understanding of infection transmission under the ever more common heat waves.

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