Mosquitoes require a bloodmeal to initiate a reproductive cycle and produce eggs. Within this bloodmeal are insulin, insulin-like growth factor 1, and various other factors that circulate in the blood of the human host. Our previous work demonstrated that some of these factors, including human insulin and human TGF-β1, activate mitogen-activated protein (MAP) kinase and phosphoinositide-3 kinase (PI3K) signaling cascades in the mosquito midgut 
. Here, we used transgenesis to overexpress a key component of the IIS cascade, Akt, in the A. stephensi
midgut to induce signaling independent of exogenous insulin. We observed significant reductions in both the prevalence and intensity of P. falciparum
infections in transgenic mosquitoes following the consumption of an infective bloodmeal. We also observed a reduction in lifespan consistent with that observed in insulin-fed A. stephensi 
, indicating that the mosquito midgut plays a central role in regulating lifespan.
Myristoylated Akt localized to the midgut epithelial cell membrane in transgenic A. stephensi
() where it was activated to subsequently phosphorylate the downstream effector protein FOXO1 (). This parallels FOXO1 phosphorylation in the midguts of mosquitoes fed bloodmeals containing insulin (). Taken together, these results suggest that the mechanisms of parasite and lifespan reduction observed in CP-myr-AsteAkt-HA transgenic mosquitoes are dependent on the activation of the PI3K/Akt/FOXO arm of the IIS cascade. It is noteworthy that Akt has been defined as “a critical signaling node within all cells of higher eukaryotes and one of the most important and versatile protein kinases at the core of human physiology and disease 
.” Akt has more than 100 experimentally verified substrates and broad crosstalk between a variety of biologically important signal transduction pathways. Thus, the mechanisms through which tissue-specific Akt overexpression regulates innate immunity and lifespan are likely to be complex 
A carboxypeptidase promoter drives the myr-AsteAkt-HA transgene, so we expected expression to rise shortly after a bloodmeal was consumed and to be midgut-specific. Expression of myr-AsteAkt-HA was indeed specific to the midgut (), but the timing of expression was unexpected since both transcript and protein were observed even in the absence of a bloodmeal (). As expected for a gene regulated by a carboxypeptidase promoter, however, protein expression increased following ingestion of the bloodmeal. Leaky transgene expression has been observed with this promoter, resulting in expression prior to bloodfeeding 
or late in the reproductive cycle 
. The process of generating a transgenic mosquito strain could also explain the unexpected expression patterns. For example, the transgene may have inserted near an enhancer DNA sequence, resulting in greater gene and protein expression 
. Although this pattern of myr-AsteAkt-HA expression was unexpected, it was ultimately advantageous because increased insulin signaling is maintained for the apparent duration of adult female life and does not depend on consumption of a bloodmeal for activity. Thus, the anti-parasite activity and lifespan effects of myr-AsteAkt-HA will occur regardless of the timing and quantity of bloodmeals that are consumed by a transgenic mosquito.
Increased insulin signaling in the mosquito midgut, whether through ingestion of exogenous insulin 
or overexpression of active IIS proteins such as Akt, can significantly reduce mosquito lifespan and inhibit P. falciparum
development. Importantly, we observed that increased AsteAkt expression in the homozygous line increased parasite resistance to the point that oocyst formation on the midgut was completely blocked. Although it will likely be necessary to deploy heterozygous mosquitoes for any future transmission blocking strategy, our data suggest that an increase in myr-AsteAkt expression, possibly through manipulation of the promoter or transgene insertion site, could yield heterozygous mosquitoes that are resistant to P. falcipaurm
infection. Lifespan reduction can also impact malaria parasite prevalence based on the combined effects of a relatively short natural lifespan of A. stephensi 
and a relatively lengthy parasite development time. In particular, models of vector competence routinely demonstrate that the daily probability of survival is the single most important factor in determining how effectively a mosquito transmits a pathogen 
. All else being equal, even modest reductions in lifespan will have significant effects on disease transmission.
In summary, we have developed a novel mechanism to reduce the transmission of the human malaria parasite P. falciparum. This approach is based on the manipulation of two key physiological parameters, lifespan and innate immunity, through activation of a single signaling protein, Akt. Increased Akt activity significantly reduced infection prevalence in the mosquito host at the same time that it reduced the infective period of the mosquito lifespan. A multi-component approach to transgenesis focused on manipulation of the IIS cascade could be combined with overexpression of additional anti-parasite effectors to effectively block parasite transmission, reduce lifespan, and increase fecundity. Perhaps more importantly, a multi-component approach could prevent the escape of adaptive parasite variants, providing a powerful new tool for malaria control.