The high level of divergence between dipteran and lepidopteran apoLp-IIIs sequences may reflect the plasticity of apoLp-IIIs, as divergent amino acid sequences can fold into proteins with similar tertiary structures. Some of the divergence may also be due to adaptation to differences in biology and feeding behavior. For example, adult mosquito females rely on blood as a nutrient source for reproduction, which is very rich in proteins and lipids compared to the flower nectar or fruit juice consumed by adult moths. Mosquito apoLp-IIIs could have diverged as they adapted to meet the functional demands of blood feeding. Notably, we were unable to identify orthologs of mosquito ApoLp-IIIs in any other dipteran insect, including Drosophila, precluding a phylogenetic comparison within this order.
In G3 An. gambiae
females infected with P. berghei
AgApoLp-III expression was highly induced in response to P. berghei
infection 24 hpi, when ookinete invasion is taking place (, A and D). This induction required the presence of ookinetes, as it was no longer observed when mosquitoes fed on an infected mouse were kept at a temperature that prevents ookinete formation (28°C) (). The induction of AgApoLp-III is not a response to bacteria in the gut lumen, because ookinete invasion still induces expression after the gut microbiota was eliminated by oral administration of antibiotics (); and is not part of a general apoptotic response, because it is not observed when apoptosis of midgut epithelial cells is induced by oral administration of actinomycin D. Interestingly, in the refractory An. gambiae
L35 strain, AgApoLp-III silencing also greatly increased the number of parasites present 7 days post infection 
. In L35 females, however, all parasites are still eliminated and melanized in the ookinete-to-oocyst transition 
. Together, these data indicate that in G3 and L35 mosquitoes AgApoLp-III silencing increases the number of ookinetes that successfully invade the midgut and reach the basal lamina.
The response of An. gambiae
Yaoundé females to Plasmodium
infection is very different 
. In this strain, AgApoLpIII expression 22–25 hours post-feeding was not induced in the midguts of mosquitoes infected with P. berghei
ANKA 2.34, which generates invasive ookinetes, relative to the control group fed on mice infected with the P. berghei
ANKA 2.33 strain that lacks gametocytes and does not form ookinetes 
. In G3 An. gambiae
females, AgApoLp-III expression in infected midguts is regulated at the transcriptional level. The lack of transcriptional activation of the AgApoLp-III gene in Yaoundé females could explain why silencing AgApoLp-III in this strain had no effect on Plasmodium
. It is surprising that two An. gambiae
strains considered to be susceptible to P. berghei
infection (Yaoundé and G3) exhibit such dramatic differences in AgApoLp-III regulation when they are infected with the same parasite. Although the mechanism mediating these differences is unknown, these observations add support to the idea that there is a broad range of compatibility, defined as the extent to which the immune system limits infection, between particular strains of mosquitoes and specific parasites strains 
It is not clear why high levels of AgApoLp-III in the cytoplasm of invaded cells are detrimental to the parasite. It is possible that AgApoLp-III could be working as a cytoplasmic pathogen recognition receptor. If this were the case, one would predict that some other cellular responses such as protein nitration may not be triggered effectively when the presence of parasite-derived molecules is not detected.
We have previously shown that ookinete invasion induces high levels of nitric oxide synthase expression in the cell cytoplasm 
that is followed by peroxidase-mediated protein nitration 
and proposed that these cellular responses limit Plasmodium
survival  
. Studies in vertebrates indicate that apolipoprotein E (apoE), a mammalian homolog of insect apoLp-III, increases nitric oxide production in immune activated RAW cells by increasing arginine uptake 
. Thus, AgApoLp-III could be enhancing the production of nitric oxide in invaded midgut cells.
AgApoLp-III silencing also promotes Plasmodium
melanization (), indicating that, when present, AgApoLp-III inhibits activation of melanization responses in the mosquito. Recently, Seo et al. 
reported that lipid-bound apoLp-III from H. cunea
plays a critical role in reducing oxidative stress, whereas this antioxidant activity is absent in the lipid-free state. ApoLp-III in the hemolymph may reduce lipid oxidation and prevent activation of the phenol oxidase cascade.
Our studies revealed that, besides the known roles of ApoLp-IIIs in lipid transport and hemocyte biology, AgApoLp-III expression can be also induced in epithelial cells in response to parasite invasion. Furthermore, expression of AgApoLp-III in the cell cytoplasm enhances antiplasmodial defense. The molecular mechanism mediating these responses is under investigation.