Supplementary Material for: Anopheles gambiae Blood Feeding Initiates an Anticipatory Defense Response to Plasmodium berghei

<p>Mosquitoes have potent innate defense mechanisms that protect them from infection by diverse pathogens. Much remains unknown about how different pathogens are sensed and specific responses triggered. Leucine-Rich repeat IMmune proteins (LRIMs) are a mosquito-specific family of putative innate receptors. Although some LRIMs have been implicated in mosquito immune responses, the function of most family members is largely unknown. We screened <i>Anopheles gambiae </i>LRIMs by RNAi for effects on mosquito infection by rodent malaria and found that LRIM9 is a <i>Plasmodium berghei </i>antagonist with phenotypes distinct from family members LRIM1 and APL1C, which are key components of the mosquito complement-like pathway. LRIM9 transcript and protein levels are significantly increased after blood feeding but are unaffected by <i>Plasmodium </i>or midgut microbiota. Interestingly, LRIM9 in the hemolymph is strongly upregulated by direct injection of the ecdysteroid, 20-hydroxyecdysone. Our data suggest that LRIM9 may define a novel anti-<i>Plasmodium </i>immune defense mechanism triggered by blood feeding and that hormonal changes may alert the mosquito to bolster its defenses in anticipation of exposure to blood-borne pathogens.</p

Evaluation of drive mechanisms (including transgenes and drivers) in different environmental conditions and genetic backgrounds

Three major objectives, develop viable gene drive mechanisms, identify the epidemiologically significant vectors of pathogens in specific transmission zones, and introgress effector genes into specific populations, must be met in order to move to the field laboratory advances in genetic control strategie

What are relevant assays for refractoriness?

The engineering and testing of genes that result in refractory phenotypes are important components of the continuing effort towards the use of population replacement strategies for vector control. Both ‘endogenous’ and ‘synthetic’ refractory phenotypes are being considered. Additional research is required to determine the prevalence of such phenotypes in the various vector–pathogen combinations, and the threshold levels of activity of genes conferring transmission blocking, as well as to develop efficient methods for the evaluation of their entire spectrum of biological effect

Supplementary Material for: A Serine Protease Homolog Negatively Regulates TEP1 Consumption in Systemic Infections of the Malaria Vector Anopheles gambiae

Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of <i>Anopheles gambiae</i>, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1_cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including <i>Plasmodium berghei</i>, <i>Escherichia coli</i> and the entomopathogenic fungus <i>Beauveria bassiana</i>. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in <i>CLIPA2</i> knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections

Supplementary Material for: The Peptidoglycan Recognition Proteins PGRPLA and PGRPLB Regulate Anopheles Immunity to Bacteria and Affect Infection by Plasmodium

<p>Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito <i>Anopheles</i><i>coluzzii</i>, the peptidoglycan receptor PGRPLC activates the immune-deficiency (Imd) pathway limiting both the microbiota load and <i>Plasmodium</i> infection. Here, we carried out an RNA interference screen to examine the role of all 7 <i>Anopheles</i> PGRPs in infections with <i>Plasmodium berghei</i> and <i>P. falciparum</i>. We show that, in addition to PGRPLC, PGRPLA and PGRPS2/PGRPS3 also participate in antiparasitic defenses, and that PGRPLB promotes mosquito permissiveness to <i>P. falciparum</i>. We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respectively. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector competence.</p