Supplementary Material for: Functional Interaction between Apolipophorins and Complement Regulate the Mosquito Immune Response to Systemic Infections

<br>The complement-like protein thioester-containing protein 1 (TEP1) is the hallmark effector molecule against <i>Plasmodium</i> ookinetes in the malaria vector <i>Anopheles gambiae</i>. We have previously shown that the knockdown of the noncatalytic clip domain serine protease CLIPA2 increased TEP1-mediated killing rendering mosquitoes more resistant to <i>Plasmodium</i>, bacterial and fungal infections. Here, CLIPA2 coimmunoprecipitation from the hemolymph of <i>Beauveria bassiana</i>-infected mosquitoes followed by mass spectrometry and functional genetic analysis led to the identification of the <i>Apolipophorin-II/I</i> gene, encoding the two lipid carrier proteins Apo-I and II, as a novel negative regulator of TEP1-mediated immune response during mosquito systemic infections. <i>Apo-II/I</i> exhibits a similar RNAi phenotype as <i>CLIPA2</i> in mosquito bioassays characterized by increased resistance to <i>B. bassiana</i> and <i>Escherichia coli</i> infections. We provide evidence that this enhanced resistance to systemic infections is TEP1 dependent. Interestingly, silencing <i>Apo-II/I</i> but not <i>CLIPA2 </i>upregulated the expression of <i>TEP1</i> following systemic infections with <i>E. coli</i> and <i>B. bassiana</i> in a c-Jun N-terminal kinase pathway-dependent manner. Our results suggest that mosquito Apo-II/I plays an important immune regulatory role during systemic infections and provide novel insight into the functional interplay between lipid metabolism and immune gene regulation

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