The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.

The serine protease homolog CLIPA14 modulates the intensity of the immune response in the mosquito Anopheles gambiae.

Clip domain serine protease homologs (SPHs) are positive and negative regulators of Anopheles gambiae immune responses mediated by the complement-like protein TEP1 against Plasmodium malaria parasites and other microbial infections. We have previously reported that the SPH CLIPA2 is a negative regulator of the TEP1-mediated response by showing that CLIPA2 knockdown (kd) enhances mosquito resistance to infections with fungi, bacteria, and Plasmodium parasites. Here, we identify another SPH, CLIPA14, as a novel regulator of mosquito immunity. We found that CLIPA14 is a hemolymph protein that is rapidly cleaved following a systemic infection. CLIPA14 kd mosquitoes elicited a potent melanization response against Plasmodium berghei ookinetes and exhibited significantly increased resistance to Plasmodium infections as well as to systemic and oral bacterial infections. The activity of the enzyme phenoloxidase, which initiates melanin biosynthesis, dramatically increased in the hemolymph of CLIPA14 kd mosquitoes in response to systemic bacterial infections. Ookinete melanization and hemolymph phenoloxidase activity were further increased after cosilencing CLIPA14 and CLIPA2, suggesting that these two SPHs act in concert to control the melanization response. Interestingly, CLIPA14 RNAi phenotypes and its infection-induced cleavage were abolished in a TEP1 loss-of-function background. Our results suggest that a complex network of SPHs functions downstream of TEP1 to regulate the melanization reaction.

The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae.

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1(cut) that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.

The mosquito melanization response is implicated in defense against the entomopathogenic fungus Beauveria bassiana.

Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic fungi with more potent bio-control activities against mosquitoes.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

CLIPB4 is a central node in the protease network that regulates humoral immunity in Anopheles gambiae mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPB4 Is a Central Node in the Protease Network that Regulates Humoral Immunity in Anopheles gambiae Mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPA7 Exhibits Pleiotropic Roles in the Anopheles gambiae Immune Response.

Clip domain serine proteases and clip domain serine protease homologs (cSPHs) are key components of serine protease cascades that drive the melanization response. Despite lacking catalytic activity, cSPHs play essential roles in regulating melanization, but the spectrum of functions they catalyze within and outside these cascades is not fully understood. Aside from their classical role as cofactors for PPO activation, we have previously revealed an unprecedented complexity in the function and molecular organization of these cSPHs in the immune response of the malaria vector Anopheles gambiae. Here, we add yet another dimension to the complex roles underpinning the contributions of cSPHs to mosquito immunity by showing that CLIPA7, a member of the expanded cSPH family, defines a novel branch within the cSPH network that is essential for the melanization of Escherichia coli but not Plasmodium ookinetes or Gram-positive bacteria. Despite its dispensability for the melanization of Gram-positive bacteria, we show that CLIPA7 is required for the clearance of systemic infections with Staphylococcus aureus. CLIPA7 is produced by hemocytes and associates with the surfaces of live E. coli and S. aureus cells in vivo as well as with those of melanized cells. Based on its RNAi phenotypes and its unique domain architecture among A. gambiae cSPHs including the presence of an RGD motif, we propose that CLIPA7 exhibits pleiotropic roles in mosquito immunity that extend beyond the regulation of melanization to microbial clearance.

Anopheles gambiae innate immunity.

The successful development of Plasmodium in Anopheles mosquitoes is governed by complex molecular and cellular interactions that we are just beginning to understand. Anopheles immune system has received particular attention as genetic evidence points clearly to its critical role in eliminating the majority of parasites invading the midgut epithelium. Several factors regulating Plasmodium development have been identified and tentatively assigned to the individual steps leading to mosquito immune reactions; non-self-recognition, signal modulation, signal transduction and effector mechanisms. Detailed knowledge of these steps and their underlying molecular mechanisms may offer novel perspectives to abort Plasmodium development in the vector. Here, we summarize our current knowledge of mosquito innate immunity highlighting both, recent advances and areas where additional research is required.

The Route of Infection Influences the Contribution of Key Immunity Genes to Antibacterial Defense in Anopheles gambiae.

Insect systemic immune responses to bacterial infections have been mainly studied using microinjections, whereby the microbe is directly injected into the hemocoel. While this methodology has been instrumental in defining immune signaling pathways and enzymatic cascades in the hemolymph, it remains unclear whether and to what extent the contribution of systemic immune defenses to host microbial resistance varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infections of the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Using functional genetic studies by RNAi, we report that the two humoral immune factors, thioester-containing protein 1 and C-type lectin 4, which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection, but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Rel2 pathway. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. Transcriptomic analysis of mosquito midguts and abdomens by RNA-seq revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm. Functional classification of differentially expressed transcripts identified metabolic genes as the most represented class in response to both routes of infection, while immune genes were poorly regulated in both routes. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host antimicrobial defense but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host.

CLIPB10 is a Terminal Protease in the Regulatory Network That Controls Melanization in the African Malaria Mosquito Anopheles gambiae.

Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.

The environment and species affect gut bacteria composition in laboratory co-cultured Anopheles gambiae and Aedes albopictus mosquitoes.

The midgut microbiota of disease vectors plays a critical role in the successful transmission of human pathogens. The environment influences the microbiota composition; however, the relative mosquito-species contribution has not been rigorously disentangled from the environmental contribution to the microbiota structure. Also, the extent to which the microbiota of the adult sugar food source and larval water can predict that of the adult midgut and vice versa is not fully understood. To address these relationships, larvae and adults of Anopheles gambiae and Aedes albopictus were either reared separately or in a co-rearing system, whereby aquatic and adult stages of both species shared the larval water and sugar food source, respectively. Despite being reared under identical conditions, clear intra- and interspecies differences in midgut microbiota-composition were observed across seven cohorts, collected at different time points over a period of eight months. Fitting a linear model separately for each OTU in the mosquito midgut showed that two OTUs significantly differed between the midguts of the two mosquito species. We also show an effect for the sugar food source and larval water on the adult midgut microbiota. Our findings suggest that the mosquito midgut microbiota is highly dynamic and controlled by multiple factors.

Functional Interaction between Apolipophorins and Complement Regulate the Mosquito Immune Response to Systemic Infections.

The complement-like protein thioester-containing protein 1 (TEP1) is the hallmark effector molecule against Plasmodium ookinetes in the malaria vector Anopheles gambiae. We have previously shown that the knockdown of the noncatalytic clip domain serine protease CLIPA2 increased TEP1-mediated killing rendering mosquitoes more resistant to Plasmodium, bacterial and fungal infections. Here, CLIPA2 coimmunoprecipitation from the hemolymph of Beauveria bassiana-infected mosquitoes followed by mass spectrometry and functional genetic analysis led to the identification of the Apolipophorin-II/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. Apo-II/I exhibits a similar RNAi phenotype as CLIPA2 in mosquito bioassays characterized by increased resistance to B. bassiana and Escherichia coli infections. We provide evidence that this enhanced resistance to systemic infections is TEP1 dependent. Interestingly, silencing Apo-II/I but not CLIPA2 upregulated the expression of TEP1 following systemic infections with E. coli and B. bassiana 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.

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 Anopheles gambiae, 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 TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. 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 CLIPA2 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.

Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito.

BACKGROUND: Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF. FINDINGS: The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes. CONCLUSIONS: B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.

Insecticide resistance to organophosphates in Culex pipiens complex from Lebanon.

BACKGROUND: Analysis of Culex pipiens mosquitoes collected from a single site in Lebanon in 2005, revealed an alarming frequency of ace-1 alleles conferring resistance to organophosphate insecticides. Following this, in 2006 the majority of municipalities switched to pyrethroids after a long history of organophosphate usage in the country; however, since then no studies have assessed the impact of changing insecticide class on the frequency of resistant ace-1 alleles in C. pipiens. METHODS: C. pipiens mosquitoes were captured indoors from 25 villages across the country and subjected to established methods for the analysis of gene amplification at the Ester locus and target site mutations in ace-1 gene that confer resistance to organophosphates. RESULTS: We conducted the first large-scale screen for resistance to organosphosphates in C. pipiens mosquitoes collected from Lebanon. The frequency of carboxylesterase (Ester) and ace-1 alleles conferring resistance to organophosphates were assessed among C. pipiens mosquitoes collected from 25 different villages across the country between December 2008 and December 2009. Established enzymatic assay and PCR-based molecular tests, both diagnostic of the major target site mutations in ace-1 revealed the absence of the F290V mutation among sampled mosquitoes and significant reduction in the frequency of G119S mutation compared to that previously reported for mosquitoes collected from Beirut in 2005. We also identified a new duplicated ace-1 allele, named ace-1D13, exhibiting a resistant phenotype by associating a susceptible and a resistant copy of ace-1 in a mosquito line sampled from Beirut in 2005. Fisher's exact test on ace-1 frequencies in the new sample sites, showed that some populations exhibited a significant excess of heterozygotes, suggesting that the duplicated allele is still present. Starch gel electrophoresis indicated that resistance at the Ester locus was mainly attributed to the Ester2 allele, which exhibits a broad geographical distribution. CONCLUSIONS: Our analysis suggests that the frequency of resistant ace-1 alleles in mosquito populations can be downshifted, and in certain cases (F290V mutation) even eliminated, by switching to a different class of insecticides, possibly because of the fitness cost associated with these alleles.

Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections.

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.