Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes

<p>Abstract</p> <p>Background</p> <p>Functional screens based on dsRNA-mediated gene silencing identified several <it>Anopheles gambiae </it>genes that limit <it>Plasmodium berghei </it>infection. However, some of the genes identified in these screens have no effect on the human malaria parasite <it>Plasmodium falciparum</it>; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different <it>Plasmodium </it>species.</p> <p>Results</p> <p>Four new <it>An. gambiae </it>(G3) genes were identified that, when silenced, have a different effect on <it>P. berghei </it>(Anka 2.34) and <it>P. falciparum </it>(3D7) infections. Orthologs of these genes, as well as <it>LRIM1 </it>and <it>CTL4</it>, were also silenced in <it>An. stephensi </it>(Nijmegen Sda500) females infected with <it>P. yoelii </it>(17XNL). For five of the six genes tested, silencing had the same effect on infection in the <it>P. falciparum-An. gambiae </it>and <it>P. yoelii-An. stephensi </it>parasite-vector combinations. Although silencing <it>LRIM1 </it>or <it>CTL4 </it>has no effect in <it>An. stephensi </it>females infected with <it>P. yoelii</it>, when <it>An. gambiae </it>is infected with the same parasite, silencing these genes has a dramatic effect. In <it>An. gambiae </it>(G3), TEP1, LRIM1 or LRIM2 silencing reverts lysis and melanization of <it>P. yoelii</it>, while <it>CTL4 </it>silencing enhances melanization.</p> <p>Conclusion</p> <p>There is a broad spectrum of compatibility, the extent to which the mosquito immune system limits infection, between different <it>Plasmodium </it>strains and particular mosquito strains that is mediated by TEP1/LRIM1 activation. The interactions between highly compatible animal models of malaria, such as <it>P. yoelii </it>(17XNL)-<it>An. stephensi </it>(Nijmegen Sda500), is more similar to that of <it>P. falciparum </it>(3D7)-<it>An. gambiae </it>(G3).</p

A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies

<p>Abstract</p> <p>Background</p> <p>Malaria transmission is now recognized as a key target for intervention. Evaluation of the <it>Plasmodium </it>oocyst burden in the midguts of <it>Anopheles spp</it>. is important for many of assays investigating transmission. However, current assays are very time-consuming, manually demanding and patently subject to observer-observer variation.</p> <p>Methods</p> <p>This report presents the development of a method to rapidly, accurately and consistently determine oocyst burdens on mosquito midguts using GFP-expressing <it>Plasmodium berghei </it>and a custom-written macro for ImageJ. The counting macro was optimized and found to be fit-for-purpose by performing gametocyte membrane feeds with parasite infected blood. Dissected midguts were counted both manually and using the automated macro, then compared. The optimized settings for the macro were then validated by using it to determine the transmission blocking efficacies of two anti-malarial compounds - dehydroepiandrosterone sulphate and lumefantrine, in comparison to manually determined analysis of the same experiment.</p> <p>Results</p> <p>Concurrence of manual and macro counts was very high (R²= 0.973) and reproducible. Estimated transmission blocking efficacies between manual and automated analysis were highly concordant, indicating that dehydroepiandrosterone sulphate has little or no transmission blocking potential, whilst lumefantrine strongly inhibits sporogony.</p> <p>Conclusion</p> <p>Recognizing a potential five-fold increase in throughput, the resulting reduction in personnel costs, and the absence of inter-operator/laboratory variation possible with this approach, this counting macro may be a benefit to the malaria community.</p

Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved

A sticky situation: When trypanosomatids attach to insect tissues.

Transmission of trypanosomatids to their mammalian hosts requires a complex series of developmental transitions in their insect vectors, including stable attachment to an insect tissue. While there are many ultrastructural descriptions of attached cells, we know little about the signaling events and molecular mechanisms involved in this process. Each trypanosomatid species attaches to a specific tissue in the insect at a particular stage of its life cycle. Attachment is mediated by the flagellum, which is modified to accommodate a filament-rich plaque within an expanded region of the flagellar membrane. Attachment immediately precedes differentiation to the mammal-infectious stage and in some cases a direct mechanistic link has been demonstrated. In this review, we summarize the current state of knowledge of trypanosomatid attachment in insects, including structure, function, signaling, candidate molecules, and changes in gene expression. We also highlight remaining questions about this process and how the field is poised to address them through modern approaches

Genetic Evidence That Drosophila frizzled Controls Planar Cell Polarity and Armadillo Signaling by a Common Mechanism

The frizzled (fz) gene in Drosophila controls two distinct signaling pathways: it directs the planar cell polarization (PCP) of epithelia and it regulates cell fate decisions through Armadillo (Arm) by acting as a receptor for the Wnt protein Wingless (Wg). With the exception of dishevelled (dsh), the genes functioning in these two pathways are distinct. We have taken a genetic approach, based on a series of new and existing fz alleles, for identifying individual amino acids required for PCP or Arm signaling. For each allele, we have attempted to quantify the strength of signaling by phenotypic measurements. For PCP signaling, the defect was measured by counting the number of cells secreting multiple hairs in the wing. We then examined each allele for its ability to participate in Arm signaling by the rescue of fz mutant embryos with maternally provided fz function. For both PCP and Arm signaling we observed a broad range of phenotypes, but for every allele there is a strong correlation between its phenotypic strength in each pathway. Therefore, even though the PCP and Arm signaling pathways are genetically distinct, the set of signaling-defective fz alleles affected both pathways to a similar extent. This suggests that fz controls these two different signaling activities by a common mechanism. In addition, this screen yielded a set of missense mutations that identify amino acids specifically required for fz signaling function

The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.

The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for microbial melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of melanotic mosquito excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response

Transmission Blocking Immunity in the Malaria Non- Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to contro