The voltage-gated sodium channel, para, limits Anopheles coluzzii vector competence in a microbiota dependent manner

Abstract The voltage-gated sodium channel, para, is a target of DDT and pyrethroid class insecticides. Single nucleotide mutations in para, called knockdown resistant or kdr, which contribute to resistance against DDT and pyrethroid insecticides, have been correlated with increased susceptibility of Anopheles to the human malaria parasite Plasmodium falciparum. However, a direct role of para activity on Plasmodium infection has not yet been established. Here, using RNA-mediated silencing, we provide in vivo direct evidence for the requirement of wild-type (wt) para function for insecticide activity of deltamethrin. Depletion of wt para, which is susceptible to insecticide, causes deltamethrin tolerance, indicating that insecticide-resistant kdr alleles are likely phenocopies of loss of para function. We then show that normal para activity in An. coluzzii limits Plasmodium infection prevalence for both P. falciparum and P. berghei. A transcriptomic analysis revealed that para activity does not modulate the expression of immune genes. However, loss of para function led to enteric dysbiosis with a significant increase in the total bacterial abundance, and we show that para function limiting Plasmodium infection is microbiota dependent. In the context of the bidirectional “enteric microbiota-brain” axis studied in mammals, these results pave the way for studying whether the activity of the nervous system could control Anopheles vector competence

Rasputin/G3BP mediates o’nyong-nyong virus subversion of antiviral immunity in Anopheles coluzzii

Posted November 23, 2022 on bioRxiv.The G3BP proteins in vertebrates and Aedes mosquito ortholog, Rasputin, are essential for alphavirus infection, but the underlying mechanism of Rasputin/G3BP proviral activity is poorly understood. It has been suggested that G3BP could influence host immune signaling, but this has not been functionally demonstrated. Here, we find that depletion of Rasputin activity in Anopheles mosquitoes, the primary vectors of the alphavirus o’nyong-nyong (ONNV), provokes dysregulation of the antiviral Imd, JAK/STAT and RNAi pathways, indicating that Rasputin is required for expression of normal basal immunity in uninfected mosquitoes. Depletion of Rasputin during ONNV bloodmeal infection causes increased transcript abundance of genes in the Imd pathway including positive regulator Rel2, and decreases ONNV infection in mosquitoes. Loss of Rasputin is complemented by co-depletion of Imd pathway positive regulator, Rel2, which restores normal ONNV infection levels. Thus, the presence of Rasputin is required for ONNV inhibition of Imd activity, and viral inhibition of Imd explains much of the Rasputin proviral activity. The viral non-structural protein 3 (nsP3) binds to Rasputin and alters the profile of cellular proteins binding to Rasputin. In the presence of nsP3, 48 Rasputin-binding proteins are unchanged but seven binding proteins are excluded and eight new proteins bind Rasputin. The Rasputin binding partners altered by nsP3 are candidate factors for ONNV immune manipulation and subversion through Rasputin. Overall, these results are consistent with and strongly suggest a mechanism in which ONNV, probably nsP3, co-opts the normal Rasputin function assuring basal cellular immune activity in order to inhibit antiviral immunity and promote infection. These observations may be generalizable for Rasputin function during alphavirus infection of other mosquitoes, as well as for G3BP function in the mammalian host, and could offer a target for vector-based control of arbovirus transmission

Genetics and immunity of Anopheles response to the entomopathogenic fungus Metarhizium anisopliae overlap with immunity to Plasmodium

International audienceEntomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae . A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum . Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae , as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium . However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium , suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence

Cinnamic acid, an autoinducer of its own biosynthesis, is processed via Hca enzymes in Photorhabdus luminescens.

International audiencePhotorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes

The Anopheles leucine-rich repeat protein APL1C is a pathogen binding factor recognizing Plasmodium ookinetes and sporozoites

We thank the Institut Pasteur core facility, the Center for the Production and Infection of Anopheles (CEPIA) for rearing and infection of mosquitoes; the UtechS Photonic BioImaging facility (Imagopole C2RT), Institut Pasteur for confocal microscopy instruments and assistance, and the Image Analysis Hub, Institut Pasteur for assistance with image analysis.International audienceLeucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum , and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P . berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P . berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P . berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition

Dual role of the Anopheles coluzzii Venus Kinase Receptor in both larval growth and immunity

International audienceVector-borne diseases and especially malaria are responsible for more than half million deaths annually. The increase of insecticide resistance in wild populations of Anopheles malaria vectors emphasises the need for novel vector control strategies as well as for identifying novel vector targets. Venus kinase receptors (VKRs) constitute a Receptor Tyrosine Kinase (RTK) family only found in invertebrates. In this study we functionally characterized Anopheles VKR in the Gambiae complex member, Anopheles coluzzii. Results showed that Anopheles VKR can be activated by L-amino acids, with L-arginine as the most potent agonist. VKR was not required for the fecundity of A. coluzzii, in contrast to reports from other insects, but VKR function is required in both Anopheles males and females for development of larval progeny. Anopheles VKR function is also required for protection against infection by Plasmodium parasites, thus identifying a novel linkage between reproduction and immunity in Anopheles. The insect specificity of VKRs as well as the essential function for reproduction and immunity suggest that Anopheles VKR could be a potentially druggable target for novel vector control strategies

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution

International audienceBackground: The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined.Methods: The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis.Results: APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus.Conclusions: APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa

Exposure of Anopheles mosquitoes to trypanosomes reduces reproductive fitness and enhances susceptibility to Plasmodium

International audienceDuring a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas

APL1C binds to extracellular <i>P</i>. <i>berghei</i> ookinetes.

XZ and YZ orthogonal views of the confocal stack images link APL1C protein binding with parasite spatial localization in mosquito midguts. Yellow lines depict the location of the parasite, for which spatial localization is presented on the sides of the stack picture. Orientation of the midgut epithelium (lu, lumen side, ba, basolateral side) is indicated by labeled arrows on the upper left panel and applies to all panels shown. Parasites in the panels bounded by the red line were external to the basolateral side of the midgut and are labeled by APL1C protein (APL1C, red; GFP, green). Parasites in the panels bounded by the green line remained in the lumen or within epithelial cells of the mosquito midgut are not labelled with APL1C (GFP, green). The scale bar is 5 μm. (PDF)</p

TEP1 protein is efficiently depleted by gene silencing in 4a3A cells.

A. Western blot analysis of TEP1 protein in the 4a3A cell line. The efficiency of TEP1 gene silencing was monitored 6 d after treatment with dsTEP1 or dsGFP in cells transfected with the constructs APL1C-V5, LRIM1-V5 or both. Detection used anti-TEP1 antibody with anti-alpha tubulin antibody as a loading control. B. Quantitative analysis of TEP1 protein immunoblotting. Expression ratio of TEP1 and tubulin loading control protein levels were quantified by densitometry. For each condition, the ratio of protein levels TEP1/tubulin was calculated and the percentage of TEP1 signal reduction relative to tubulin was quantified in cells treated with dsTEP1 as compared to dsGFP. (PDF)</p