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

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.

ePeak: from replicated chromatin profiling data to epigenomic dynamics.

We present ePeak, a Snakemake-based pipeline for the identification and quantification of reproducible peaks from raw ChIP-seq, CUT&RUN and CUT&Tag epigenomic profiling techniques. It also includes a statistical module to perform tailored differential marking and binding analysis with state of the art methods. ePeak streamlines critical steps like the quality assessment of the immunoprecipitation, spike-in calibration and the selection of reproducible peaks between replicates for both narrow and broad peaks. It generates complete reports for data quality control assessment and optimal interpretation of the results. We advocate for a differential analysis that accounts for the biological dynamics of each chromatin factor. Thus, ePeak provides linear and nonlinear methods for normalisation as well as conservative and stringent models for variance estimation and significance testing of the observed marking/binding differences. Using a published ChIP-seq dataset, we show that distinct populations of differentially marked/bound peaks can be identified. We study their dynamics in terms of read coverage and summit position, as well as the expression of the neighbouring genes. We propose that ePeak can be used to measure the richness of the epigenomic landscape underlying a biological process by identifying diverse regulatory regimes.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii.

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences. Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

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

During 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.

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii.

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii, we identified candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancers segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity. Deletion mutagenesis and functional testing revealed a fine structure of positive and negative regulatory elements that modulate activity of the enhancer core. Enhancer polymorphisms segregate in wild A. coluzzii populations in West Africa. Thus, enhancer variants that modify target gene expression leading to likely phenotypic consequences are frequent in nature. These results demonstrate the existence of naturally polymorphic A. coluzzii enhancers, which may help explain important differences between individuals or populations for malaria transmission efficiency and vector adaptation to the environment.

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

Vector-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.

Experimental Adaptation of the Yellow Fever Virus to the Mosquito Aedes albopictus and Potential risk of urban epidemics in Brazil, South America.

Despite the availability of an efficient vaccine, Yellow fever (YF), a viral disease transmitted by mosquitoes, is still a threat. In Brazil, the yellow fever virus (YFV) has been restricted to a jungle cycle for more than 70 years. However, YFV has recently invaded populated cities in the Southeast such as Rio de Janeiro where the opportunistic mosquito Aedes albopictus is well established. Using in vivo passages of YFV in Ae. albopictus, we have selected viral strains presenting substitutions in NS1 gene. We did 10 passages of YFV-74018 on two distinct Ae. albopictus populations: (i) Manaus collected from a YFV-endemic area in Amazonia and (ii) PNMNI from a YFV-free area in the state of Rio de Janeiro. Full viral genomes were deep sequenced at each passage. We obtained two YFV strains presenting a non-synonymous substitution in the NS1 gene. Interestingly, they intervened at two different positions in NS1 gene according to the mosquito population: I2772T in Ae. albopictus Manaus and S3303N in Ae. albopictus PNMNI. Both substitutions reached fixation at the passage 10. Our data suggest that YFV has the potential for adaption to Ae. albopictus thereby posing a threat to most cities in South America where this mosquito is present.

The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus.

RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.

Highly focused transcriptional response of Anopheles coluzzii to O'nyong nyong arbovirus during the primary midgut infection.

BACKGROUND: Anopheles mosquitoes are efficient vectors of human malaria, but it is unknown why they do not transmit viruses as well as Aedes and Culex mosquitoes. The only arbovirus known to be consistently transmitted by Anopheles mosquitoes is O'nyong nyong virus (ONNV, genus Alphavirus, family Togaviridae). The interaction of Anopheles mosquitoes with RNA viruses has been relatively unexamined. RESULTS: We transcriptionally profiled the African malaria vector, Anopheles coluzzii, infected with ONNV. Mosquitoes were fed on an infectious bloodmeal and were analyzed by Illumina RNAseq at 3 days post-bloodmeal during the primary virus infection of the midgut epithelium, before systemic dissemination. Virus infection triggers transcriptional regulation of just 30 host candidate genes. Most of the regulated candidate genes are novel, without known function. Of the known genes, a significant cluster includes candidates with predicted involvement in carbohydrate metabolism. Two candidate genes encoding leucine-rich repeat immune (LRIM) factors point to possible involvement of immune protein complexes in the mosquito antiviral response. The primary ONNV infection by bloodmeal shares little transcriptional response in common with ONNV infection by intrathoracic injection, nor with midgut infection by the malaria parasites, Plasmodium falciparum or P. berghei. Profiling of A. coluzzii microRNA (miRNA) identified 118 known miRNAs and 182 potential novel miRNA candidates, with just one miRNA regulated by ONNV infection. This miRNA was not regulated by other previously reported treatments, and may be virus specific. Coexpression analysis of miRNA abundance and messenger RNA expression revealed discrete clusters of genes regulated by Imd and JAK/STAT, immune signaling pathways that are protective against ONNV in the primary infection. CONCLUSIONS: ONNV infection of the A. coluzzii midgut triggers a remarkably limited gene regulation program of mostly novel candidate genes, which likely includes host genes deployed for antiviral defense, as well as genes manipulated by the virus to facilitate infection. Functional dissection of the ONNV-response candidate genes is expected to generate novel insight into the mechanisms of virus-vector interaction.

The Anopheles gambiae 2La chromosome inversion is associated with susceptibility to Plasmodium falciparum in Africa.

Chromosome inversions suppress genetic recombination and establish co-adapted gene complexes, or supergenes. The 2La inversion is a widespread polymorphism in the Anopheles gambiae species complex, the major African mosquito vectors of human malaria. Here we show that alleles of the 2La inversion are associated with natural malaria infection levels in wild-captured vectors from West and East Africa. Mosquitoes carrying the more-susceptible allele (2L+a) are also behaviorally less likely to be found inside houses. Vector control tools that target indoor-resting mosquitoes, such as bednets and insecticides, are currently the cornerstone of malaria control in Africa. Populations with high levels of the 2L+a allele may form reservoirs of persistent outdoor malaria transmission requiring novel measures for surveillance and control. The 2La inversion is a major and previously unappreciated component of the natural malaria transmission system in Africa, influencing both malaria susceptibility and vector behavior.

Correction: An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

[This corrects the article DOI: 10.1371/journal.ppat.1005306.].

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.

An assessment of bacterial small RNA target prediction programs.

Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as earlier programs for RNA-RNA hybrid prediction. Moreover, we consider how the definition of mRNA boundaries can impact overall predictions. Recent algorithms that exploit both conservation of targets and accessibility information offer improved accuracy over previous software. However, even with the best predictors, the number of true biological targets with low scores and non-targets with high scores remains puzzling.