Using capillary electrophoresis to identify Anopheline species in routine sampling sites.

In the Anopheles genus, various mosquito species are able to transmit the Plasmodium parasites responsible for malaria, while others are non-vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected in the field is an essential but problematic task. Morphological identification requires expertise and cannot be checked after processing samples in a destructive treatment, while sequencing of numerous samples is costly. Here, we introduce a method of Species identification via Simple Observation Coupled with Capillary Electrophoresis Technology (SOCCET). This molecular technique of species identification is based on precise determination of ITS2 length combined with a simple visual observation, the colour of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analysed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus colour, which allows discrimination of the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of ITS2 amplicons. Using the SOCCET method, we characterised the composition of Anopheles populations in five locations of French Guiana, where we detected a total of nine species. Anopheles braziliensis and Anopheles darlingi were detected in four locations each and represented 13 and 67% of our samples, respectively. The SOCCET method can be particularly useful when working with routine sampling sites with a moderate species diversity, that is, when the number of local species is too high to define species-specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may be further implemented for other mosquito genera.

Involvement of Microbiota in Insect Physiology: Focus on B Vitamins.

Insects are highly successful in colonizing a wide spectrum of ecological niches and in feeding on a wide diversity of diets. This is notably linked to their capacity to get from their microbiota any essential component lacking in the diet such as vitamins and amino acids. Over a century of research based on dietary analysis, antimicrobial treatment, gnotobiotic rearing, and culture-independent microbe detection progressively generated a wealth of information about the role of the microbiota in specific aspects of insect fitness. Thanks to the recent increase in sequencing capacities, whole-genome sequencing of a number of symbionts has facilitated tracing of biosynthesis pathways, validation of experimental data and evolutionary analyses. This field of research has generated a considerable set of data in a diversity of hosts harboring specific symbionts or nonspecific microbiota members. Here, we review the current knowledge on the involvement of the microbiota in insect and tick nutrition, with a particular focus on B vitamin provision. We specifically question if there is any specificity of B vitamin provision by symbionts compared to the redundant yet essential contribution of nonspecific microbes. We successively highlight the known aspects of microbial vitamin provision during three main life stages of invertebrates: postembryonic development, adulthood, and reproduction.

Three new species of Culex (Melanoconion) (Diptera: Culicidae) from French Guiana based on morphological and molecular data.

Culex mosquitoes of the subgenus Melanoconion Theobald, 1903 of the genus Culex Linnaeus, 1758 include numerous species recognized as vectors of viruses affecting humans. This subgenus is the most speciose among the entire mosquito fauna of the Americas. Despite decades of taxonomic research, many species remain undiscovered, especially in the Amazonian biome. In this article, we provide the description of three new species of Culex (Melanoconion) recently discovered in a biological reserve in French Guiana. Culex (Mel.) sallumae n. sp., Cx. (Mel.) hutchingsae n. sp. and Cx. (Mel.) lucakermanni n. sp. are described based on both morphological features of the male genitalia and molecular barcodes obtained from type specimens. Diagnostic characters to assist their identification are provided and their placement within the infrasubgeneric classification of the subgenus Melanoconion is discussed.

Analyzing gut microbiota composition in individual Anopheles mosquitoes after experimental treatment.

The microbiota of Anopheles mosquitoes influences malaria transmission. Antibiotics ingested during a blood meal impact the mosquito microbiome and malaria transmission, with substantial differences between drugs. Here, we assessed if amoxicillin affects the gut mosquito microbiota. We collected Anopheles larvae in Burkina Faso, kept them in semi-field conditions, and offered a blood meal to adult females. We tested the impact of blood supplementation with two alternative amoxicillin preparations on microbiota composition, determined by high-throughput sequencing in individual gut samples. Our analysis detected four major genera, Elizabethkingia, Wigglesworthia, Asaia, and Serratia. The antibiotic treatment significantly affected overall microbiota composition, with a specific decrease in the relative abundance of Elizabethkingia and Asaia during blood digestion. Besides its interest on the influence of amoxicillin on the mosquito microbiota, our study proposes a thorough approach to report negative-control data of high-throughput sequencing studies on samples with a reduced microbial load.

Linking microbiota composition with antimalarial antibody response.

Microbiota composition recently arose as a factor correlating with malaria infection. Mandal et al. showed, via cecal transplant and antibacterial treatment, that the mouse microbiota modulates parasitemia by affecting spleen germinal centers where B cells are matured. They further identified correlations between microbiota composition and malaria severity in Ugandan children.

The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota.

Serratia marcescens is a bacterial species widely found in the environment, which very efficiently colonizes mosquitoes. In this study, we isolated a red-pigmented S. marcescens strain from our mosquito colony (called S. marcescens VA). This red pigmentation is caused by the production of prodigiosin, a molecule with antibacterial properties. To investigate the role of prodigiosin on mosquito-S. marcescens interactions, we produced two white mutants of S. marcescens VA by random mutagenesis. Whole genome sequencing and chemical analyses suggest that one mutant has a nonsense mutation in the gene encoding prodigiosin synthase, while the other one is deficient in the production of several types of secondary metabolites including prodigiosin and serratamolide. We used our mutants to investigate how S. marcescens secondary metabolites affect the mosquito and its microbiota. Our in vitro tests indicated that S. marcescens VA inhibits the growth of several mosquito microbiota isolates using a combination of prodigiosin and other secondary metabolites, corroborating published data. This strain requires secondary metabolites other than prodigiosin for its proteolytic and hemolytic activities. In the mosquito, we observed that S. marcescens VA is highly virulent to larvae in a prodigiosin-dependent manner, while its virulence on adults is lower and largely depends on other metabolites.

Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions.

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

Antibiotic Treatment in Anopheles coluzzii Affects Carbon and Nitrogen Metabolism.

The mosquito microbiota reduces the vector competence of Anopheles to Plasmodium and affects host fitness; it is therefore considered as a potential target to reduce malaria transmission. While immune induction, secretion of antimicrobials and metabolic competition are three typical mechanisms of microbiota-mediated protection against invasive pathogens in mammals, the involvement of metabolic competition or mutualism in mosquito-microbiota and microbiota-Plasmodium interactions has not been investigated. Here, we describe a metabolome analysis of the midgut of Anopheles coluzzii provided with a sugar-meal or a non-infectious blood-meal, under conventional or antibiotic-treated conditions. We observed that the antibiotic treatment affects the tricarboxylic acid cycle and nitrogen metabolism, notably resulting in decreased abundance of free amino acids. Linking our results with published data, we identified pathways which may participate in microbiota-Plasmodium interactions via metabolic interactions or immune modulation and thus would be interesting candidates for future functional studies.

Functional analysis of the three major PGRPLC isoforms in the midgut of the malaria mosquito Anopheles coluzzii.

Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

The tripartite interactions between the mosquito, its microbiota and Plasmodium.

The microbiota of Anopheles mosquitoes interferes with mosquito infection by Plasmodium and influences mosquito fitness, therefore affecting vectorial capacity. This natural barrier to malaria transmission has been regarded with growing interest in the last 20 years, as it may be a source of new transmission-blocking strategies. The last decade has seen tremendous progress in the functional characterisation of the tripartite interactions between the mosquito, its microbiota and Plasmodium parasites. In this review, we provide insights into the effects of the mosquito microbiota on Plasmodium infection and on mosquito physiology, and on how these aspects together influence vectorial capacity. We also discuss three current challenges in the field, namely the need for a more relevant microbiota composition in experimental mosquitoes involved in vector biology studies, for a better characterisation of the non-bacterial microbiota, and for further functional studies of the microbiota present outside the gut.

[Mosquito microbiota and its influence on disease vectorial transmission]. / Le microbiote de moustique et son influence sur la transmission vectorielle.

Mosquitoes (Diptera: Culicidae) are found worldwide. Around 100 among 3500 mosquito species are known to be vectors of parasites and viruses, responsible for infectious diseases including malaria and dengue. Mosquitoes host diverse microbial communities that influence disease transmission, either by direct interference or via affecting host immunity and physiology. These microbial communities are present within diverse tissues, including the digestive tract, and vary depending on the sex of the mosquito, its developmental stage, and ecological factors. This review summarizes the current knowledge about the mosquito microbiota, defined as a community of commensal, symbiotic or pathogenic microbes harboured by a host. We first describe the current knowledge on the diversity of the microbiota, that includes bacteria, fungi, parasites and viruses and on its modes of acquisition throughout the mosquito life cycle. We then focus on microbial interactions within the mosquito gut, which notably affect vector competence, and on host-microbe interactions affecting mosquito fitness. Finally, we discuss current or potential methods based on the use of microbes or microbial products to interfere with pathogen transmission or to reduce mosquito lifespan and reproduction.

A Swiss Army Knife to Cut Malaria Transmission.

The mosquito microbiota is known to naturally limit malaria transmission, acting directly on parasites and via effects on mosquito immunity and fitness. Using genetically modified bacteria and mosquitoes, two studies uncover new potential of this multipotent prospective tool to reduce disease transmission.

The Peptidoglycan Recognition Proteins PGRPLA and PGRPLB Regulate Anopheles Immunity to Bacteria and Affect Infection by Plasmodium.

Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito Anophelescoluzzii, the peptidoglycan receptor PGRPLC activates the immune-deficiency (Imd) pathway limiting both the microbiota load and Plasmodium infection. Here, we carried out an RNA interference screen to examine the role of all 7 Anopheles PGRPs in infections with Plasmodium berghei and P. falciparum. We show that, in addition to PGRPLC, PGRPLA and PGRPS2/PGRPS3 also participate in antiparasitic defenses, and that PGRPLB promotes mosquito permissiveness to P. falciparum. We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respectively. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector competence.

Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes.

Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.

Larval diet affects mosquito development and permissiveness to Plasmodium infection.

The larval stages of malaria vector mosquitoes develop in water pools, feeding mostly on microorganisms and environmental detritus. Richness in the nutrient supply to larvae influences the development and metabolism of larvae and adults. Here, we investigated the effects of larval diet on the development, microbiota content and permissiveness to Plasmodium of Anopheles coluzzii. We tested three fish diets often used to rear mosquitoes in the laboratory, including two pelleted diets, Dr. Clarke's Pool Pellets and Nishikoi Fish Pellets, and one flaked diet, Tetramin Fish-Flakes. Larvae grow and develop faster and produce bigger adults when feeding on both types of pellets compared with flakes. This correlates with a higher microbiota load in pellet-fed larvae, in agreement with the known positive effect of the microbiota on mosquito development. Larval diet also significantly influences the prevalence and intensity of Plasmodium berghei infection in adults, whereby Nishikoi Fish Pellets-fed larvae develop into adults that are highly permissive to parasites and survive longer after infection. This correlates with a lower amount of Enterobacteriaceae in the midgut microbiota. Together, our results shed light on the influence of larval feeding on mosquito development, microbiota and vector competence; they also provide useful data for mosquito rearing.

Differential Effects of Azithromycin, Doxycycline, and Cotrimoxazole in Ingested Blood on the Vectorial Capacity of Malaria Mosquitoes.

Background. The gut microbiota of malaria vector mosquitoes grows after a blood meal and limits Plasmodium infection. We previously showed that penicillin and streptomycin in the ingested blood affect bacterial growth and positively impact mosquito survival and permissiveness to Plasmodium. In this study, we examine the effects of doxycycline, azithromycin, and co-trimoxazole. All 3 antibiotics are used in mass drug administration programs and have antimicrobial activities against bacteria and various stages of malaria parasites. Methods. The effects of blood meal supplementation with antibiotics on the mosquito microbiota, lifespan, and permissiveness to Plasmodium falciparum were assessed. Results. Ingestion of any of the 3 antibiotics significantly affected the mosquito microbiota. Azithromycin decreased P falciparum infection load and mosquito lifespan, whereas at high concentrations, doxycycline increased P falciparum infection load. Co-trimoxazole negatively impacted infection intensity but had no reproducible effect on mosquito lifespan. Conclusions. Our data suggest that the overall effect of antibiotic treatment on parameters critical for mosquito vectorial capacity is drug specific. The negative effect of azithromycin on malaria transmission is consistent with current efforts for disease elimination, whereas additional, larger scale investigations are required before conclusions can be drawn about doxycycline.

Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana.

Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings.

Antibiotics in ingested human blood affect the mosquito microbiota and capacity to transmit malaria.

Malaria reduction is most efficiently achieved by vector control whereby human populations at high risk of contracting and transmitting the disease are protected from mosquito bites. Here, we identify the presence of antibiotics in the blood of malaria-infected people as a new risk of increasing disease transmission. We show that antibiotics in ingested blood enhance the susceptibility of Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota. This effect is confirmed in a semi-natural setting by feeding mosquitoes with blood of children naturally infected with Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity, which are known to augment vectorial capacity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass drug administration programs against communicable diseases may necessitate increased vector control.

Functional analysis of PGRP-LA in Drosophila immunity.

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.