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.
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.
Gene drives for mosquito population modification are novel tools for malaria control. Strategies to safely test antimalarial effectors in the field are required. Here, we modified the Anopheles ...gambiae zpg locus to host a CRISPR/Cas9 integral gene drive allele (zpgD) and characterized its behaviour and resistance profile. We found that zpgD dominantly sterilizes females but can induce efficient drive at other loci when it itself encounters resistance. We combined zpgD with multiple previously characterized non-autonomous payload drives and found that, as zpgD self-eliminates, it leads to conversion of mosquito cage populations at these loci. Our results demonstrate how self-eliminating drivers could allow safe testing of non-autonomous effector-traits by local population modification. They also suggest that after engendering resistance, gene drives intended for population suppression could nevertheless serve to propagate subsequently released non-autonomous payload genes, allowing modification of vector populations initially targeted for suppression.
Key behaviours, physiologies and gene expressions in Anopheles mosquitoes impact the transmission of Plasmodium. Such mosquito factors are rhythmic to closely follow diel rhythms. Here, we set to ...explore the impact of the mosquito circadian rhythm on the tripartite interaction between the vector, the parasite and the midgut microbiota, and investigate how this may affect the parasite infection outcomes. We assess Plasmodium falciparum infection prevalence and intensity, as a proxy for gametocyte infectivity, in Anopheles gambiae mosquitoes that received a gametocyte-containing bloodfeed and measure the abundance of the midgut microbiota at different times of the mosquito rearing light-dark cycle. Gametocyte infectivity is also compared in mosquitoes reared and maintained under a reversed light-dark regime. The effect of the circadian clock on the infection outcome is also investigated through silencing of the CLOCK gene that is central in the regulation of animal circadian rhythms. The results reveal that the A. gambiae circadian cycle plays a key role in the intensity of infection of P. falciparum gametocytes. We show that parasite gametocytes are more infectious during the night-time, where standard membrane feeding assays (SMFAs) at different time points in the mosquito natural circadian rhythm demonstrate that gametocytes are more infectious when ingested at midnight than midday. When mosquitoes were cultured under a reversed light/dark regime, disrupting their natural physiological homeostasis, and infected with P. falciparum at evening hours, the infection intensity and prevalence were significantly decreased. Similar results were obtained in mosquitoes reared under the standard light/dark regime upon silencing of CLOCK, a key regulator of the circadian rhythm, highlighting the importance of the circadian rhythm for the mosquito vectorial capacity. At that time, the mosquito midgut microbiota load is significantly reduced, while the expression of lysozyme C-1 (LYSC-1) is elevated, which is involved in both the immune response and microbiota digestion. We conclude that the tripartite interactions between the mosquito vector, the malaria parasite and the mosquito gut microbiota are finely tuned to support and maintain malaria transmission. Our data add to the knowledge framework required for designing appropriate and biologically relevant SMFA protocols.
The malaria parasite replicates asexually in the red blood cells of its vertebrate host employing epigenetic mechanisms to regulate gene expression in response to changes in its environment. We used ...chromatin immunoprecipitation followed by sequencing in conjunction with RNA sequencing to create an epigenomic and transcriptomic map of the developmental transition from asexual blood stages to male and female gametocytes and to ookinetes in the rodent malaria parasite Plasmodium berghei. Across the developmental stages examined, heterochromatin protein 1 associates with variantly expressed gene families localised at subtelomeric regions and variant gene expression based on heterochromatic silencing is observed only in some genes. Conversely, the euchromatin mark histone 3 lysine 9 acetylation (H3K9ac) is abundant in non-heterochromatic regions across all developmental stages. H3K9ac presents a distinct pattern of enrichment around the start codon of ribosomal protein genes in all stages but male gametocytes. Additionally, H3K9ac occupancy positively correlates with transcript abundance in all stages but female gametocytes suggesting that transcription in this stage is independent of H3K9ac levels. This finding together with known mRNA repression in female gametocytes suggests a multilayered mechanism operating in female gametocytes in preparation for fertilization and zygote development, coinciding with parasite transition from host to vector.
After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium ...parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito–parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.
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.
Understanding malaria vector's population dynamics and their spatial distribution is important to define when and where the largest infection risks occur and implement appropriate control strategies. ...In this study, the seasonal spatio-temporal dynamics of the malaria vector population and transmission intensity along intermittent rivers in a semi-arid area of central Ethiopia were investigated.
Mosquitoes were collected monthly from five clusters, 2 close to a river and 3 away from a river, using pyrethrum spray catches from November 2014 to July 2016. Mosquito abundance was analysed by the mixed Poisson regression model. The human blood index and sporozoite rate was compared between seasons by a logistic regression model.
A total of 2784 adult female Anopheles gambiae sensu lato (s.l.) were collected during the data collection period. All tested mosquitoes (n = 696) were identified as Anopheles arabiensis by polymerase chain reaction. The average daily household count was significantly higher (P = 0.037) in the clusters close to the river at 5.35 (95% CI 2.41-11.85) compared to the clusters away from the river at 0.033 (95% CI 0.02-0.05). Comparing the effect of vicinity of the river by season, a significant effect of closeness to the river was found during the dry season (P = 0.027) and transition from dry to wet season (P = 0.032). Overall, An. arabiensis had higher bovine blood index (62.8%) as compared to human blood index (23.8%), ovine blood index (9.2%) and canine blood index (0.1%). The overall sporozoite rate was 3.9% and 0% for clusters close to and away from the river, respectively. The overall Plasmodium falciparum and Plasmodium vivax entomologic inoculation rates for An. arabiensis in clusters close to the river were 0.8 and 2.2 infective bites per person/year, respectively.
Mosquito abundance and malaria transmission intensity in clusters close to the river were higher which could be attributed to the riverine breeding sites. Thus, vector control interventions including targeted larval source management should be implemented to reduce the risk of malaria infection in the area.
Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium ...parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.
Recognition of peptidoglycan (PGN) is paramount for insect antibacterial defenses. In the fruit fly Drosophila melanogaster, the transmembrane PGN Recognition Protein LC (PGRP-LC) is a receptor of ...the Imd signaling pathway that is activated after infection with bacteria, mainly Gram-negative (Gram-). Here we demonstrate that bacterial infections of the malaria mosquito Anopheles gambiae are sensed by the orthologous PGRPLC protein which then activates a signaling pathway that involves the Rel/NF-kappaB transcription factor REL2. PGRPLC signaling leads to transcriptional induction of antimicrobial peptides at early stages of hemolymph infections with the Gram-positive (Gram+) bacterium Staphylococcus aureus, but a different signaling pathway might be used in infections with the Gram- bacterium Escherichia coli. The size of mosquito symbiotic bacteria populations and their dramatic proliferation after a bloodmeal, as well as intestinal bacterial infections, are also controlled by PGRPLC signaling. We show that this defense response modulates mosquito infection intensities with malaria parasites, both the rodent model parasite, Plasmodium berghei, and field isolates of the human parasite, Plasmodium falciparum. We propose that the tripartite interaction between mosquito microbial communities, PGRPLC-mediated antibacterial defense and infections with Plasmodium can be exploited in future interventions aiming to control malaria transmission. Molecular analysis and structural modeling provided mechanistic insights for the function of PGRPLC. Alternative splicing of PGRPLC transcripts produces three main isoforms, of which PGRPLC3 appears to have a key role in the resistance to bacteria and modulation of Plasmodium infections. Structural modeling indicates that PGRPLC3 is capable of binding monomeric PGN muropeptides but unable to initiate dimerization with other isoforms. A dual role of this isoform is hypothesized: it sequesters monomeric PGN dampening weak signals and locks other PGRPLC isoforms in binary immunostimulatory complexes further enhancing strong signals.
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