Characterization of Anopheles gambiae D7 salivary proteins as markers of human–mosquito bite contact

Background Malaria is transmitted when infected Anopheles mosquitoes take a blood meal. During this process, the mosquitoes inject a cocktail of bioactive proteins that elicit antibody responses in humans and could be used as biomarkers of exposure to mosquito bites. This study evaluated the utility of IgG responses to members of the Anopheles gambiae D7 protein family as serological markers of human–vector contact. Methods The D7L2, D7r1, D7r2, D7r3, D7r4 and SG6 salivary proteins from An. gambiae were expressed as recombinant antigens in Escherichia coli. Antibody responses to the salivary proteins were compared in Europeans with no prior exposure to malaria and lifelong residents of Junju in Kenya and Kitgum in Uganda where the intensity of malaria transmission is moderate and high, respectively. In addition, to evaluate the feasibility of using anti-D7 IgG responses as a tool to evaluate the impact of vector control interventions, we compared responses between individuals using insecticide-treated bednets to those who did not in Junju, Kenya where bednet data were available. Results We show that both the long and short forms of the D7 salivary gland antigens elicit a strong antibody response in humans. IgG responses against the D7 antigens reflected the transmission intensities of the three study areas, with the highest to lowest responses observed in Kitgum (northern Uganda), Junju (Kenya) and malaria-naïve Europeans, respectively. Specifically, the long form D7L2 induced an IgG antibody response that increased with age and that was lower in individuals who slept under a bednet, indicating its potential as a serological tool for estimating human–vector contact and monitoring the effectiveness of vector control interventions. Conclusions This study reveals that D7L2 salivary antigen has great potential as a biomarker of exposure to mosquito bites and as a tool for assessing the efficacy of vector control strategies such as bednet use. Graphical abstract

A global Anopheles gambiae gene co-expression network constructed from hundreds of experimental conditions with missing values

Gene co-expression networks can be used to determine gene regulation and attribute gene function to biological processes. Different high throughput technologies, including one and two-channel microarrays and RNA-sequencing, allow evaluating thousands of gene expression data simultaneously, but these methodologies provide results that cannot be directly compared. Thus, it is complex to analyze coexpression relations between genes, especially when there are missing values arising for experimental reasons. Networks are a helpful tool for studying gene co-expression, where nodes represent genes and edges represent co-expression of pairs of genes. In this paper, we propose a method for constructing a gene co-expression network for the Anopheles gambiae transcriptome from 257 unique studies obtained with different methodologies and experimental designs. We introduce the sliding threshold approach to select node pairs with high Pearson correlation coefficients. The robustness of the method was verified by comparing edge weight distributions under random removal of conditions. The properties of the constructed network are studied in this paper, including node degree distribution, coreness, and community structure. The network core is largely comprised of genes that encode components of the mitochondrial respiratory chain and the ribosome, while different communities are enriched for genes involved in distinct biological processes. This suggests that the overall network structure is driven to maximize the integration of essential cellular functions, possibly allowing the flexibility to add novel functions.

Resting Behavior of Malaria Vectors in Ghana and Its Implication on Vector Control.

Background: In Sub-Saharan Africa, there is widespread use of long-lasting insecticidal nets (LLINs) and Indoor residual spraying (IRS) to help control the density of malaria vectors and decrease the incidence of malaria in communities. An understanding of the interactions between increased insecticide use and resting behaviour patterns of malaria mosquitoes is important for an effective vector control programme. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of increasing insecticide resistance in malaria vectors in sub-saharan Africa.Methods: Indoor and outdoor resting Anopheline mosquitoes were sampled during the dry and rainy seasons in five sites that were in 3 ecological landscapes [Sahel savannah (Kpalsogou, Pagaza, Libga), Coastal savannah (Anyakpor) and Forest (Konongo) zones] using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, pit shelter and Prokopack for outdoor collections. PCR based molecular diagnostics were used to determine mosquito speciation, genotype for knockdown resistance mutations (L1014S and L1014F), G119S Ace-1 mutation, specific host blood meal origins and sporozoite infection in field collected mosquitoes.Results: Anopheles gambiae s. l. was the predominant species (89.95%, n = 1,718), followed by An. rufipes (8.48%, n=162), and An. funestus s. l. (1.57%, n = 30). Sibling species of the Anopheles gambiae revealed An. coluzzii accounted for 63% (95% CI: 57.10 – 68.91), followed by An. gambiae s. s [27% (95% CI: 21.66 – 32.55)], and An. arabiensis [9% (95% CI: 6.22 – 13.57)]. The mean resting density of An. gambiae s. l. was higher outdoors (79.63%; 1,368/1,718) than indoors (20.37%; 350/1,718) (z = -4.815, p< 0.0001). The kdr west L1014F and the Ace-1 mutations were highest in indoor resting An. coluzzii and An. gambiae in the sahel-savannah sites compared to the forest and coastal savannah sites. Overall, the blood meal analyses revealed a large proportion of the malaria vectors preferred feeding on humans (70.2 %) than animals (29.8%) in all sites. The sporozoite rates was only detected in indoor resting An. coluzzii from the sahel savannah (5.0%) and forest (2.5%) zones.Conclusion: The study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and persistence of malaria transmission indoors despite the use of LLINs and IRS. Continuous monitoring of changes in resting behavior of mosquitoes and implementation of complementary malaria control interventions are needed to target outdoor resting Anopheles mosquitoes in Ghana.

Molecular Characterization and Genotype Distribution of Thioester-containing Protein 1 Gene, A Key Regulator of Malaria Transmission in An. Gambiae Mosquitoes in Western Kenya

BackgroundEvolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodiumparasites.These forcesmay elevate the introduction of new species genotypes that adapt to new breeding habitats which could have serious implications on malaria transmission.Thioester-containing protein 1 (TEP1) of Anopheles gambiaeplays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms determining vector competence and subsequently malaria transmission in western Kenya. MethodsAnopheles gambiaeadult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectivelyfrom Homa Bay, Kakamega, Bungoma, and Kisumu countiesbetween 2017 and 2020.Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR.TEP1 alleles were determined using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of alleles was sequenced.ResultsTwo TEP1 alleles (TEP1*S1 and TEP1*R2)and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2)were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively bothin An. gambiaeand An. arabiensis. There was no significant difference detected among the population and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (FST= 0.019) across all sites corresponded to an effective migration index (Nm= 12.571) and lowNei’s genetic distance values (<0.500) among the subpopulation.The comparative fixation index values revealed minimal genetic differentiation between speciesand high levels of gene flow among populations.ConclusionThere is a low genetic diversity and population structure in western Kenya. TEP1* R2 and TEP1*S1 were the most common alleles in both species which may have been maintained through generations in time, However, the TEP1*R2 allele was in low frequencies and may be used to estimatemalaria prevalence. Continued surveillance of the distribution of TEP1 is essential for monitoring the population dynamics of local vectors and their implications on malaria transmission and hence designing targeted vector interventions.

Toll signaling enhances mosquito antiplasmodial immunity by promoting differentiation of hemocytes to the Megacyte lineage

Activation of Toll signaling in Anopheles gambiae, by silencing Cactus, eliminates Plasmodium ookinetes by enhancing local release of hemocytes-derived microvesicles that promote activation of the mosquito complement-like system. A new effector hemocyte subpopulation of large granulocytes, the megacytes, was recently identified. We report that Cactus silencing dramatically increases the proportion of megacytes, from 5 to 79% of circulating granulocytes. Transcriptomic and morphological analysis, as well as in situ hybridization and expression of cell-specific markers, indicate that Cactus silencing triggers granulocyte differentiation into megacytes. Megacytes are very plastic cells that can extend long filopodia and tend to form clusters in vivo. Moreover, megacytes are massively recruited to the basal midgut surface in response to bacterial feeding and Plasmodium infection. We propose that Toll signaling promotes differentiation of granulocytes to the megacyte lineage, a major cellular effector of antibacterial and antiplasmodial immunity.

Entomological Indicators of Malaria Transmission Prior to a Cluster Randomised Controlled Trial of a “Lethal House Lure” Intervention in Central Côte D’Ivoire

Background: A study was conducted prior to implementing a cluster randomised controlled trial (CRT) of a lethal house lure strategy in central Côte d’Ivoire and aimed to provide baseline information on malaria vectors in 40 village clusters. Methods: Human landing catches (HLC) was performed between November-December 2016, capturing mosquitoes indoor and outdoor between 18.00-08.00. Mosquitoes were processed for entomological indicators of malaria transmission (human biting rates, parity rates, sporozoite infection rates and the entomological inoculation rates (EIR)). Species composition and allelic frequencies of Kdr-w and Ace-1R mutations were also investigated within the Anopheles gambiae complex. Results: Overall, 15,632 mosquitoes were captured. Anopheles gambiae s.l. and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for Anopheles gambiae s.l. (66.2%) compared to Anopheles funestus (10.3%). The mean biting rate for An. gambiae s.l. was almost 5 times higher than that for An. funestus s.l.(19.8 bites per person per night for An. gambiae s. l. vs 4.3 bites per person per night for An. funestus s. l.) and this was evident indoor and outdoor. An. funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (1.6% (1,373) for An. gambiae vs 4.7 % (722) for An. funestus s.l.). There was no significant difference between the proportion infected outdoor and indoor for An. gambiae s.l. (1.6% vs 1.5%; OR=1.11[0.65-1.9]; P=0.676), but for An. funestus, more mosquitoes were infected outdoor (6.4%) than indoor (3.5%) (OR=1.86 [1.07-3.23]; P=0.0249). The majority of both infected vectors with malaria parasites harboured P. falciparum (90.6% for An. gambiae s. l. and 97, 8% for An. funestus s. l.). The EIR for both vectors (0.43 infected bites per night) were similar and there were no significant differences for transmission occurring outdoor and indoor for both species. Of the An. gambiae s.l. analysed, only An. gambiae (14.1%) and An. coluzzii (85.9%) were found. The allelic frequencies of Kdr and Ace-1R were higher in An. gambiae (0.97 for Kdr and 0.19 for Ace-1R) than in An. coluzzii (0.86 for Kdr and 0.10 for Ace-1R) (P<0.001).Conclusion: Despite universal coverage of long-lasting insecticidal nets (LLINs) in the area, there was an abundance of malaria vectors in the study in area in central Côte d’Ivoire, specifically highly resistant An. gambiae s.l. as well as An. funestus s.l.. The malaria sporozoite rate was higher in An. funestus s.l than An. gambiae s.l.. but EIR rates in these two species were similarly high, both indoor and outdoor. Novel tools or strategies are urgently needed to further reduce malaria transmission in this area.

Assessment of Plasmodium falciparum Infection and Fitness of Genetically Modified Anopheles gambiae Aimed at Mosquito Population Replacement

Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY QUANTIFICATION OF OLEANOLIC ACID IN LAUNAEA TARAXACIFOLIA AND LARVICIDAL ACTIVITY AGAINST ANOPHELES GAMBIAE

Objective: One of the measures used to prevent malaria is the management of breeding sites. For preventive and ecologically profitable control, the use of bio-larvicides made from active plant extracts would be an asset for the control of malaria vectors, in particular Anopheles gambiae. Advances in pharmacognosy have revealed the benefits of several phytochemicals with very rich and varied therapeutic effects. Among the latter, oleanolic acid (OA) is quite remarkable because of its various and multiple properties, much of which is demonstrated with the leaves of Launaea taraxacifolia. Methods: After a liquid-liquid fractionation with different organic solvents of the hydro-methanolic extract of Launaea taraxacifolia, we obtained three fractions named Fhex (hexane fraction), FDCM (dichloromethane fraction) and FHM (hydro-methanolic fraction) which were tested on 3rd instar Anopheles gambiae larvae. Results: Fhex proved to be the most active with LC50 of 120.11 ppm and 69.50 ppm respectively in 24 and 48 hours of contact. We then developed a new method of Ultra-Violet High Performance Liquid Chromatography (HPLC / UV) method and determined the quantity of oleanolic acid in the Fhex and FDCM fractions to be respectively 0.46% and 0.23% . Conclusion: Launaea taraxacifolia has a larvicidal potential due to the presence of oleanolic acid whose inhibitory effect against Anopheles gambiae larvae.