Important Malaria Vector Research Articles

Evidence of endogenous Non-retroviral RNA Virus Sequences into the genome and transcriptome of the malaria vector Anopheles darlingi.

The characterization of non-retroviral integrated RNA virus sequences (NIRVS) in mosquitoes has emerged as a significant area of research that could yield insight into virus-host interactions. This study aimed to characterize NIRVS in the Anopheles darlingi reference genome and identify putative transcribed NIRVS in field-collected mosquitoes from Colombia. The An. darlingi reference genome was analyzed to identify and characterize NIRVS by conducting a BLAST query with all the virus sequences previously identified in arthropods available in the NCBI-virus repository. In addition, An. darlingi field-collected mosquitoes were examined for NIRVS using a metatranscriptomic approach. As a result, 44 NIRVS were identified in the An. darlingi genome, constituting integrations of negative single-stranded RNA viruses (ssRNA-) from the families Rhabdoviridae, Chuviridae and Phasmaviridae, and integrations of double-stranded RNA viruses (dsRNA) from the families Partitiviridae and Sedoreoviridae. These NIRVS were not randomly distributed but clustered in specific regions of the genome enriched with BEL/Pao and Ty3/Gypsy long terminal repeat elements. Furthermore, putative NIRVS-like sequences were present in the transcriptomic data from all the Colombian An. darlingi natural populations. This study is significant as it represents the first identification of NIRVS in the most important malaria vector of the Neotropics. The findings help in understanding the intricate relationship between the mosquito and its virome, and the regulation of viruses' mechanisms in the Anopheles genus.

Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran

BackgroundAnopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes.MethodsMosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes.ResultsThe susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi.ConclusionBased on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

Attributes of Anopheles gambiae swarms in South Central Uganda

BackgroundAnopheles gambiae continues to be widespread and an important malaria vector species complex in Uganda. New approaches to malaria vector control are being explored including population suppression through swarm reductions and genetic modification involving gene drives. Designing and evaluating these new interventions require good understanding of the biology of the target vectors. Anopheles mosquito swarms have historically been hard to locate in Uganda and therefore have remained poorly characterized. In this study we sought to identify and characterize An. gambiae s.l mosquito swarms in three study sites of high An. gambiae s.l prevalence within Central Uganda.MethodsNine sampling visits were made to three villages over a 2-year period. Sampling targeted both wet and dry seasons and was done for 2 days per village during each trip, using sweep nets. All swarm data were analysed using the JMP 14 software (SAS Institute, Inc., Cary, NC, USA), parametrically or non-parametrically as appropriate.ResultsMost of the An. gambiae s.s. swarms sampled during this study were single-species swarms. However, some mixed An. gambiae s.s. and Culex spp. mosquito swarms were also observed. Swarms were larger in the wet season than in the dry season. Mean swarm height ranged from 2.16 m to 3.13 m off the ground and only varied between villages but not by season. Anopheles gambiae mosquitoes were present in all three villages, preferred to swarm over bare ground markers, and could be effectively sampled by field samplers.ConclusionsThis study demonstrated that An. gambiae s.l swarms could be effectively located and sampled in South Central Uganda and provided in-depth descriptions of hitherto poorly understood aspects of An. gambiae local swarm characteristics. Swarms were found close to inhabited households and were greater in size and number during the rainy season. Anopheles gambiae s.s swarms were significantly associated with bare ground markers and were sometimes at heights over 4 m above the ground, showing a necessity to develop tools suitable for swarm sampling at these heights. While mixed species swarms have been reported before elsewhere, this is the first documented instance of mixed genus swarms found in Uganda and should be studied further as it could have implications for swarm sampling explorations where multiple species of mosquitoes exist.Graphical

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

Distribution and insecticide resistance profile of the major malaria vector Anopheles funestus group across the African continent.

There has been significant progress in malaria control in the last 2 decades, with a decline in mortality and morbidity. However, these gains are jeopardised by insecticide resistance, which negatively impacts the core interventions, such as insecticide-treated nets (ITN) and indoor residual spraying (IRS). While most malaria control and research efforts are still focused on Anopheles gambiae complex mosquitoes, Anopheles funestus remains an important vector in many countries and, in some cases, contributes to most of the local transmission. As countries move towards malaria elimination, it is important to ensure that all dominant vector species, including An. funestus, an important vector in some countries, are targeted. The objective of this review is to compile and discuss information related to A. funestus populations' resistance to insecticides and the mechanisms involved across Africa, emphasising the sibling species and their resistance profiles in relation to malaria elimination goals. Data on insecticide resistance in An. funestus malaria vectors in Africa were extracted from published studies. Online bibliographic databases, including Google Scholar and PubMed, were used to search for relevant studies. Articles published between 2000 and May 2023 reporting resistance of An. funestus to insecticides and associated mechanisms were included. Those reporting only bionomics were excluded. Spatial variation in species distribution and resistance to insecticides was recorded from 174 articles that met the selection criteria. It was found that An. funestus was increasingly resistant to the four classes of insecticides recommended by the World Health Organisation for malaria vector control; however, this varied by country. Insecticide resistance appears to reduce the effectiveness of vector control methods, particularly IRS and ITN. Biochemical resistance due to detoxification enzymes (P450s and glutathione-S-transferases [GSTs]) in An. funestus was widely recorded. However, An. funestus in Africa remains susceptible to other insecticide classes, such as organophosphates and neonicotinoids. This review highlights the increasing insecticide resistance of An. funestus mosquitoes, which are important malaria vectors in Africa, posing a significant challenge to malaria control efforts. While An. funestus has shown resistance to the recommended insecticide classes, notably pyrethroids and, in some cases, organochlorides and carbamates, it remains susceptible to other classes of insecticides such as organophosphates and neonicotinoids, providing potential alternative options for vector control strategies. The study underscores the need for targeted interventions that consider the population structure and geographical distribution of An. funestus, including its sibling species and their insecticide resistance profiles, to effectively achieve malaria elimination goals.

Larvacidal effects of methanolic and ethanolic leaf extracts of <i>Xienemia americana</i> against female <i>Anopheles gambiae</i> S.L. (Culicidae: Diptera)

Anopheles gambiae complex is the most important vector of malaria in sub-Saharan Africa. It is thought that Nigeria comprises about 45 species of Anopheles mosquitoes of which Anopheles gambiae is the most prevalent. Extract of different concentrations were evaluated at the level of 2.00 3.00, 4.00, 5.00 and 0.00 (Control) mg/ml. Distilled water only was used as control. Twenty (20) larvae were put into four (4) cups containing 100ml of the test solution of each concentration. Larval to pupa mortality were recorded and calculated after 24, 48 and 72h after treatment. Mortality results were used to determine LC50 and LC90 using probit analysis. The larvicidal activity of methanol and Ethanolic extracts of the X. Americana were evaluated on 4th instar larvae of Anopheles gambiae. From the results concentration of X. americana with ethanol has shown high larvicidal effect with 10%, 12%, 3% adult emergence when compare with control. While Methanol concentration reveals low larvicidal effect with 16 % adult emergence. Treatment group X. Americana showed highest survival from pupa to adult (28, 18% and 6%) recorded, this means concentration of ethanol has larvicidal effect against A. gambiae. However, methanol concentration shows low activity with 45, 21 and 16% followed by X. Americana with 18, 15 and 10% recorded the highest larvicidal activity among all the concentrations with methanol respectively. Concentration of 5.0 mL, 12.0 mL, 20.0 mL, 25.0, 30.0mL compare to control group. All the extracts proved to be toxic to the test organism, though there was a remarkable difference in the concentrations and timing of their activity.

Mitotic metaphase karyotype of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae) from Kassala State, eastern Sudan

The mosquito Anopheles arabiensis Patton is the most important malaria vector in Sudan. The study was conducted for the first time to describe numerically the karyotype of An. arabiensis from Kassala State, eastern Sudan. Adults An. arabiensis were caught from human dwellings during the rainy season of 2022. We examined for the first time the utility of brain ganglia tissues of adult mosquitoes for mitotic chromosomal preparations using Giemsa stain - spreading technique. High-quality chromosomal preparations were examined and photographed. Chromosome measurements were carried out using computer software and analyzed statistically using SPSS® software. The diploid mitotic chromosome complement of An. arabiensis consists of three pairs of chromosomes, two pairs of metacentric autosomes (chromosome II and chromosome III) and one acrocentric dot-shaped pair, sex chromosome, which is homomorphic in females (XX) and heteromorphic in males (XY). Chromosome II was described as the longest (2.61±0.07) of the complement and constitute 44.39% of the total length (5.88 μm) of the haploid chromosomes set, while chromosome I (X=1.39 ±0.04; Y=1.04±0.04) as the shortest chromosome. Chromosome X appears in the males significantly larger than chromosome Y (P = 0.00). Chromosome III has an intermediate length (1.88±0.06) compared with the other chromosomes. Comparison of the average lengths of the three chromosome pairs by ANOVA test revealed highly statistical significant differences between them (P < 0.00). The study establishes a strong cytogentic data, which can contribute to accurate identification of the mosquito An. arabiensis and to planning human malaria vector control programs in Kassala State, eastern Sudan.

Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations

The mosquito Anopheles gambiae s.s. is a primary malaria vector throughout sub-Saharan Africa including the islands of the Comoros archipelago (Anjouan, Grande Comore, Mayotte and Mohéli). These islands are located at the northern end of the Mozambique Channel in eastern Africa. Previous studies have shown a relatively high degree of genetic isolation between the Comoros islands and mainland populations of A. gambiae, but the origin of the island populations remains unclear. Here, we analyzed phylogenetic relationships among island and mainland populations using complete mitochondrial genome sequences of individual A. gambiae specimens. This work augments earlier studies based on analysis of the nuclear genome. We investigated the source population of A. gambiae for each island, estimated the number of introductions, when they occurred and explored evidence for contemporary gene flow between island and mainland populations. These studies are relevant to understanding historical patterns in the dispersal of this important malaria vector and provide information critical to assessing their potential for the exploration of genetic-based vector control methods to eliminate this disease. Phylogenetic analysis and haplotype networks were constructed from mitogenome sequences of 258 A. gambiae from the four islands. In addition, 112 individuals from seven countries across sub-Saharan Africa and Madagascar were included to identify potential source populations. Our results suggest that introduction events of A. gambiae into the Comoros archipelago were rare and recent events and support earlier claims that gene flow between the mainland and these islands is limited. This study is concordant with earlier work suggesting the suitability of these oceanic islands as appropriate sites for conducting field trial releases of genetically engineered mosquitoes (GEMs).

Spatial distribution and ecological niche modeling of geographical spread of Anopheles gambiae complex in Nigeria using real time data

The need for evidence-based data, to inform policy decisions on malaria vector control interventions in Nigeria, necessitated the establishment of mosquito surveillance sites in a few States in Nigeria. In order to make evidence-based-decisions, predictive studies using available data becomes imperative. We therefore predict the distribution of the major members of the Anopheles gambiae s.l. in Nigeria. Immature stages of Anopheles were collected from 72 study locations which span throughout the year 2020 resulted in the identification of over 60,000 Anopheline mosquitoes. Of these, 716 breeding sites were identified with the presence of one or more vector species from the An. gambiae complex and were subsequently used for modelling the potential geographical distribution of these important malaria vectors. Maximum Entropy (MaxEnt) distribution modeling was used to predict their potentially suitable vector habitats across Nigeria. A total of 23 environmental variables (19 bioclimatic and four topographic) were used in the model resulting in maps of the potential geographical distribution of three dominant vector species under current climatic conditions. Members of the An. gambiae complex dominated the collections (98%) with Anopheles stephensi, Anopheles coustani, Anopheles funestus, Anopheles moucheti, Anopheles nilli also present. An almost equal distribution of the two efficient vectors of malaria, An. gambiae and Anopheles coluzzii, were observed across the 12 states included in the survey. Anopheles gambiae and Anopheles coluzzii had almost equal, well distributed habitat suitability patterns with the latter having a slight range expansion. However, the central part of Nigeria (Abuja) and some highly elevated areas (Jos) in the savannah appear not suitable for the proliferation of these species. The most suitable habitat for Anopheles arabiensis was mainly in the South-west and North-east. The results of this study provide a baseline allowing decision makers to monitor the distribution of these species and establish a management plan for future national mosquito surveillance and control programs in Nigeria.

Characterization of lab-based swarms of Anopheles gambiae mosquitoes using 3D-video tracking

Mosquito copulation is a crucial determinant of its capacity to transmit malaria-causing Plasmodium parasites as well as underpinning several highly-anticipated vector control methodologies such as gene drive and sterile insect technique. For the anopheline mosquitoes responsible for African malaria transmission, mating takes place within crepuscular male swarms which females enter solely to mate. However, the mechanisms that regulate swarm structure or that govern mate choice remain opaque. We used 3D-video tracking approaches and computer vision algorithms developed for the study of other complex biological systems to document swarming behavior of a lab-adapted Anopheles gambiae line in a lab-based setting. By reconstructing trajectories of individual mosquitoes lasting up to 15.88 s, in swarms containing upwards of 200 participants, we documented swarm-like behavior in both males and females. In single sex swarms, encounters between individuals were fleeting (< 0.75 s). By contrast, in mixed swarms, we were able to detect 79 ‘brief encounters’ (> 0.75 s; < 2.5 s) and 17 longer-lived encounters (> 2.5 s). We also documented several examples of apparent male-male mating competition. These findings represent the first steps towards a more detailed and quantitative description of swarming and courtship behavior in one of the most important vectors of malaria.

Mosquito Larvae and Pupae Transport from the Field.

Laboratory study of field-collected mosquitoes can allow researchers to better understand the ways variation within and among mosquito populations shapes burdens of mosquito-borne disease. The Anopheles gambiae complex comprises the most important vectors of malaria, but it can be challenging to keep in the laboratory. For some species of mosquitoes, especially An. gambiae, it is very difficult to bring viable eggs into the laboratory. Instead, it is preferable to collect larvae or pupae and then transport them as carefully as possible back to the laboratory. This simple protocol allows a researcher to start new laboratory colonies from larvae or pupae collected from natural breeding sites or proceed directly to their planned experiments. The use of natural breeding sites provides additional reassurance that the resulting colonies are representative of natural populations.

Genetic Diversity and Population Structure of Anopheles funestus in Western Kenya Based on Mitochondrial DNA Marker COII.

The mitochondrial marker, COII, was employed to assess the genetic structure and diversity of Anopheles funestus, a very important malaria vector in Africa that adapt and colonize different ecological niches in western Kenya. Mosquitoes were collected using mechanical aspirators in four areas (Bungoma, Port Victoria, Kombewa, and Migori) in western Kenya. Following morphological identification, PCR was used to confirm the species. The COII gene was amplified, sequenced, and analyzed to determine genetic diversity and population structure. A total of 126 (Port Victoria-38, Migori-38, Bungoma-22, and Kombewa-28) sequences of COII were used for population genetic analysis. Anopheles funestus had a high haplotype diversity (Hd = 0.97 to 0.98) but low nucleotide diversity (Π = 0.004 to 0.005). The neutrality test revealed negative Tajima's D and Fs values indicating an excess of low-frequency variation. This could be attributed to either population expansion or negative selection pressure across all the populations. No genetic or structural differentiation (Fst = -0.01) and a high level of gene flow (Gamma St, Nm = 17.99 to 35.22) were observed among the populations. Population expansion suggests the high adaptability of this species to various ecological requirements, hence sustaining its vectorial capacity and malaria transmission.

Proof-of-concept study for a long-acting formulation of ivermectin injected in cattle as a complementary malaria vector control tool

BackgroundDomesticated animals play a role in maintaining residual transmission of Plasmodium parasites of humans, by offering alternative blood meal sources for malaria vectors to survive on. However, the blood of animals treated with veterinary formulations of the anti-helminthic drug ivermectin can have an insecticidal effect on adult malaria vector mosquitoes. This study therefore assessed the effects of treating cattle with long-acting injectable formulations of ivermectin on the survival of an important malaria vector species, to determine whether it has potential as a complementary vector control measure.MethodsEight head of a local breed of cattle were randomly assigned to either one of two treatment arms (2 × 2 cattle injected with one of two long-acting formulations of ivermectin with the BEPO® technology at the therapeutic dose of 1.2 mg/kg), or one of two control arms (2 × 2 cattle injected with the vehicles of the formulations). The lethality of the formulations was evaluated on 3–5-day-old Anopheles coluzzii mosquitoes through direct skin-feeding assays, from 1 to 210 days after treatment. The efficacy of each formulation was evaluated and compared using Cox proportional hazards survival models, Kaplan–Meier survival estimates, and log-logistic regression on cumulative mortality.ResultsBoth formulations released mosquitocidal concentrations of ivermectin until 210 days post-treatment (hazard ratio > 1). The treatments significantly reduced mosquito survival, with average median survival time of 4–5 days post-feeding. The lethal concentrations to kill 50% of the Anopheles (LC50) before they became infectious (10 days after an infectious blood meal) were maintained for 210 days post-injection for both formulations.ConclusionsThis long-lasting formulation of ivermectin injected in cattle could complement insecticide-treated nets by suppressing field populations of zoophagic mosquitoes that are responsible, at least in part, for residual malaria transmission. The impact of this approach will of course depend on the field epidemiological context. Complementary studies will be necessary to characterize ivermectin withdrawal times and potential environmental toxicity.Graphical

Phytochemical Composition and Bioassay on Iranian Teucrium Polium Extracts against Anopheles Stephensi (Diptera: Culicidae).

Anopheles stephensi is an important malaria vector mosquito in Iran and other western Asian countries. In many human communities, plant products have been used traditionally instead of synthetic pesticides for mosquito control due to their minimal hazardous effects. Teucrium polium, known popularly as felty germander, has been introduced in Persian Medicine (PM) as an insect repellent from a long time ago. The present study was undertaken to evaluate repellent and larvicidal activity of dichloromethane (DCMETP) and ethanolic extracts (EE-TP) of T. polium against An. stephensi under laboratory conditions. The possible chemical components of the extracts were also investigated through gas chromatography/mass spectrometry (GC-MS) technique. Based on the results, DCME-TP showed better repellent activity than EE-TP with 56.67 and 28.33 % protection, respectively. Larvicidal activity of DCME-TP with 49.41% mortality was also higher than EE-TP (20.24%). The main identified constituents of DCME-TP were long chain alkanes, phenol, aromatic ester, oxaspiro and triterpenoid. While phenolic and aliphatic acid were only the identified components in EE-TP. It is notable that lupeol was detected in DCME of T. polium for the first time. DCME-TP can be considered as a new herbal candidate to control An. stephensi mosquitoes. Further studies are required on this extract for the fractionation and identification of the active compounds, and the evaluation of their bioactivity in the laboratory and field.

Population genomics reveal distinct and diverging populations of An. minimus in Cambodia

Anopheles minimus is an important malaria vector throughout its wide geographic range across Southeast Asia. Genome sequencing could provide important insights into the unique malaria transmission dynamics in this region, where many vector species feed and rest outdoors. We describe results from a study using Illumina deep whole-genome sequencing of 302 wild-caught An. minimus collected from three Cambodian provinces over several years (2010, 2014, 2016) and seasons to examine the level of population structure and genetic diversity within this species. These specimens cluster into four distinct populations of An. minimus s.s., with two populations overlapping geographically. We describe the underlying genetic diversity and divergence of these populations and investigated the genetic variation in genes known to be involved in insecticide resistance. We found strong signals of selection within these An. minimus populations, most of which were present in the two Northeastern Cambodian populations and differ from those previously described in African malaria vectors. Cambodia is the focus of the emergence and spread of drug-resistant malaria parasites, so understanding the underlying genetic diversity and resilience of the vectors of these parasites is key to implementing effective malaria control and elimination strategies. These data are publicly available as part of the MalariaGEN Vector Observatory, an open access resource of genome sequence data.

Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric morphometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (COI) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%–0.63%) and high interspecific distances (range, 1.92%–3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.

Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum

BackgroundThe colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon.MethodsLaboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load.ResultsSignificant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation.ConclusionsColonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

The effect of light and ventilation on house entry by Anopheles arabiensis sampled using light traps in Tanzania: an experimental hut study

BackgroundIn sub-Saharan Africa, house design and ventilation affects the number of malaria mosquito vectors entering houses. This study hypothesized that indoor light from a CDC-light trap, visible from outside a hut, would increase entry of Anopheles arabiensis, an important malaria vector, and examined whether ventilation modifies this effect.MethodsFour inhabited experimental huts, each situated within a large chamber, were used to assess how light and ventilation affect the number of hut-entering mosquitoes in Tanzania. Each night, 300 female laboratory-reared An. arabiensis were released inside each chamber for 72 nights. Nightly mosquito collections were made using light traps placed indoors. Temperature and carbon dioxide concentrations were measured using data loggers. Treatments and sleepers were rotated between huts using a randomized block design.ResultsWhen indoor light was visible outside the huts, there was an 84% increase in the odds of collecting mosquitoes indoors (Odds ratio, OR = 1.84, 95% confidence intervals, 95% CI 1.74–1.95, p < 0.001) compared with when it was not. Although the odds of collecting mosquitoes in huts with closed eaves (OR = 0.54, 95% CI 0.41–0.72, p < 0.001) was less than those with open eaves, few mosquitoes entered either type of well-ventilated hut. The odds of collecting mosquitoes was 99% less in well-ventilated huts, compared with poorly-ventilated traditional huts (OR = 0.01, 95% CI 0.01–0.03, p < 0.001). In well-ventilated huts, indoor temperatures were 1.3 °C (95% CI 0.9–1.7, p < 0.001) cooler, with lower carbon dioxide (CO2) levels (mean difference = 97 ppm, 77.8–116.2, p < 0.001) than in poorly-ventilated huts.ConclusionAlthough light visible from outside a hut increased mosquito house entry, good natural ventilation reduces indoor carbon dioxide concentrations, a major mosquito attractant, thereby reducing mosquito-hut entry.

Size and shape variations in wing morphology of Anopheles maculipennis s.s. Meigen, 1818 (Diptera: Culicidae) from northeastern Turkey

Anopheles maculipennis Meigen, 1818 (Diptera: Culicidae) complex was discovered as the first sibling species complex among mosquito species and identified as a highly important malaria vector in the Middle East and Europe. Anopheles maculipennis s.s. is the nominotypical member species of the complex, and widely spread across the whole of Europe. Body size and shape are the most important characters of organisms and is related to numerous variables. Biological size and shape may be affected by altitude and altitudinal differences. In this study, the variation in wing size and shape of An. maculipennis s.s. populations collected from four sampling stations in Iğdır Province (Mürşitali, Sürmeli, Yukarıçıyrıklı and Zülfikarköy) and two sampling stations in Kars Province (Kötek and Kozlu) at different altitudes and in different habitats from northeastern Turkey in 2019 were investigated. It was assumed that altitude and the environmental differences related with altitude may affect the wing (body) size or shape of An. maculipennis s.s. This is the first comparative geometric morphometric study of An. maculipennis s.s. populations in Turkey and the results indicate size and shape differences among some populations. While centroid size did not show a linear association with altitude, samples from the highest altitude population had larger wings than the other populations.