Genotype distribution and allele frequency of thioester-containing protein 1(Tep1) and its effect on development of Plasmodium oocyst in populations of Anopheles arabiensis in Ethiopia.

BACKGROUND: Thioester-containing protein 1 (TEP1) is a crucial component of mosquitoes' natural resistance to parasites. To effectively combat malaria, there is a need to better understand how TEP1 polymorphism affects phenotypic traits during infections. Therefore, the purpose of this study was to determine the Tep1 genotype frequency in malaria vector populations from south-western Ethiopia and investigate its effect on Plasmodium oocyst development in Anopheles arabiensis populations. METHODS: Using standard dippers, Anopheles mosquito larvae were collected from aquatic habitats in Asendabo, Arjo Dedessa, and Gambella in 2019 and 2020. Collected larvae were reared to adults and identified morphologically. Female An. gambiae s.l. were allowed to feed on infected blood containing the same number of gametocytes obtained from P. falciparum and P. vivax gametocyte-positive individuals using indirect membrane feeding methods. Polymerase Chain Reaction (PCR) was used to identify An. gambiae s.l. sibling species. Three hundred thirty An. gambiae s.l. were genotyped using Restricted Fragment Length Polymorphism (RFLP) PCR and sub samples were sequenced to validate the TEP1 genotyping. RESULTS: Among the 330 samples genotyped, two TEP1 alleles, TEP1*S1 (82% frequency) and TEP1*R1 (18% frequency), were identified. Three equivalent genotypes, TEP1*S1/S1, TEP1*R1/R1, and TEP1*S1/R1, had mean frequencies of 65.15%, 2.12%, and 32.73%, respectively. The nucleotide diversity was ranging from 0.36554 to 0. 46751 while haplotype diversity ranged from 0.48871 to 0.63161, across all loci. All sample sites had positive Tajima's D and Fu's Fs values. There was a significant difference in the TEP1 allele frequency and genotype frequency among mosquito populations (p < 0.05), except populations of Anopheles arabiensis from Asendabo and Gambella (p > 0.05). In addition, mosquitoes with the TEP1 *RR genotype were susceptible and produced fewer Plasmodium oocysts than mosquitoes with the TEP1 *SR and TEP1 *SS genotypes. CONCLUSION: The alleles identified in populations of An. arabiensis were TEP1*R1 and TEP1*S1. There was no significant variation in TEP1*R1 allele frequency between the high and low transmission areas. Furthermore, An. arabiensis carrying the TEP1*R1 allele was susceptible to Plasmodium infection. Further studies on vector-parasite interactions, particularly on the TEP1 gene, are required for vector control techniques.

Molecular xenomonitoring reveals Anopheles funestus and An. rivulorum as the primary vectors of lymphatic filariasis in coastal Kenya.

BACKGROUND: Lymphatic filariasis (LF) is an infectious neglected tropical disease caused by mosquito-borne nematodes such as Wuchereria bancrofti, Brugia malayi, and Brugia timori. Globally, LF affects 51 million people, with approximately 863 million at risk in 47 countries. In Kenya, filariasis is endemic along the entire coastal strip, and more recently, at the Kenya-Ugandan border. The World Health Organization (WHO) recommends mass drug administration to reduce disease transmission and morbidity. Monitoring the effectiveness of such interventions relies on robust surveillance, achieved through microscopic examination of microfilariae in nighttime blood, detection of circulating filarial antigens (CFA), and molecular xenomonitoring. We focused on molecular xenomonitoring along the Kenyan coast due to its noninvasive nature and the opportunity to identify new vectors. METHODS: In 2022, mosquitoes were collected from Kilifi, Kwale, and Taita-Taveta counties located within the LF endemic region in Kenya. Subsequently, genomic deoxyribonucleic acid (gDNA) was extracted from these mosquitoes for speciation and analysis of Wuchereria bancrofti infection rates. The impact of sociodemographic and household attributes on infection rates was assessed using generalized estimating equations. RESULTS: A total of 18,121 mosquitoes belonging to Culicinae (63.0%, n = 11,414) and Anophelinae (37.0%, n = 6707) subfamilies were collected. Morphological identification revealed that Anopheline mosquitoes were dominated by An. funestus (45.4%, n = 3045) and An. gambiae (42.8%, n = 2873). Wuchereria bancrofti infection rates were highest in Kilifi (35.4%; 95% CI 28.0-43.3%, n = 57/161) and lowest in Taita Taveta (5.3%; 95% CI 3.3-8.0%, n = 22/412). The major vectors incriminated are An. rivulorum, An. funestus sensu stricto, and An. arabiensis. Mosquitoes of the An. funestus complex were significantly associated with LF transmission (OR 18.0; 95% CI 1.80-180; p = 0.014). Additionally, a higher risk of transmission was observed outdoors (OR 1.74; 95% CI 1.08-2.82; p = 0.024) and in homesteads that owned livestock (OR 2.00; 95% CI 1.09-3.66; p = 0.025). CONCLUSIONS: In this study, we identified An. funestus s.l. sibling species, An. rivulorum and An. funestus s.s., as the primary vectors of lymphatic filariasis along the Kenyan coast. These findings also highlight that a significant portion of disease transmission potentially occurs outdoors where indoor-based vector control tools, including long-lasting insecticidal nets and indoor residual spray, may not be effective. Therefore, control measures targeting outdoor resting mosquitoes such as zooprophylaxis, larval source management, and attractive sugar baits may have potential for LF transmission reduction.

Urban malaria and its determinants in Eastern Ethiopia: the role of Anopheles stephensi and urbanization.

BACKGROUND: Malaria prevention and control strategies have been hampered by urbanization and the spread of Anopheles stephensi. The spread of this vector into Africa further complicates the already complex malaria situation, that could put about 126 million Africans at risk of infection. Hence, this study aimed to assess the determinants of urban malaria, focusing on the role of urbanization and the distribution of An. stephensi in Eastern Ethiopia. METHODS: A matched case control study was conducted among febrile urban residents of Dire Dawa (malaria positive as cases and negative as a control). A capillary blood sample was collected for parasite identification using microscopic examination and an interviewer administered questionnaire was used to collect additional data. Centers for Disease Control and Prevention miniature light traps (CDC-LT) and Prokopack aspirator were used to collect adult mosquito vectors from the selected cases and control houses to identify the mosquito vector species. Then, the data were exported to STATA for analysis. Conditional logistic regression was done to identify determinants, and principal component Analysis (PCA) was done for some independent variables. RESULTS: This study enrolled 132 cases and 264 controls from urban setting only. Of the 132 cases, 90 cases were positive for Plasmodium falciparum, 34 were positive for Plasmodium vivax and 8 had mixed infections. All cases and controls were similar with regard to their respective age and sex. Travel history (AOR: 13.1, 95% CI 2.8-61.4), presence of eves and holes on walls (AOR: 2.84, 95% CI 1.5-5.5), history of malaria diagnosis (AOR: 2.4, 95% CI 1.1-5.3), owning any livestock (AOR: 7.5, 95% CI 2.4-22.8), presence of stagnant water in the area (AOR: 3.2, 95% CI 1.7-6.1), sleeping under bed net the previous night (AOR: 0.21, 95% CI 0.1-0.6) and knowledge on malaria and its prevention (AOR: 2.2, 95% CI 1.2-4.1) were determinants of urban malaria infection. About 34 adult Anopheles mosquitoes were collected and identified from those selected cases and control houses and 27 of them were identified as An. stephensi. CONCLUSION: Among the cases, the dominant species were P. falciparum. This study identified travel history, house condition, past infection, livestock ownership, stagnant water, bed net use, and malaria knowledge as determinants of infection. This study also found the dominance of the presence of An. stephensi among the collected mosquito vectors. This suggests that the spread of An. stephensi may be impacting malaria infection in the study area. Hence, strengthening urban-targeted malaria interventions should be enhanced to prevent and control further urban malaria infection and spread.

Feeding habits and malaria parasite infection of Anopheles mosquitoes in selected agroecological areas of Northwestern Ethiopia.

BACKGROUND: Surveillance of the host-anopheline mosquitoes' interaction is important for assessing malaria transmission risk and guiding vector control. We assume that changes in malaria vector species' feeding habits, as well as the surrounding environment, have a substantial impact on varied malaria transmission. In this study, we determined the vertebrate host feeding patterns of anopheline mosquitoes to characterize entomologic risk factors for malaria in Jabi Tehnan, Northwestern Ethiopia. METHODS: Blood-fed anophelines surveyed during malaria surveillance in Jabi Tehnan district of northwestern Ethiopia were utilized in this study. They were collected using Centers for Disease Control and Prevention (CDC) light traps deployed in selected households per village, placed indoors and outdoors, spanning three agroecological settings (dry mountain, plateau, and semiarid highlands) between June 2020 and May 2021. The engorged mosquitoes were analyzed for host blood meal sources and Plasmodium infection via polymerase chain reaction (PCR) and/or sequencing. Infection rates and bovine and human blood indices were calculated and compared for abundant species; between indoors and outdoors and between agroecology using a chi-squared test for equality of proportion in R package at a significant level of p ≤ 0.05. RESULTS: A total of 246 mosquitoes were successfully typed (indoor, 121; outdoor, 125), with greater relative abundance indoors in mountain and plateau highlands, and outdoors in semiarid areas. Despite ecological differences in blood-fed capture rates, cattle served as the most utilized blood meal source by 11 anopheline species with an overall bovine blood index (BBI) of 74.4%. This trend was dictated by Anopheles gambiae s.l. (198/246; BBI = 73.7%), which exhibited the most plastic feeding habits that included humans (human blood index = 15.7%) and other livestock and rodents. A total of five anopheline species (An. gambiae s.l., An. funestus s.l., An. coustani s.l., An. pretoriensis, and An. pharoensis) fed on humans, of which the first three were found infected with Plasmodium parasites. Most of the infected specimens were An. arabiensis (5.6%, 11/198) and had recently fed mainly on cattle (72.7%, 8/11); one each of infected An. funestus s.l. and An. coustani s.l. had fed on humans and cattle, respectively. CONCLUSIONS: The results demonstrate communal feeding on cattle by anophelines including primary and secondary malaria vectors. This study also indicates the importance of cattle-targeted interventions for sustainable control of malaria vectors in the study areas.

Human-to-Anopheles dirus mosquito transmission of the anthropozoonotic malaria parasite, Plasmodium knowlesi.

BACKGROUND: Plasmodium knowlesi, identified as the fifth human malaria parasite, has rapidly spread across various Southeast Asian countries, yet uncertainties persist regarding its human-mosquito-human transmission. Therefore, this study aims to explore the transmission potential of P. knowlesi from human blood to mosquitoes. METHODS: A direct membrane-feeding assay was conducted by infecting laboratory-reared female Anopheles dirus mosquitoes with P. knowlesi-infected human blood from a single patient presenting with febrile malaria. Mosquitoes were dissected 7 days post-infection under a stereomicroscope to detect oocysts in the midgut, stained with mercurochrome. Salivary glands were examined 14 days post-infection for the presence of sporozoites. Malaria diagnosis employed microscopy by expert microscopists and nested PCR assays. RESULTS: Upon dissecting 745 out of 1439 blood-fed An. dirus mosquitoes on day 7 post-infection, two oocysts were identified in the midguts of two mosquitoes (0.27%). An additional 694 mosquitoes were dissected for salivary glands on day 14 post-infection, with three mosquitoes (0.43%) exhibiting sporozoites. Further confirmation by nested-PCR assay verified these sporozoites as belonging to the P. knowlesi species. CONCLUSIONS: The findings underscore the potential transmission of P. knowlesi from human blood to mosquitoes. The significance of these findings necessitates further investigation, such as repeating similar experiments among natural vectors, to gain deeper insights into the transmission dynamics of P. knowlesi in Southeast Asia.

Why does malaria transmission continue at high levels despite universal vector control? Quantifying persistent malaria transmission by Anopheles funestus in Western Province, Zambia.

BACKGROUND: Some settings continue to experience a high malaria burden despite scale-up of malaria vector control to high levels of coverage. Characterisation of persistent malaria transmission in the presence of standard control measures, also termed residual malaria transmission, to understand where and when individuals are exposed to vector biting is critical to inform refinement of prevention and control strategies. METHODS: Secondary analysis was performed using data collected during a phase III cluster randomized trial of attractive targeted sugar bait stations in Western Province, Zambia. Two seasonal cohorts of children aged 1-14 years were recruited and monitored monthly during the malaria transmission season, concurrent with entomological surveillance using a combination of human landing catch (HLC) and Centres for Disease Control (CDC) light traps at randomly selected households in study clusters. Behavioural data from cohort participants were combined with measured Anopheles funestus landing rates and sporozoite positivity to estimate the human behaviour-adjusted entomological inoculation rate (EIR). RESULTS: Behavioural data from 1237 children over 5456 child-visits in 20 entomology surveillance clusters were linked with hourly landing rates from 8131 female An. funestus trapped by HLC. Among all An. funestus tested by enzyme-linked immunosorbent assay (ELISA), 3.3% were sporozoite-positive. Mean EIR directly measured from HLC was 0.07 infectious bites per person per night (ib/p/n). When accounting for child locations over the evening and night, the mean behaviour-adjusted EIR was 0.02 ib/p/n. Children not sleeping under insecticide-treated nets (ITNs) experienced 13.6 infectious bites per person per 6 month season, 8% of which occurred outdoors, while ITN users received 1.3 infectious bites per person per 6 month season, 86% of which were received outdoors. Sleeping under an ITN can prevent approximately 90% of potential An. funestus bites among children. CONCLUSIONS: In this setting ITNs have a high personal protective efficacy owing to peak An. funestus biting occurring indoors while most individuals are asleep. However, despite high household possession of ITNs (>90%) and high individual use (>70%), children in this setting experience more than one infectious bite per person per 6 month transmission season, sufficient to maintain high malaria transmission and burden. New tools and strategies are required to reduce the malaria burden in such settings.

Dihydroartemisinin suppresses the susceptibility of Anopheles stephensi to Plasmodium yoelii by activating the Toll signaling pathway.

BACKGROUND: Malaria is a serious public health concern. Artemisinin and its derivatives are first-line drugs for the treatment of Plasmodium falciparum malaria. In mammals, artemisinin exhibits potent anti-inflammatory and immunoregulatory properties. However, it is unclear whether artemisinin plays a regulatory role in the innate immunity of mosquitoes, thereby affecting the development of Plasmodium in Anopheles when artemisinin and its metabolites enter mosquitoes. This study aims to determine the effect of dihydroartemisinin (DHA), a first-generation semisynthetic derivative of artemisinin, on innate immunity and malaria vector competence of Anopheles stephensi. METHODS: Anopheles stephensi was fed Plasmodium-infected mice treated with DHA via gavage, Plasmodium-infected blood containing DHA in vitro, or DHA-containing sugar, followed by Plasmodium yoelii infection. The engorged female mosquitoes were separated and dissected 8 and 17 days after infection. Plasmodium oocysts and sporozoites were counted and compared between the control and DHA-treated groups. Additionally, total RNA and proteins were extracted from engorged mosquitoes 24 and 72 h post infection (hpi). Real-time polymerase chain reaction (PCR) and western blotting were performed to detect the transcriptional levels and protein expression of immune molecules in mosquitoes. Finally, the Toll signaling pathway was inhibited via RNA interference and the infection density was analyzed to confirm the role of the Toll signaling pathway in the effect of DHA on the vector competence of mosquitoes. RESULTS: DHA treatment via different approaches significantly reduced the number of Plasmodium oocysts and sporozoites in mosquitoes. The transcriptional levels of anti-Plasmodium immune genes (including TEP1, LRIM1, and APL1C), Toll pathway genes (including Tube, MyD88, and Rel1), and the effector defensin 1 were upregulated by DHA treatment at 24 and 72 hpi. TEP1 and Rel1 protein expression was significantly induced under DHA treatment. However, Rel1 knockdown in DHA-treated mosquitoes abrogated DHA-mediated refractoriness to Plasmodium infection. CONCLUSIONS: DHA treatment effectively inhibited the development of P. yoelii in A. stephensi by upregulating mosquitoes' Toll signaling pathway, thereby influencing the susceptibility of Anopheles to Plasmodium.

The expansion of an invasive malaria vector: Anopheles stephensi emergence in Arba Minch town in the southern Rift Valley of Ethiopia.

Urban areas in malaria-endemic countries in East Africa are experiencing a significant increase in malaria cases, with the establishment of an "exotic" urban malaria vector, Anopheles stephensi, increasing the risk of urban malaria. To this end, the present study aimed to investigate the emergence of this species in Arba Minch, Ethiopia. Following the detection of An. stephensi in other parts of Ethiopia, 76 artificial containers (55 discarded tyres, 18 concrete water storage, and three plastic containers) were sampled in 21 locations in Arba Minch town, for immature Anopheles mosquito stages, using the standard dipping technique. Larvae were reared into adults which were morphologically identified at the species level 2-3 days after emergence. Morphological identification results were confirmed by species-specific polymerase chain reaction. Of the examined containers, 67 (88%) had at least one Anopheles larva. Thirty-two of the adults emerged were morphologically identified as An. stephensi, with 26 (81%) confirmed by molecular analysis. This is the first study to report An. stephensi from Arba Minch, one of South Ethiopia's largest towns, highlighting the need for increased vigilance. The planned and ongoing study in and around Arba Minch will contribute to understanding the bionomics and role of An. stephensi in malaria parasite transmission, helping develop a strategy to address the impending risk of urban malaria in Ethiopia.

Insights into malaria vectors-plant interaction in a dryland ecosystem.

Improved understanding of mosquito-plant feeding interactions can reveal insights into the ecological dynamics of pathogen transmission. In wild malaria vectors Anopheles gambiae s.l. and An. funestus group surveyed in selected dryland ecosystems of Kenya, we found a low level of plant feeding (2.8%) using biochemical cold anthrone test but uncovered 14-fold (41%) higher rate via DNA barcoding targeting the chloroplast rbcL gene. Plasmodium falciparum positivity was associated with either reduced or increased total sugar levels and varied by mosquito species. Gut analysis revealed the mosquitoes to frequently feed on acacia plants (~ 89%) (mainly Vachellia tortilis) in the family Fabaceae. Chemical analysis revealed 1-octen-3-ol (29.9%) as the dominant mosquito attractant, and the sugars glucose, sucrose, fructose, talose and inositol enriched in the vegetative parts, of acacia plants. Nutritional analysis of An. longipalpis C with high plant feeding rates detected fewer sugars (glucose, talose, fructose) compared to acacia plants. These results demonstrate (i) the sensitivity of DNA barcoding to detect plant feeding in malaria vectors, (ii) Plasmodium infection status affects energetic reserves of wild anopheline vectors and (iii) nutrient content and olfactory cues likely represent potent correlates of acacia preferred as a host plant by diverse malaria vectors. The results have relevance in the development of odor-bait control strategies including attractive targeted sugar-baits.

Investigating the association between household exposure to Anopheles stephensi and malaria in Sudan and Ethiopia: A case-control study protocol.

BACKGROUND: Endemic African malaria vectors are poorly adapted to typical urban ecologies. However, Anopheles stephensi, an urban malaria vector formerly confined to South Asia and the Persian Gulf, was recently detected in Africa and may change the epidemiology of malaria across the continent. Little is known about the public health implications of An. stephensi in Africa. This study is designed to assess the relative importance of household exposure to An. stephensi and endemic malaria vectors for malaria risk in urban Sudan and Ethiopia. METHODS: Case-control studies will be conducted in 3 urban settings (2 in Sudan, 1 in Ethiopia) to assess the association between presence of An. stephensi in and around households and malaria. Cases, defined as individuals positive for Plasmodium falciparum and/or P. vivax by microscopy/rapid diagnostic test (RDT), and controls, defined as age-matched individuals negative for P. falciparum and/or P. vivax by microscopy/RDT, will be recruited from public health facilities. Both household surveys and entomological surveillance for adult and immature mosquitoes will be conducted at participant homes within 48 hours of enrolment. Adult and immature mosquitoes will be identified by polymerase chain reaction (PCR). Conditional logistic regression will be used to estimate the association between presence of An. stephensi and malaria status, adjusted for co-occurrence of other malaria vectors and participant gender. CONCLUSIONS: Findings from this study will provide evidence of the relative importance of An. stephensi for malaria burden in urban African settings, shedding light on the need for future intervention planning and policy development.

Molecular xenomonitoring as an indicator of microfilaraemia prevalence for lymphatic filariasis in Samoa in 2019.

BACKGROUND: Lymphatic filariasis (LF) is a globally significant, vector-borne, neglected tropical disease that can result in severe morbidity and disability. As the World Health Organization (WHO) Global Programme to Eliminate Lymphatic Filariasis makes progress towards LF elimination, there is greater need to develop sensitive strategies for post-intervention surveillance. Molecular xenomonitoring (MX), the detection of pathogen DNA in vectors, may provide a sensitive complement to traditional human-based surveillance techniques, including detection of circulating filarial antigen and microfilaraemia (Mf). This study aims to explore the relationship between human Mf prevalence and the prevalence of polymerase chain reaction (PCR)-positive mosquitoes using MX. METHODS: This study compared Mf and MX results from a 2019 community-based survey conducted in 35 primary sampling units (PSUs) in Samoa. This study also investigated concordance between presence and absence of PCR-positive mosquitoes and Mf-positive participants at the PSU level, and calculated sensitivity and negative predictive values for each indicator using presence of any Mf-positive infection in humans or PCR-positive mosquitoes as a reference. Correlation between prevalence of filarial DNA in mosquitoes and Mf in humans was estimated at the PSU and household/trap level using mixed-effect Bayesian multilevel regression analysis. RESULTS: Mf-positive individuals were identified in less than half of PSUs in which PCR-positive mosquito pools were present (13 of 28 PSUs). Prevalence of PCR-positive mosquitoes (each species separately) was positively correlated with Mf prevalence in humans at the PSU level. Analysed at the species level, only Aedes polynesiensis demonstrated strong evidence of positive correlation (r) with human Mf prevalence at both PSU (r: 0.5, 95% CrI 0.1-0.8) and trap/household levels (r: 0.6, 95% CrI 0.2-0.9). CONCLUSIONS: Findings from this study demonstrate that MX can be a sensitive surveillance method for identifying residual infection in low Mf prevalence settings. MX identified more locations with signals of transmission than Mf-testing. Strong correlation between estimated PCR-positive mosquitoes in the primary vector species and Mf in humans at small spatial scales demonstrates the utility of MX as an indicator for LF prevalence in Samoa and similar settings. Further investigation is needed to develop MX guidelines to strengthen the ability of MX to inform operational decisions.

Analysis of diversity and an updated catalog of mosquito species (Diptera: Culicidae) in the Capivari-Monos Environmental Protection Area, São Paulo, Brazil.

The Capivari-Monos Environmental Protection Area (EPA) is located in the southern part of the São Paulo city Green Belt. Since the 1950s, this region has been affected by uncontrolled urban sprawl, resulting in a change in the ecological habits of some vector mosquitoes. Over the last two decades, cases of autochthonous bromeliad malaria associated with the presence of anopheline mosquitoes in the EPA have been recorded. Anopheles cruzii, the primary vector of plasmodia in the region, is abundant and found naturally infected with both Plasmodium vivax and Plasmodium malariae. In light of this, the present study sought to update the catalog of mosquito fauna in this EPA, analyze mosquito diversity among sites with different degrees of conservation and compare species using different collection techniques. Field collections were carried out from March, 2015 to April, 2017. A total of 20,755 specimens were collected, distributed in 106 different taxa representing 16 genera. Analysis of the diversity among the sites based on the Shannon and Simpson indices showed that the most preserved of them had the lowest indices because of the dominance of An. cruzii. The results highlight the increase in the number of different taxa collected as different mosquito collection techniques were included, confirming the importance of using several strategies to ensure adequate sampling of a local mosquito fauna when exploring a greater number of ecotopes. Furthermore, the survey produced the most recent and complete list of mosquito species in the Capivari-Monos EPA, a refuge and shelter for native and introduced mosquito species where new biocenoses, including pathogens, vertebrate hosts, and vectors can form, allowing zoonotic outbreaks in the local human population to occur.

Leucinostatins from fungal extracts block malaria transmission to mosquitoes.

BACKGROUND: Malaria is a mosquito-transmitted disease that kills more than half a million people annually. The lack of effective malaria vaccines and recently increasing malaria cases urge innovative approaches to prevent malaria. Previously, we reported that the extract from the soil-dwelling fungus Purpureocillium lilacinum, a common fungus from the soil, reduced Plasmodium falciparum oocysts in Anopheles gambiae midguts after mosquitoes contacted the treated surface before feeding. METHODS: We used liquid chromatography to fraction fungal crude extract and tract the active fraction using a contact-wise approach and standard membrane feeding assays. The purified small molecules were analyzed using precise mass spectrometry and tandem mass spectrometry. RESULTS: We isolated four active small molecules from P. lilacinum and determined them as leucinostatin A, B, A2, and B2. Pre-exposure of mosquitoes via contact with very low-concentration leucinostatin A significantly reduced the number of oocysts. The half-maximal response or inhibition concentration (EC50) via pre-exposure was 0.7 mg/m2, similar to atovaquone but lower than other known antimalarials. The inhibitory effect of leucinostatin A against P. falciparum during intraerythrocytic development, gametogenesis, sporogonic development, and ookinete formation, with the exception of oocyst development, suggests that leucinostatins play a part during parasite invasion of new cells. CONCLUSIONS: Leucinostatins, secondary metabolites from P. lilacinum disrupt malaria development, particular transmission to mosquitoes by contact. The contact-wise malaria control as a nonconventional approach is highly needed in malaria-endemic areas.

Malaria burden and residual transmission: two thirds of mosquito bites may not be preventable with current vector control tools on Bioko Island, Equatorial Guinea.

OBJECTIVES: This study assesses exposure to malaria vector mosquitos that is nonpreventable through use of nets, the contribution of outdoor and indoor biting towards residual vector exposure, and the risk factors for being bitten and for being infected with malaria parasites on Bioko Island, Equatorial Guinea. METHODS: Human behavior and malaria infection data were collected from 13,735 randomly selected residents during cross-sectional surveys, concomitantly with entomological human landing catches, indoors and outdoors, in 20 locations on the Island. Self-reported time of going indoors, going to bed and whether using a net were analyzed to impute for each respondent the number of bites received outdoors and indoors during the night before the survey. RESULTS: On average, each person received 2.7 (95% CI: 2.6-2.8) bites per night outdoors, 8.5 (8.3 to 8.7) bites indoors if not using a net, and 4.7 (4.5 to 4.8) bites indoors if using a net. Malaria infection was associated with more bites, regardless of whether received indoors or outdoors. Older age, male gender, not using a net, rural location, and going indoors later increased the risk of being bitten. The proportion of bites not averted by using a net was estimated as 66% (61 to 71). CONCLUSIONS: A large proportion of biting, mostly indoors, may not be preventable by bednets. Tools targeting indoor biting should be prioritized in Bioko. Novel vector control tools are urgently needed to reduce overall exposure to mosquito bites.

Anopheles gambiae lacking AgTRIO probe inefficiently on a mammalian host.

Malaria is initiated as Plasmodium sporozoites are injected into the dermis when an infected mosquito probes on a vertebrate host for a blood meal. Factors in the mosquito saliva, such as AgTRIO, can alter the ability of Anopheles gambiae to transmit Plasmodium. We therefore used CRISPR-Cas9-mediated genome editing to generate AgTRIO knockout (KO) A. gambiae and examined the ability of these mosquitoes to probe on a vertebrate host. AgTRIO KO mosquitoes showed a diminished host probing capacity and required repetitive probing to locate a blood resource to complete a blood meal. This increased probing resulted in enhanced Plasmodium transmission to the vertebrate host. Our data demonstrate the importance of the A. gambiae saliva protein AgTRIO in probing and its influence on the ability of mosquitoes to transmit malaria.

Mosquito prevalence, resting habitat preference, and Plasmodium infection status of anophelines in coastal Karnataka during the declining phase of malaria-an exploratory study.

Malaria has a historical presence in the Dakshina Kannada (D.K.) and Udupi districts of Karnataka, India. To understand the potential involvement of anopheline fauna in malaria transmission, we conducted an exploratory entomological survey. The study is crucial given the decreasing malaria incidence in these districts in recent years. From September 2022 to August 2023, we collected indoor resting mosquitoes using a manual aspirator at 27 randomly chosen sites within three distinct resting habitats (human dwellings, cattle sheds, and construction sites) in the urban areas of Udupi and Dakshina Kannada districts. Mosquitoes were morphologically identified, and anopheline specimens were tested for the presence of malarial parasite by polymerase chain reaction (PCR) analysis. We collected a total of 1810 mosquitoes, comprising 21 species distributed across five genera. Culex emerged as the predominant genus, constituting 84.4% of the collected specimens, while Anopheles accounted for 5.4%. Among the observed species, Culex quinquefasciatus was predominant, comprising 77.9% of the mosquito specimens collected in this study. Two malaria vectors, An. stephensi and An. subpictus complex, constituted 16.3% and 1.0% of the total anophelines collected, respectively. None of the 96 female anophelines was tested positive for Plasmodium infection. Our findings suggest that Anopheles mosquitoes prefer resting in cattle sheds over human dwellings. While our study identified two malaria vectors, they were present at low densities. To gain a more comprehensive understanding of the dynamics of these vector mosquitoes, it is essential to conduct long-term surveillance to monitor their prevalence and role in malaria transmission.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations.

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA-LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10-100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Host-specific effects of a generalist parasite of mosquitoes.

Microsporidians are obligate parasites of many animals, including mosquitoes. Some microsporidians have been proposed as potential agents for the biological control of mosquitoes and the diseases they transmit due to their detrimental impact on larval survival and adult lifespan. To get a more complete picture of their potential use as agents of biological control, we measured the impact of Vavraia culicis on several life-history traits of Aedes aegypti and Anopheles gambiae. We measured the infection dynamics and clearance rate for the two species, and we assessed sexual dimorphism in infection dynamics within each species. Our results show differences in infection dynamics, with Ae. aegypti life-history traits being more affected during its aquatic stage and exhibiting higher clearance of the infection as adults. In contrast, An. gambiae was unable to clear the infection. Additionally, we found evidence of sexual dimorphism in parasite infection in An. gambiae, with males having a higher average parasite load. These findings shed light and improve our knowledge of the infection dynamics of V. culicis, a microsporidian parasite previously recognized as a potential control agent of malaria.

Long-term blood-free rearing of Anopheles mosquitoes with no effect on fitness, Plasmodium infectivity nor microbiota composition.

Mosquito-borne diseases kill millions of people each year. Therefore, many innovative research and population control strategies are being implemented but, most of them require large-scale production of mosquitoes. Mosquito rearing depends on fresh blood from human donors, experimentation animals or slaughterhouses, which constitutes a strong drawback since high blood quantities are needed, raising ethical and financial constraints. To eliminate blood dependency and the use of experimentation animals, we previously developed BLOODless, a patented diet that represents an important advance towards sustainable mosquito breeding in captivity. BLOODless diet was used to maintain a colony of Anopheles stephensi for 40 generations. Bloodmeal appetite, fitness, Plasmodium berghei infectivity, whole genome sequencing and microbiota were evaluated over time. Here we show that BLOODless can be implemented in Anopheles insectaries since it allows long-term rearing of mosquitoes in captivity, without a detectable effect on their fitness, infectivity, nor on their midgut and salivary microbiota composition.

Gene expression analysis of Anopheles Meigen, 1818 (Diptera: Culicidae) innate immunity after Plasmodium Marchiafava & Celli, 1885 (Apicomplexa) infection: Toward developing new malaria control strategies.

Despite the critical role of the Anopheles innate immune system in defending against Plasmodium infection, there is still limited information about the key immune mechanisms in Anopheles. This review assesses recent findings on the expression characteristics of immune-related genes in Anopheles following exposure to Plasmodium. A literature review, unrestricted by publication date, was conducted to evaluate immune-related gene expression in different organs of Anopheles after Plasmodium infection. Mosquito immune responses in the midgut are essential for reducing parasite populations. Additionally, innate immune responses in the salivary glands and hemocytes circulating in the hemocoel play key roles in defense against the parasite. Transcriptomic analysis of the mosquito's innate immune response to Plasmodium infection provides valuable insights into key immune mechanisms in mosquito defense. A deeper understanding of immune mechanisms in different organs of Anopheles following Plasmodium infection will aid in discovering critical targets for designing novel control strategies.