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For successful transmission, the malaria parasite must traverse tissue epithelia and survive attack from the insect's innate immune system. Hemocytes play a multitude of roles in mosquitoes, including defense against invading pathogens. Here, we show that hemocytes of the major malaria vector Anopheles gambiae promote Plasmodium falciparum infection by maintaining midgut epithelial integrity by controlling cell proliferation upon blood feeding. The mosquito's hemocytes also control the midgut microbiota and immune gene expression. Our study unveils novel hemocyte functions that are exploited by the human malaria parasite to evade the mosquito's immune system., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Larval source management (LSM) has a long history of advocacy and successes but is rarely adopted where funds are limited. The World Health Organization (WHO) guidelines on malaria prevention recommend the use of LSM as a supplementary intervention to the core vector control methods (insecticide-treated nets and indoor residual spraying), arguing that its feasibility in many settings can be limited by larval habitats being numerous, transient, and difficult to find or treat. Another key argument is that there is insufficient high-quality evidence for its effectiveness to support wide-scale implementation. However, the stagnation of progress towards malaria elimination demands that we consider additional options to the current emphasis on insecticidal commodities targeting adult mosquitoes inside homes. This letter is the result of a global, crossdisciplinary collaboration comprising: (a) detailed online expert discussions, (b) a narrative review of countries that have eliminated local malaria transmission, and (c) a mathematical modeling exercise using two different approaches. Together, these efforts culminated in seven key recommendations for elevating larval source management as a strategy for controlling malaria and other mosquito-borne diseases in Africa (Box 1). LSM encompasses the use of larvicide (a commodity) as well as various environmental sanitation measures. Together, these efforts lead to the long-term reduction of mosquito populations, which benefits the entire community by controlling both disease vector and nuisance mosquitoes. In this paper, we argue that the heavy reliance on large-scale cluster-randomized controlled trials (CRTs) to generate evidence on epidemiological endpoints restricts the recommendation of approaches to only those interventions that can be measured by functional units and deliver relatively uniform impact and, therefore, are more likely to receive financial support for conducting these trials. The explicit impacts of LSM may be better captured by using alternative evaluation approaches, especially high-quality operational data and a recognition of locally distinct outcomes and tailored strategies. LSM contributions are also evidenced by the widespread use of LSM strategies in nearly all countries that have successfully achieved malaria elimination. Two modelling approaches demonstrate that a multifaceted strategy, which incorporates LSM as a central intervention alongside other vector control methods, can effectively mitigate key biological threats such as insecticide resistance and outdoor biting, leading to substantial reductions in malaria cases in representative African settings. This argument is extended to show that the available evidence is sufficient to establish the link between LSM approaches and reduced disease transmission of mosquito-borne illnesses. What is needed now is a significant boost in the financial resources and public health administration structures necessary to train, employ and deploy local-level workforces tasked with suppressing mosquito populations in scientifically driven and ecologically sensitive ways. In conclusion, having WHO guidelines that recognize LSM as a key intervention to be delivered in multiple contextualized forms would open the door to increased flexibility for funding and aid countries in implementing the strategies that they deem appropriate. Financially supporting the scale-up of LSM with high-quality operations monitoring for vector control in combination with other core tools can facilitate better health. The global health community should reconsider how evidence and funding are used to support LSM initiatives., Competing Interests: Declarations. Competing interests: Sarah Jane Moore (SJM) conducts evaluation of vector control products including larvicides for a number of manufacturers via funding for contract research to her employer without personal payment or consultancy. Therefore, there is no conflict of interest. Arnon Houri Yafin (AHY) is the CEO of ZzappMalaria, a company that develops software to support larval source management (LSM) operations. Silas Majambere (SM) works for Valent BioSciences LLC, a manufacturer of biological larvicides. William Jany (WJ) works for Clarke International, USA, a global provider of vector control devices. No other authors have conflicts of interest to declare. Ethical approval and consent to participate: Not applicable., (© 2025. The Author(s).)

Background: Plasmodium vivax is a major cause of malaria, particularly outside Africa, necessitating effective strategies for public health management. Transmission-blocking vaccines (TBVs) have shown the potential to inhibit malaria transmission by targeting antigens expressed in sexual-stage parasites. Pbg37, a conserved protein expressed in sexual stages from gametocyte to ookinete in the rodent parasite P. berghei , is a viable target for TBV development., Methods and Findings: In this study, we constructed a transgenic strain, TrPvg37Pb , expressing Pvg37 using the P. berghei ΔPbg37 strain. Initial findings demonstrated that the replacement of Pbg37 with the exogenous Pvg37 did not impact parasite growth or development. Notably, Pvg37 was expressed during the gametocyte to ookinete development and was associated with the plasmic membrane, similar to Pbg37. To evaluate the potential of Pvg37 as a TBV candidate, we synthesized two Pvg37 polypeptides and immunized rabbits to generate antibodies. In vitro experiments demonstrated that anti-Pvg37-P2 antibodies significantly inhibited the formation of male gametes and ookinetes in the transgenic TrPvg37Pb parasite. Additionally, in mosquito feeding assays, mosquitos feeding on TrPvg37Pb -infected mice passively transferred with anti-Pvg37-P2 antibodies showed a significant 80.2% decrease in oocyst density compared to the control group. Furthermore, in direct membrane feeding experiments using four clinical P. vivax isolates, the anti-Pvg37 antibodies significantly reduced oocyst density by 28.6-50.4%., Conclusion: Pvg37 is a promising candidate for P. vivax TBV development, deserving further research and optimization to enhance its immunogenicity and transmission-blocking activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Zhang, Zhao, Liu, Liu, Liu, Zhang, Wu, Roobsoong, Bantuchai, Thongpoon, Sripoorote, Wang, Cui and Cao.)

Mosquitoes are important vectors for the transmission of some major infectious diseases of humans, i.e., malaria, dengue, West Nile Virus and Zika virus. The burden of these diseases is different for different regions, being highest in tropical and subtropical areas, which have high annual rainfall, warm temperatures, and less pronounced seasonality. The life cycle of mosquitoes consists of four distinct stages: eggs, larvae, pupae, and adults. These life stages have different mortality rates and only adults can reproduce. Seasonal weather may affect the population dynamics of mosquitoes, and the relative abundance of different mosquito stages. We developed a stage-structured model that considers laboratory experiments describing how temperature and rainfall affects the reproduction, maturation and survival of different Anopheles mosquito stages, the species that transmits the parasite that causes malaria. We consider seasonal temperature and rainfall patterns and describe the stage-structured population dynamics of the Anopheles mosquito in Ain Mahbel, Algeria, Cape Town, South Africa, Nairobi, Kenya and Kumasi, Ghana. We find that neglecting seasonality leads to significant overestimation or underestimation of mosquito abundance. We find that depending on the region, mosquito abundance: peaks one, two or four times a year, periods of low abundance are predicted to occur for durations ranging from six months (Ain Mahbel) to not at all (Nairobi); and seasonal patterns of relative abundance of stages are substantially different. The region with warmer temperatures and higher rainfall across the year, Kumasi, Ghana, is predicted to have higher mosquito abundance, which is broadly consistent with reported malaria deaths relative to the other countries considered by our study. Our analysis reveals distinct patterns in mosquito abundance across different months and regions. Control strategies often target one specific life stage, for example, applying larvicides to kill mosquito larvae, or spraying insecticides to kill adult mosquitoes. Our findings suggest that differences in seasonal weather affect mosquito stage structure, and that the best approaches to vector control may differ between regions in timing, duration, and efficacy., (© 2025. The Author(s), under exclusive licence to the Society for Mathematical Biology.)

There are three Anopheles mosquito species in East Africa that are responsible for the majority of malaria transmission, posing a significant public health concern. Understanding the vector competence of different mosquito species is crucial for targeted and cost-effective malaria control strategies. This study investigated the vector competence of laboratory reared strains of East African An. gambiae sensu stricto, An. funestus s.s., and An. arabiensis mosquitoes towards local isolates of Plasmodium falciparum infection. Mosquito feeding assays using gametocytaemic blood from local donors revealed significant differences in both prevalence and intensity of oocyst and sporozoite infections among the three vectors. An. funestus mosquitoes presented the highest sporozoite prevalence 23.5% (95% confidence interval (CI) 17.5-29.6) and intensity of infection 6-58138 sporozoites. Relative to An. funestus, the odds ratio for sporozoites prevalence were 0.46 (95% CI 0.25-0.85) in An. gambiae and 0.19 (95% CI 0.07-0.51) in An. arabiensis, while the incidence rate ratio for sporozoite intensity was 0.31 (95% CI 0.14-0.69) in An. gambiae and 0.66 (95% CI 0.16-2.60) in An. arabiensis. Our findings indicate that all three malaria vector species may contribute to malaria transmission in East Africa, with An. funestus demonstrating superior vector competence. In conclusion, there is a need for comprehensive malaria control strategies targeting major malaria vector species, an update of malaria transmission models to consider vector competence and evaluation of malaria transmission blocking interventions in assays that include An. funestus mosquitoes., Competing Interests: Declarations. Ethics approval and consent to participate: All adult participants provided written informed consent, while for children under 18 years old, consent was obtained from their parent or guardian, with the children’s assent also sought for their participation. All study volunteers received artemisinin-lumefantrine treatment within 24 h of diagnosis, following the Tanzania Guidelines for Diagnosis and Treatment of Malaria81, administered by a qualified nurse. No adverse effects were reported among the participants during the study period. The study activities were reviewed and approved by the Institutional Review Board of IHI (IHI/IRB/No: 44–2020) and the National Institute for Medical Research Tanzania (NIMR/HQ/R.8a/Vol.IX/3595). All research was performed in accordance to relevant guidelines and regulations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Although the World Health Organization (WHO) certified Taiwan as being malaria-free in 1965, there are reports of a few imported cases each year by travelers who visit malaria-endemic areas. This study examined the epidemiology of imported malaria cases in Taiwan from 2014 to 2020, utilizing national surveillance data from the Taiwan Centers for Disease Control. Malaria cases were confirmed through the application of standard laboratory methods. Passenger data came from the Tourism Bureau, Ministry of Transportation and Communication, Taiwan (TBMTC). All data were analyzed using SPSS version 21. The analysis included a dataset comprising 64 cases of imported malaria. Of the total cases, 77.8% were acquired from Africa, and 17.5% from Asia. Plasmodium falciparum was responsible for more than half (57.1%) of the cases, Plasmodium vivax malaria for 25.4% of cases, Plasmodium malariae malaria for 6.3%, Plasmodium ovale malaria for 4.8%, and unspecified pathogen malaria for 6.3% of the cases. Majority of the patients were male (75%) and were predominantly aged 20 to 59 years (70.3%). Most cases of imported malaria occurred during the fall season, and 51.6% of cases occurred in 8 cities during the period of 2014 to 2020. No evidence exists to indicate that indigenous malaria transmission occurs in Taiwan. Anopheles minimus was found in 4 cities (counties), namely Tainan City and Pingtung County in Southern Taiwan; Hualien County and Taitung County in Eastern Taiwan. The findings of this study highlight the necessity for robust surveillance systems, effective vector control measures, and targeted interventions for travelers and immigrants to prevent malaria outbreaks and maintain Taiwan's malaria-free status., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2025 the Author(s). Published by Wolters Kluwer Health, Inc.)

Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests, (© 2025. The Author(s).)

In tropical countries, malaria transmission is the major health issue. To eradicate malaria, health communities depend on the control measure that affects economy and environment of the countries. To overcome these burdens, there is a great need to develop vaccine against malaria, but there is no vaccine to control malaria effectively. Transmission blocking vaccine (TBV) is the best alternative approach to control malaria, which inhibits the malaria transmission. Mosquito-based TBVs, blocks the transmission of Plasmodium developmental stages in mosquito. There are some potential candidate antigens for mosquito-based TBV, e.g., AgCPB1, AnAPN1, AgFREP1, etc. AcSGU is one of the most potential candidates for TBVs. AcSGU protein is glycol-anchored protein in Anopheles culicifacies which is highly expressed after blood feeding. In the present study acsgu gene was amplified from genomic DNA, sequenced (GenBank id: MN402758) and characterised. The sequence of acsgu (gene and protein) was analysed by different immuno-informatics tools to confirm its potentiality as a candidate antigen. The target protein was cloned, isolated and immunised for immunogenic response analysis. The acsgu gene is single exonic which encodes for AcSGU protein with single functional MBF2 domain. It is conserved in most of the Anopheles species. Bioinformatics analysis confirmed the stability and immunogenic nature of the protein. Protein-Protein interaction revealed effective interaction of AcSGU with Pf47 and TLR4 molecules. AcSGU protein was expressed in E. coli BL21 (DE3) by using expression vector pLATE51. The immunogenic response in AcSGU protein was remarkable in the rabbit. This study confirmed that AcSGU protein is the potential candidate for transmission blocking vaccine to generate anti-midgut immunity against plasmodium. It can be used as a candidate for the development of multistage targeting vaccines against malaria., Competing Interests: Compliance with ethical standards. Ethical approval: All the experiments conducted on animals were performed as per the IAEC guideline, MDU Rohtak (Reg No:1767/RE/S/14/CPCSEA). Experimental work has been approved by the IAEC committee) (Letter No: 153–165/17–12-B). Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Plasmodium, the causative agents of malaria, are obtained by mosquitoes from an infected human. Following Plasmodium acquisition by Anopheles gambiae, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT) plays a critical role in its subsequent sporogony in the mosquito. A critical location for this development is the midgut, a tissue we show expresses mosGILT. Using membrane-feeding and murine infection models, we demonstrate that antibodies against mosGILT reduce the number of P. falciparum and P. berghei oocysts in the midgut and the infection prevalence of both species in the mosquito. mosGILT antibodies act in the mosquito midgut, specifically impacting the Plasmodium oocyst stage. Targeting mosGILT can therefore interfere with the Plasmodium life cycle in the mosquito and potentially serve as a transmission-blocking vaccine., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Mosquitoes are important drivers of infectious diseases transmission, with Anopheles mosquitoes being responsible of malaria transmission. In Cambodia, where malaria is prevalent in forested regions, understanding the ecology of these vectors is crucial. This study aimed to investigate the abundance, distribution, seasonal patterns, biting behaviour of Anopheles mosquitoes, and prevalence of Plasmodium, in Mondulkiri province, Northeastern Cambodia., Methods: Conducted in 9 sites, seven in forested and two in neighbouring villages, over one year, the collection of Anopheles mosquitoes was made hourly for a 72-h period every month, using a human-baited double net trap. Each mosquito was collected using a mouth-aspirator and identified morphologically, and screened for the presence of Plasmodium., Results: Primary vectors, including Anopheles baimaii, Anopheles dirus, Anopheles maculatus, and Anopheles minimus, constituted 11.1% of all female mosquitoes, while 12 secondary vector species represented 29.4% of the overall collection. Anopheles species were more prevalent during the late rainy season (August to November), with year-round activity observed. Primary vectors were predominantly found in forest sites, while other vector species were found in both village and forest environments. Notably, primary vectors exhibited a preference for nocturnal biting, yet a significant proportion (19.2%) displayed daytime activity, highlighting a potential risk of daytime malaria transmission. Among 5,056 Anopheles specimens tested, only 36 Plasmodium spp.-infected samples were detected, mainly in forest sites (94%), and in specimens collected at night. This study provides essential insights into the ecology of Anopheles in Mondulkiri Forest., Conclusions: The identification of primary and secondary vectors, their seasonal dynamics, and biting behaviour contribute to enhances our understanding of malaria transmission risks in these areas, guiding future strategies toward effective and context-specific control measures, while stressing the need for individual protection during daytime., Competing Interests: Declarations. Ethics approval and consent to participate: The study protocol was reviewed and approved by the National Ethics Committee for Health research, Ministry of Health, Kingdom of Cambodia (306NECHR, 100NECHR). The study procedures and risks were explained to the collectors and their informed consent was obtained. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Rios et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The Lihir Islands of Papua New Guinea, located in an area with high burden of malaria and hosting a large mining operation, offer a unique opportunity to study transmission. There, we investigated human and vector factors influencing malaria transmission., Methods: In 2019, a cross-sectional study was conducted on 2,914 individuals assessing malaria prevalence through rapid diagnostic tests (RDT), microscopy, and quantitative PCR (qPCR). A logistic regression analysis identified infection-associated factors. Anopheles species distribution, biting behaviours, and sporozoite carriage were assessed through human landing catches and larval surveys., Results: Overall malaria prevalence (any species) was 3.6% by RDT, 4.5% by microscopy, and 15.0% by qPCR. P. vivax accounted for 37.1% of infections, P. falciparum for 34.6%, P. malariae for 3.0%, P. ovale 0.2%, and mixed infections for 24.5%. Prevalence (qPCR) varied across geographic areas, from 8.5% in the mine-impacted zone (MIZ) to 27.0% in the non-MIZ. Other factors independently associated with infection risk included cohabiting with an infected individual (aOR = 1.94, 95%CI: 1.56-2.42), and residing in traditional housing (aOR = 1.65, 95%CI: 1.21-2.25). Children had double the infection risk compared to adults, and the use of long-lasting insecticidal-treated nets did not decrease risk of infection. An. punctulatus was the major vector in one of the four geographical areas; while An. farauti was predominant in the rest of them, both with an early biting behaviour but with different biting intensities by geographical area. Entomological inoculation rates ranged from 26.9 (95%CI: 12.3-45.2) infective bites per person-year in the MIZ to 441.3 (95%CI: 315.7-572.1) in the non-MIZ., Conclusions: Malaria transmission and infection was lower in the MIZ compared to other areas. Measures focusing on at-risk groups, including vector-control and transmission interruption methods, could be taken into account by the mine and the healthcare authorities to reduce malaria burden outside the MIZ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Millat-Martínez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: The World Health Organization recommends three drug anti-malarial combinations: cloroquine+primaquine, artesiminin+primaquine, and cloroquine+tafenoquine. These combinations aim to eradicate Plasmodium by disrupting its life cycle within the human body. We evaluated the effect of these medications on the vectorial competence of two main vectors in the New World., Methods: We recruited patients diagnosed with malaria vivax from a primary care in Manaus, Amazonas. To determine how different treatments affected vectors, we collected blood samples prior to treatment at the hospital and at six intervals over the next 68 hours at the patient's homes. These samples were used to infect Anopheles darlingi and Anopheles aquasalis. To assess the potential for Plasmodium transmission by bite to a new human host, we analyzed the infection intensity, infection rate, and presence of parasites in the salivary gland of the mosquitoes., Results: The results show the infection of the mosquitoes fed with the patient's blood during the first days of treatment with all three drug combinations. However, the cloroquine+tafenoquine combination was the least effective while artesiminin+primaquine was the most effective., Conclusions: In the first few days of treatment, two main vectors continue to spread malaria vivax from patients, potentially contributing to the ongoing transmission in malaria-endemic regions., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Malaria caused by Plasmodium parasites remains a large health burden. One approach to combat this disease involves vaccinating individuals with whole sporozoites that have been genetically modified to arrest their development at a specific stage in the liver by targeted gene deletion, resulting in a genetically attenuated parasite (GAP). Through a comprehensive phenotyping screen, we identified the hscb gene, encoding a putative iron-sulfur protein assembly chaperone, as crucial for liver stage development, making it a suitable candidate gene for GAP generation. Parasites lacking Plasmodium berghei HscB (PbHscB) exhibited normal sporozoite production in mosquitoes, but their liver stage development was severely impaired, characterized by slow growth and delayed expression of merozoite surface protein 1 (MSP1). In vivo experiments demonstrated that PbHscB-deficient parasites exhibited a delay in prepatency of 2-4 days, emphasizing the significance of PbHscB for exo-erythrocytic development. Although knockout of PbHscB alone allowed breakthrough infections, it is a potent candidate for a dual gene deletion strategy. PlasMei2, an RNA-binding protein, was previously found to be crucial for the completion of liver stage development. We generated a PbHscB-PbMei2-double attenuated parasite line, serving as a late liver stage-arresting replication-competent (LARC) GAP, providing a solid block of liver-to-blood stage transition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schmid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The epidemiological behavior of Plasmodium vivax malaria occurs across spatial scales including within-host, population, and metapopulation levels. On the within-host scale, P. vivax sporozoites inoculated in a host may form latent hypnozoites, the activation of which drives secondary infections and accounts for a large proportion of P. vivax illness; on the metapopulation level, the coupled human-vector dynamics characteristic of the population level are further complicated by the migration of human populations across patches with different malaria forces of (re-)infection. To explore the interplay of all three scales in a single two-patch model of Plasmodium vivax dynamics, we construct and study a system of eight integro-differential equations with periodic forcing (arising from the single-frequency sinusoidal movement of a human sub-population). Under the numerically-informed ansatz that the limiting solutions to the system are closely bounded by sinusoidal ones for certain regions of parameter space, we derive a single nonlinear equation from which all approximate limiting solutions may be drawn, and devise necessary and sufficient conditions for the equation to have only a disease-free solution. Our results illustrate the impact of movement on P. vivax transmission and suggest a need to focus vector control efforts on forest mosquito populations. The three-scale model introduced here provides a more comprehensive framework for studying the clinical, behavioral, and geographical factors underlying P. vivax malaria endemicity., Competing Interests: Declarations. Conflict of interest: The authors have no conflict of interest to declare., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Malaria remains a threat in sub-Saharan Africa, particularly in Côte d'Ivoire, where it is endemic and represents the leading cause of hospital consultations, morbidity and mortality. The strong climatic variations that exist between coastal and savannah areas of Côte d'Ivoire suggest that vector control interventions should be scheduled according to the eco-epidemiological diversity. This study evaluates bioecological parameters of vectors and malaria transmission in two health districts, one coastal and one central of Côte d'Ivoire., Methods: The study was conducted in the coastal (Jacqueville) and savannah (Béoumi) areas of Côte d'Ivoire from November 2018 to March 2019. Human Landing Catches (HLC) were conducted monthly at the study sites to determine Anopheles vector species composition, biting behaviour as well as entomological parameters of malaria transmission. Mosquitoes were collected over 12 h, from 6:00 pm to 6:00 am during 2 days per month. Mosquitoes infectivity was revealed by enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum circumsporozoite protein. A random sample of 100 Anopheles gambiae sensu lato (s.l.) including all CSP-positive females, were further classified by polymerase chain reaction (PCR) at the species and molecular form levels., Results: In Jacqueville, 853 (99.7%) An. gambiae s.l., and 3 (0.35%) Anopheles pharoensis were collected. In Béoumi, 811 (96.3%) An. gambiae s.l., 23 (2.73%) Anopheles funestus and 8 (0. 95%) An. pharoensis have been found. Anopheles coluzzii represented the only species of the An. gambiae complex in Jacqueville. Among the An. gambiae s.l. samples tested in Beoumi, 29 (58%) were An. coluzzii and the rest 21 (42%) was An. gambiae sensu stricto. The human biting rate (HBR) in Jacqueville increased from 5.7 (b/p/n) in November to 17.3 (b/p/n) in March. Conversely in Béoumi the HBR decreased from 16.4 (b/p/n) in November to 0.69 (b/p/n) in March. In Jacqueville, the entomological inoculation rate (EIR) varies from 0.21 to 0.56 (ib/p/n) with the pic of 0.56 (ib/p/n) in February. In Béoumi no infection was detected in the parous An. gambiae s.l. samples tested during the study period., Conclusions: This study evaluates bioecological parameters of vectors and malaria transmission in two health districts, one coastal and one central of Côte d'Ivoire., Competing Interests: Declarations. The findings and conclusions expressed herein are those of the author(s) and do not necessarily represent the official position of neither USAID nor PMI, nor those of Centers for Disease Control and Prevention (CDC). Ethics approval and consent to participate: The study protocol has been approved by the Côte d'Ivoire national ethics committee prior to the start of the experiments. The study protocol has been approved by the local health and administrative authorities. Before starting the study, the village chiefs gave their oral informed consent. In addition, all entomological surveys and sample collections carried out on private land or private residential areas were carried out with the authorization and informed written consent of the residents. Volunteers participating in the HLC have given written informed consent for their participation. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: The high burden of malaria in Africa is largely due to the presence of competent and adapted Anopheles vector species. With invasive Anopheles stephensi implicated in malaria outbreaks in Africa, understanding the genomic basis of vector-parasite compatibility is essential for assessing the risk of future outbreaks due to this mosquito. Vector compatibility with P. falciparum arises from ancient coevolution and involves genes such as Pfs47 in P. falciparum and P47Rec in Anopheles. Questions remain about whether sub-continental vector variation is a selective pressure on current Plasmodium populations., Methods: We analyzed the genetic diversity in parasite-vector interaction genes in P. falciparum and An. gambiae from 9 and 15 countries in Africa, respectively. Specifically, we looked for evidence of malaria vector-mediated selection within three P. falciparum genes (Pfs47, Pfs16, Pfs37) and conducted association analyses with occurrence probabilities of prominent malaria vectors., Results: Higher protein haplotype diversities of Pfs47 and Pfs16 were associated with the probability of occurrence of An. arabiensis and An. funestus together. Only Pfs16 carried a signature of positive selection consistently (average Tajima's D = -2.96), which was associated with the probability of occurrence of An. funestus. These findings support vector-mediated selection on the basis of vector species diversity that may be occurring within Africa. We also employed phylogenetic analyses of An. gambiae interaction genes (P47Rec, APN1, HPX15) to identify significant subspecies diversity as a prerequisite to vector-population-mediated selection. Anopheles gambiae HPX15 revealed significant within-species differentiation (multiple branches bootstrap > 70) compared with absence of variation in P47Rec, suggesting that further investigation into subspecies-mediated selection on the basis of HPX15 is needed. Finally, we observed five amino acid changes at P47Rec in invasive An. stephensi compared with dominant African Anopheles species, calling for further investigation of the impact these distinct P47Rec variants might have on local African P. falciparum Pfs47 diversity., Conclusions: Overall, these findings suggest that vector variation within Africa could influence P. falciparum diversity and lay a genomic framework for future investigation of invasive An. stephensi's impact on African malaria., Competing Interests: Declarations. Ethics approval and consent to participate: Wild mosquitoes used for this study were collected from dwellings and animal houses, following homeowners’ verbal consent. The study protocol for the collection was reviewed by the Centers for Disease Control and Prevention, USA, and determined to be research not involving human subjects (2017-227). A material transfer agreement was established with Baylor University and the US PMI/Abt Associates for molecular analysis of mosquitoes. Competing interests: The authors declare no competing interests. Consent for publication: Not applicable., (© 2024. The Author(s).)

Background: Ethiopia continues to grapple with a persistent malaria burden, characterized by ongoing transmission and recurrent outbreaks. Human behavior influences both malaria exposure and the effectiveness of vector interventions, complicating malaria control efforts. Implementing tailored strategies that account for the complex interplay between human activities and vector behavior remains a challenge in both high- and low-transmission areas in Ethiopia, particularly for vulnerable highland populations and temporary labor migrants, due to lack of data. The aim of this study was to examine the spatiotemporal patterns of human-mosquito interactions and evaluate the effectiveness and suitability of long-lasting insecticidal nets (LLINs) in settings involving lowland resident populations, seasonal migrant workers and highland communities., Methods: Concurrent human and vector behavior data were collected from high-transmission lowlands (residents and temporary migrant workers) and vulnerable highlands populations. Hourly human behavior observations (HBOs), which examined LLIN use, indoor versus outdoor human presence and sleeping patterns, were paired in a crossover design with mosquito sampling using US Centers for Disease Control light traps (CDC LT) as a proxy for mosquito biting behavior. The study was conducted during the peak (October-December 2022) and minor (March-May 2023) malaria transmission seasons ('peak' and 'minor') for a total of 368 nights. In the highlands, four villages consisting of eight households per village were selected for surveillance; in the lowlands, four villages consisting of two resident villages and two farm sites with migrant workers, with eight households/structures per village or farm, were used for data collection. CDC LT and HBO data were integrated to evaluate HBO-adjusted human biting rates (HBO-adjusted HBR) of Anopheles mosquitoes., Results: In the highland villages, residents predominantly engaged in indoor activities, with their peak activity overlapping with the peak biting hours (1800-2200 hours). A substantial proportion of inhabitants slept indoors without LLINs in the peak and minor seasons (42.8% and 39.2%, respectively). Highland residents were significantly more exposed to malaria vectors indoors (88.4% peak, 88.6% minor) than outdoors during both transmission seasons. In lowland villages, both resident and seasonal migrant worker populations exhibited predominantly outdoor activity, particularly during peak biting hours (1800-2300 hours). Both residents and temporary migrants were significantly more exposed to Anopheles mosquitoes outdoors (resident: 65.0% peak, 67.1% minor; migrant: 70.5% peak, 80.0% minor) than indoors during both transmission seasons. LLIN usage was minimal and offered limited protection, with < 16.63% of person-time spent under nets by resident populations and 10.7% by migrant workers., Conclusions: Malaria control in Ethiopia requires context-specific strategies tailored to diverse ecological settings that consider the impact of human behavior on exposure to Anopheles mosquitoes. Limited LLIN effectiveness, human activities coinciding with peak biting times and minimal LLIN usage create significant protection gaps. Comprehensive control necessitates supplemental tools addressing exposure in all locations and times. In the Ethiopian highlands, where indoor activities predominate, increased LLIN usage combined with targeted indoor residual spraying could reduce transmission. In lowland areas, both residents and seasonal migrant workers face relatively higher outdoor exposure risks, requiring additional measures, such as topical and spatial repellents. We recommend implementing data-driven, hyperlocal approaches based on specific human-vector interactions to enhance malaria control effectiveness across the Ethiopian highlands and lowlands., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval for the study was obtained from the National Research Ethical Review Committee (NRERC), Addis Ababa, Ethiopia (reference number: 02/256/630/14), AHRI/ALERT Ethics Review Committee (protocol number: P0-08-22) and Aklilu Lemma Institute of Pathobiology Institutional Research Ethics Review Committee (reference number: ALIPB IRERC/111/2015/23). The study received a non-research determination from PATH. Participation in the study was voluntary. Permission was obtained from the household owners and the mosquito collectors of each study village. All methods used in this study were performed in accordance with the relevant guidelines and regulations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Lipid metabolism is an essential component in reproductive physiology. While lipid mobilization has been implicated in the growth of Plasmodium falciparum malaria parasites in their Anopheles vectors, the role of this process in the reproductive biology of these mosquitoes remains elusive. Here, we show that impairing lipolysis in Anopheles gambiae, the major malaria vector, leads to embryonic lethality. Embryos derived from females in which we silenced the triglyceride lipase AgTL2 or the lipid storage droplet AgLSD1 develop normally during early embryogenesis but fail to hatch due to severely impaired metabolism. Embryonic lethality is efficiently recapitulated by exposing adult females to broad-spectrum lipase inhibitors prior to blood feeding, unveiling lipolysis as a potential target for inducing mosquito sterility. Our findings provide mechanistic insights into the importance of maternal lipid mobilization in embryonic health that may inform studies on human reproduction., Competing Interests: A patent application covering the concept of the application of lipase inhibitors to induce sterility in mosquitoes is being processed on behalf of F.C. and M.A.I. by the President and Fellows of Harvard University. The authors state that they have no other competing interests., (Copyright: © 2024 Itoe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Despite consecutive decades of success in reducing malaria transmission, Ethiopia went off track towards its goal of malaria elimination by 2030, as outlined in the NMCP malaria strategy. Recent malaria outbreaks in Ethiopia are attributed to the emergence and spread of diagnostic and drug-resistant Plasmodium falciparum, increased insecticide resistance in major vectors and the spread of invasive Anopheles stephensi. The effects of the COVID-19 pandemic, environmental anomalies and internal conflicts have also potentially played a role in increasing malaria transmission. This study aimed to evaluate the contribution of environmental factors and An. stephensi to the spatiotemporal trends of recent malaria cases in Ethiopia., Methods: Clinical malaria case data reported weekly between January 2013 and January 2023 were obtained from the Ethiopian Public Health Institute (EPHI), Addis Ababa. A negative binomial regression model was used to explain the variability and potential overdispersion in the weekly number of malaria cases reported across Ethiopian administrative zones. This model incorporated fixed effects for selected environmental factors and random effects to capture temporal trends, zone specific seasonal patterns, spatial trends at the zone level, and the presence of An. stephensi and its impact., Results: Our negative binomial regression model highlighted 56% variability in the data and slightly more than half (55%) was due to environmental factors, while the remainder was captured by random effects. A significant nationwide decline in malaria risk was observed between 2013 and 2018, followed by a sharp increase in early 2022. Malaria risk was higher in western and northwestern zones of Ethiopia compared to other zones. Zone-specific seasonal patterns, not explained by environmental factors, were grouped into four clusters of seasonal behaviours. The presence of An. stephensi was not shown to have any significant impact on malaria risk., Conclusions: Understanding the spatial and temporal drivers of malaria transmission and therefore identifying more appropriate malaria control strategies are key to the success of any malaria elimination and eradication programmes in Ethiopia. Our study found that approximately 50% of malaria risk variability could be explained by environmental, temporal, and spatial factors included in the analysis, while the remaining variation was unexplained and may stem from other factors not considered in this study. This highlights the need for a better understanding of underlying factors driving local malaria transmission and outbreaks, to better tailor regional programmatic responses., Competing Interests: Declarations. Ethics approval and consent to participate: The study is part of the CEASE (Controlling emergent An. stephensi in Sudan and Ethiopia), the umbrella Project received approval from the National (Ref: 02/256/630/14) and the ALERT/AHRI (Ref: P0-08-22) research ethics committees, Addis Ababa, Ethiopia. This manuscript used case data made readily available by the Ethiopian Federal Ministry of Health malaria programme (PHEM), thus consent was not necessary. Consent for publication: Not applicable. Competing interests: No competing interests were declared by the authors., (© 2024. The Author(s).)

Vector mosquito control is an integral part of malaria control. The global emergence of insecticide resistance in malaria-transmitting Anophelines has become an impediment and has created an urgent need for novel mosquito control approaches. Here, we show that a biopesticide derived from the soil-dwelling bacterium Chromobacterium sp. Panama ( Csp&#95;P ) kills insecticide-resistant Anopheles mosquitoes, regardless of their resistance mechanisms. In addition, sublethal dose of Csp&#95;P acts as a synergist to now used chemical insecticides across multiple classes. Moreover, Csp&#95;P reduces host-seeking behavior and malaria parasite infection in vector mosquitoes in ways that further decrease transmission. Mosquito glutathione S -transferases are essential for Csp&#95;P 's mosquito-killing mechanism. Enclosed field trials in Burkina Faso, conducted in diverse ecological settings and supported by a mathematical model, have now demonstrated its potential for malaria control in settings with widespread insecticide resistance.

Background: Residual malaria transmissions in Africa may be associated with improved coverage of insecticide-treated nets, house features, and livestock husbandry. These human-land use activities may drive the ecology and behaviour of malaria vectors which sustain residual malaria transmission. This study was conducted to assess changes in the ecology and behaviour of Anopheles funestus and Anopheles arabiensis in villages with high coverage of insecticide-treated nets to guide the selection of complementary vector control strategies against residual malaria transmission., Methods: Mosquitoes were collected using a CDC-light trap, miniaturized double net trap, and Prokopack aspirator from 222 households in three villages (Ebuyu, Chirombora, and Mzelezi) within Kilombero Valley. Anopheles mosquitoes were morphologically identified to their physiological status and species-complex levels. A sub-sample of Anopheles mosquitoes was exposed to laboratory analyses of sibling species, host preference, and sporozoite rates. Additionally, the local houses were geo-referenced using Global Positioning Systems (GPS) devise, and house features were recorded and associated with vector abundance., Results: The population of An. funestus s.s was abundant with high Plasmodium sporozoite rates inside houses compared to An. arabiensis. However, these vector species equally blood-fed on humans inside houses, but they also flexibly mixed human and animal blood meal. Fewer An. funestus were caught in houses with metal- than grass roofs and houses with and without animals. Contrastingly, fewer An. arabiensis were caught from houses with screened eaves compared to houses with open eaves., Conclusions: This study confirms that An. funestus dominates residual malaria transmission over An. arabiensis. These vector species exhibit anthropophily and opportunistic blood-feeding behaviour in areas with high coverage of insecticide-treated nets, but they numerically respond differently to local house improvements. These results imply that integrating mosquito-proof houses, improved insecticide-treated nets, and livestock-based interventions could effectively reduce and eventually eliminate residual malaria transmission., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Limwagu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Ethiopia faces several severe challenges in terms of malaria elimination, including drug resistance and diagnostic evasion in the Plasmodium falciparum parasite, insecticide resistance in the primary Anopheles malaria vector, and, most recently, the invasion of the Asian malaria vector Anopheles stephensi. Novel malaria control methods are therefore needed, and in this paper, we describe the evaluation of a larval source management (LSM) strategy implemented in response to An. stephensi. The primary outcome was the malaria incidence rate compared between intervention and non-intervention sites in the presence of An. stephensi., Methods: Intervention (Batu and Dire Dawa) and control (Metehara) towns were selected, and weekly malaria passive case detection data collected between 2014 and 2023 were obtained from the Oromia regional state and Dire Dawa City Administration Health Bureau. In addition, data regarding intervention were obtained from the President's Malaria Initiative (PMI) reports. Weekly malaria passive case data were used to evaluate the change in the estimated malaria incidence rate and trends of temporal patterns of the estimated malaria incidence rate before and after interventions. An interrupted time series model with a cyclic second-order random walk structure periodic seasonal term was used to assess the impact of LSM on malaria incidence rate in the intervention and control settings., Results: An upsurge in malaria cases occurred after 2020 at both the intervention and control sites. The temporal patterns of malaria incidence rate showed an increasing trend after the intervention. The ITS model depicted that the LSM has no impact in reducing the malaria incidence rate at both intervention site Dire Dawa [immediate impact = 1.462 (0.891, 2.035)], [Lasting impact = 0.003 (- 0.012, 0.018)], and Batu [Immediate impact 0.007 (- 0.235, 0.249), [Lasting impact = 0.008 (- 0.003, 0.013)]., Conclusions: An overall increasing trend in the malaria incidence rate was observed irrespective of the implementation of LSM in the urban settings of Ethiopia, where An. stephensi has been found. Further investigations and validations of the incorporation of LSM into control activities are warranted., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Urbanization and microclimate variation in cities can influence mosquito behavior and parasite development, thus affecting malaria transmission. This study investigates how the impact of microclimate variations due to household roof types can aid in the survival of Anopheles stephensi and the development of Plasmodium vivax in an urban slum setting. Understanding these vital environmental interactions is essential for devising effective control strategies to achieve malaria elimination. Anopheles stephensi (F1) mosquitoes were membrane-fed with blood collected from P. vivax-infected patients before (day 0) and during (day 1) antimalarial treatment. The parasite development and mosquito survival were monitored in simulated microclimatic conditions of a variety of household roof types (thatched, asbestos, tiled) against standard laboratory conditions. Mosquito dissections were undertaken to detect oocysts and sporozoites in An. stephensi mosquitoes (oocyst: day 3-5, sporozoites: day 7-11). The maximum number of oocysts were detected in infected mosquitoes in thatched-roof conditions, whereas the largest oocyst was in the asbestos roof type. Circumsporozoite-ELISA results indicated the presence of sporozoites in infected mosquitoes for up to 29 days under standard conditions, 18 days in thatched-roof and asbestos roof conditions, and 14 days in tiled conditions. The univariate binary logistic regression model indicated a significant influence of microclimatic conditions of thatched roofs on parasite development. The Kaplan-Meier survival analysis revealed that the median survival of P. vivax-infected An. stephensi in thatched-roof conditions was 14 days, followed by asbestos (11 days) and tiled (10 days) roof conditions. In conclusion, thatched-roof houses were favorable for the development and survival of P. vivax-infected An. stephensi.

Malaria remains one of the most important infectious diseases globally due to its high incidence and mortality rates. The influx of infected cases from endemic to non-endemic malaria regions like Europe has resulted in a public health concern over sporadic local outbreaks. This is facilitated by the continued presence of competent Anopheles vectors in non-endemic countries.We modelled the potential distribution of the main malaria vector across Spain using the ensemble of eight modelling techniques based on environmental parameters and the Anopheles maculipennis s.l. presence/absence data collected from 2000 to 2020. We then combined this map with the number of imported malaria cases in each municipality to detect the geographic hot spots with a higher risk of local malaria transmission.The malaria vector occurred preferentially in irrigated lands characterized by warm climate conditions and moderate annual precipitation. Some areas surrounding irrigated lands in northern Spain (e.g. Zaragoza, Logroño), mainland areas (e.g. Madrid, Toledo) and in the South (e.g. Huelva), presented a significant likelihood of A. maculipennis s.l. occurrence, with a large overlap with the presence of imported cases of malaria.While the risk of malaria re-emergence in Spain is low, it is not evenly distributed throughout the country. The four recorded local cases of mosquito-borne transmission occurred in areas with a high overlap of imported cases and mosquito presence. Integrating mosquito distribution with human incidence cases provides an effective tool for the quantification of large-scale geographic variation in transmission risk and pinpointing priority areas for targeted surveillance and prevention.

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.

Background: The use of microsporidia as a disease-transmission-blocking tool has garnered significant attention. Microsporidia sp. MB, known for its ability to block malaria development in mosquitoes, is an optimal candidate for supplementing malaria vector control methods. This symbiont, found in Anopheles mosquitoes, can be transmitted both vertically and horizontally with minimal effects on its mosquito host. Its genome, recently sequenced from An. arabiensis, comprises a compact 5.9 Mbp., Results: Here, we analyze the Microsporidia sp. MB genome, highlighting its major genomic features, gene content, and protein function. The genome contains 2247 genes, predominantly encoding enzymes. Unlike other members of the Enterocytozoonida group, Microsporidia sp. MB has retained most of the genes in the glycolytic pathway. Genes involved in RNA interference (RNAi) were also identified, suggesting a mechanism for host immune suppression. Importantly, meiosis-related genes (MRG) were detected, indicating potential for sexual reproduction in this organism. Comparative analyses revealed similarities with its closest relative, Vittaforma corneae, despite key differences in host interactions., Conclusion: This study provides an in-depth analysis of the newly sequenced Microsporidia sp. MB genome, uncovering its unique adaptations for intracellular parasitism, including retention of essential metabolic pathways and RNAi machinery. The identification of MRGs suggests the possibility of sexual reproduction, offering insights into the symbiont's evolutionary strategies. Establishing a reference genome for Microsporidia sp. MB sets the foundation for future studies on its role in malaria transmission dynamics and host-parasite interactions., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Despite the progress in scaling vector control interventions in Ethiopia, malaria is still a major health problem in the country. Monitoring of the local vector populations and the effectiveness of vector control strategies is necessary to guide programme decisions to optimize malaria prevention efforts. This study investigated the feeding preference, the biting behaviour and resting behaviours of Anopheles mosquitoes in selected localities of Dembiya District., Methods: Adult Anopheles mosquitoes were sampled indoors and outdoors from June 2018 to May 2019 using CDC light traps, pyrethrum spray catches, artificial pit shelters, and mouth aspirators at both Guramba Bata and Arebiya study sites. Anopheles mosquitoes were identified to the species level. Their blood meal source and Plasmodium sporozoite infections were determined using an enzyme-linked immunosorbent assay., Results: Anopheles mosquitoes belonging to 11 species were identified from 2,055 collected mosquito specimens. Anopheles pharoensis was the predominant species at both the Guramba Bata (46.5%) and Arebiya (46.2%) study sites. The CDC light traps caught the highest number of Anopheles mosquitoes in both study sites. In Guramba Bata the density of outdoor host-seeking and resting Anopheles mosquitoes were higher than indoors (P ≤ 0.05). The human blood indexes (HBI) of indoor and outdoor host-seeking Anopheles arabiensis were 17.4% and 15.3%, respectively. The entomological inoculation rate (EIR) of outdoor host-seeking An. arabiensis was 4.7 infective bites/person/year. Additionally, the outdoor EIR of host-seeking Anopheles coustani was 25.7ib/p/year., Conclusions: Anopheles mosquitoes in Dembiya district were more likely to seek a host and rest outdoors than indoors. A reevaluation of vector control strategies is needed to ensure Ethiopia remains on the path to malaria elimination. The detection of Plasmodium circumsporozoite protein in potential secondary vectors, such as An. coustani requires further investigation to substantiate their role in malaria transmission., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical clearance was obtained from Addis Ababa University by the institutional ethical review board of the College of Natural and Computational Sciences (Ref. No. CNSDO/692/10/2018). Written consent was obtained from the head of the household and other study participants. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Significant effort and resources have been invested to control malaria transmission in sub-Saharan Africa, but it remains a major public health problem. For the parasite to be transmitted, the female Anopheles vector must survive 10-14 days following an infective bite to allow Plasmodium gametocytes to develop into infectious sporozoites. The goal of this study was to assess factors associated with wild-caught Anopheles survival and infection following host-seeking and indoor resting., Methods: The study was conducted between January 2020 to March 2022 in a longitudinal cohort of 75 households in 5 villages including a total of 755 household members in Bungoma County, Kenya. Monthly adult mosquito collection was conducted by attenuated aspiration in all enrolled households, and mosquitoes were reared for 7 days. The daily mortality rate was determined through day 7. All mosquitoes were morphologically identified. Female Anopheles were dissected, and species-level members of the Anopheles gambiae complex were resolved by molecular methods. The abdomens of all samples were processed for Plasmodium falciparum oocyst detection by PCR., Results: Within a 25-month period, the total numbers of non-Anopheles and Anopheles mosquitoes collected indoors were 12,843 and 712, respectively. Anopheles gambiae and An. funestus were the major vectors, though their distributions varied between different villages; 61.2% (n = 436/712) of the Anopheles mosquitoes survived up to day 7, with the lowest mortality rate recorded on day 5 of captivity. The survival rate also varied between the different Anopheles species. Six hundred eighty-three of 712 mosquito abdomens were tested for P. falciparum; 7.8% (53/683) tested positive for P. falciparum, with An. funestus having a higher (10%) prevalence than An. gambiae s.s. (6.0%, p = 0.095, Pearson Chi-square test). The proportion of household members sleeping under a bednet the night before mosquito collection varied across time and village. Anopheles funestus survival times were refractory to household ITN usage, and An. gambaie s.s. survival was reduced only under very high (100%) ITN usage., Conclusions: Despite ITN usage, mosquitoes still acquired blood meals and P. falciparum infections. Survival differed across species and was inversely correlated with high ITN usage in the household but not oocyst development., Competing Interests: Declarations Ethics approval and consent to participate Ethical approval was granted by Moi University Institutional Research and Ethics Committee (Formal Approval No. 0001863) and Duke University Institutional Review Board (Pro00082000). Written informed consent was obtained the household heads and participants prior to data and mosquito collection. All study methods were carried out in accordance with ethical guidelines and regulations set out by both Moi University and Duke Ethical Review Boards. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Microsporidia MB is a promising candidate for developing a symbiont-based strategy for malaria control because it disrupts the capacity of An. arabiensis to transmit the Plasmodium parasite. The symbiont is predominantly localized in the reproductive organs and is transmitted vertically from mother to offspring and horizontally (sexually) during mating. Due to the contribution of both transmission routes, Microsporidia MB has the potential to spread through target vector populations and become established at high prevalence. Stable and efficient vertical transmission of Microsporidia MB is important for its sustainable use for malaria control, however, the vertical transmission efficiency of Microsporidia MB can vary. In this study, we investigate the mechanistic basis of Microsporidia MB vertical transmission in An. arabiensis. We show that vertical transmission occurs through the acquisition of Microsporidia MB by Anopheles cystocyte progenitors following the division of germline stem cells. We also show that Microsporidia MB replicates to increase infection intensity in the oocyte of developing eggs when mosquitoes take a blood meal suggesting that symbiont proliferation in the ovary is coordinated with egg development. The rate of Microsporidia MB transmission to developing eggs is on average higher than the recorded (mother to adult offspring) vertical transmission rate. This likely indicates that a significant proportion of An. arabiensis offspring lose their Microsporidia MB symbionts during development. The stability of germline stem cell infections, coordination of symbiont proliferation, and very high rate of transmission from germline stem cells to developing eggs indicate that Microsporidia MB has a highly specialized vertical transmission strategy in An. arabiensis, which may explain host specificity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Onchuru et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rwanda achieved unprecedented malaria control gains from 2000 to 2010, but cases increased 20-fold between 2011 and 2017. Vector control challenges and environmental changes were noted as potential explanations, but no studies have investigated causes of the resurgence or identified which vector species drove transmission. We conducted a retrospective study in four sites in eastern Rwanda that conducted monthly entomological surveillance and outpatient malaria care. We compared sporozoite rates, human blood index (HBI), and relative abundance of the primary vectors, Anopheles gambiae and Anopheles arabiensis, from 2017 to 2020. We then modeled the effects of vector control interventions, insecticide resistance, and temperature changes on species composition and reported malaria incidence. Sporozoite rates were 28 times higher and HBI was four times higher in An. gambiae compared with An. arabiensis. Insecticide-treated bed nets, first distributed nationally in 2010, were associated with decreased An. gambiae relative abundance. However, increased pyrethroid resistance was associated with increased An. gambiae relative abundance and malaria incidence. Epidemic malaria peaks corresponded to periods of model-predicted An. gambiae re-emergence, and increased regional air temperatures during the period were further associated with increased malaria incidence. Indoor residual spraying (IRS), implemented with non-pyrethroid insecticides later in the period, was associated with 86% reductions in An. gambiae relative abundance and 75% reductions in malaria incidence. These findings suggest that increased pyrethroid resistance and the re-emergence of An. gambiae were closely linked to the malaria resurgence in eastern Rwanda. Non-pyrethroid IRS or other control measures that effectively target An. gambiae may help prevent future resurgences.

Anophelinae mosquitoes are exposed to a variety of microbes including Plasmodium parasites that cause malaria. When infected, mosquitoes mount versatile immune responses, including the production of antimicrobial peptides. Cecropins are one of the most widely distributed families of antimicrobial peptides in insects and all previously studied Anopheles members are playing roles in adult mosquito immunity. We have identified and characterized a novel member of the Anopheles gambiae cecropin family, cecropin D (CecD), that is uniquely expressed and immune-responsive at late larval stages to promote microbial clearance through its broad-spectrum antibacterial activity during larval-pupal developmental transition. Interestingly, Cecropin D also exhibited highly potent activity against Plasmodium falciparum sporozoites, the malaria parasite stage that is transmitted from mosquitoes and infects humans and thereby holds promise as a malaria transmission-blocking agent. Finally, we have defined unequivocal cecropin-specific molecular signatures to systematically organize the diversity of the cecropin family in malaria vectors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barreto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gene drives are a promising means of malaria control with the potential to cause sustained reductions in transmission. In real environments, however, their impacts will depend on local ecological and epidemiological factors. We develop a data-driven model to investigate the impacts of gene drives that causes vector population suppression. We simulate gene drive releases in sixteen ~ 12,000 km 2 areas of west Africa that span variation in vector ecology and malaria prevalence, and estimate reductions in vector abundance, malaria prevalence and clinical cases. Average reductions in vector abundance ranged from 71.6-98.4% across areas, while impacts on malaria depended strongly on which vector species were targeted. When other new interventions including RTS,S vaccination and pyrethroid-PBO bednets were in place, at least 60% more clinical cases were averted when gene drives were added, demonstrating the benefits of integrated interventions. Our results show that different strategies for gene drive implementation may be required across different African settings., (© 2024. The Author(s).)

Malaria parasites hijack the metabolism of their mammalian host during the blood-stage cycle. Anopheles mosquitoes depend on mammalian blood to lay eggs and to transmit malaria parasites. However, it remains understudied whether changes in host metabolism affect parasite transmission in mosquitoes. In this study, we discovered that Plasmodium infection significantly decreased the levels of the tryptophan metabolite, 5-hydroxytryptamine (5-HT), in both humans and mice. The reduction led to the decrease of 5-HT in mosquitoes. Oral supplementation of 5-HT to Anopheles stephensi enhanced its resistance to Plasmodium berghei infection by promoting the generation of mitochondrial reactive oxygen species. This effect was due to the accumulation of dysfunctional mitochondria caused by 5-HT-mediated inhibition of mitophagy. Elevating 5-HT levels in mouse serum significantly suppressed parasite infection in mosquitoes. In summary, our data highlight the critical role of metabolites in animal blood in determining the capacity of mosquitoes to control parasite infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., (© 2024. The Author(s).)

The cortical cytoskeleton of subpellicular microtubules (SPMTs) supports the Plasmodium ookinete morphogenesis during mosquito transmission of malaria. SPMTs are hypothesized to function as the cytoskeletal tracks in motor-driven cargo transport for apical organelle and structure assembly in ookinetes. However, the SPMT-based transport motor has not been identified in the Plasmodium. The cytoplasmic dynein is the motor moving towards the minus end of microtubules (MTs) and likely be responsible for cargo transport to the apical part in ookinetes. Here we screen 7 putative dynein heavy chain (DHC) proteins in the P. yoelii and identify DHC3 showing peripheral localization in ookinetes. DHC3 is localized at SPMTs throughout ookinete morphogenesis. We also identify five other dynein subunits localizing at SPMTs. DHC3 disruption impairs ookinete development, shape, and gliding, leading to failure in mosquito infection of Plasmodium. The DHC3-deficient ookinetes display defective formation or localization of apical organelles and structures. Rab11A and Rab11B interact with DHC3 at SPMTs in a DHC3-dependent manner, likely functioning as the receptors for the cargoes driven by SPMT-dynein. Disturbing Rab11A or Rab11B phenocopies DHC3 deficiency in ookinete morphogenesis. Our study reveals an SPMT-based dynein motor driving the transport of Rab11A- and Rab11B-labeled cargoes in the ookinete morphogenesis of Plasmodium., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background Objectives: Anopheles funestus is playing an increasingly important role in malaria transmission in sub-Saharan Africa. Trehalase, an enzyme required for trehalose breakdown, is important for mosquito flight and stress adaptation. Hence, its inhibition has emerged as a promising malaria management strategy., Methods: A collection of 1900 natural compounds from the ZINC database were screened against the 3D modeled structure of An. funestus trehalase protein using in silico tools. ADMET-AI, a web-based platform, was used to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the selected compounds., Results: We report 5 natural compounds namely, ZINC00488388, ZINC00488525, ZINC00488566, ZINC00488304, and ZINC00488456 that demonstrated strong binding affinity to the trehalase protein. These compounds interacted with critical residues of the trehalase protein and exhibited good drug-like characteristics., Interpretation Conclusion: These compounds show promise as trehalase protein inhibitors for malaria management. Nonetheless, additional experimental studies are required to optimize these compounds as potential trehalase inhibitors., (Copyright © 2024 Journal of Vector Borne Diseases.)

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., (© 2024. The Author(s).)

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 (EC 50 ) via pre-exposure was 0.7 mg/m 2 , 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., (© 2024. The Author(s).)

The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host's hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Human serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX ™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality., Methods: Several commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity., Results: Supplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS-X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS-X supplementation., Conclusions: Serum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays., (© 2024. The Author(s).)

Anopheles jamesii, considered to be a non-malaria vector, is an abundant mosquito species found in coastal India and several countries in Asia. In Goa, western India, An. jamesii is the dominant anopheline species by numbers, and in recent epidemiological surveys, they have tested positive for Plasmodium carriage. To assess An. jamesii's capacity as a malaria vector, we carried out six controlled membrane feeding assays with the F1 generation of field-caught An. jamesii and Plasmodium vivax-infected patient blood samples. As a control, the established local vector, Anopheles stephensi, was fed in parallel to compare the oocyst and sporozoite loads. The average oocyst load of An. jamesii was 3.29, while for An. stephensi it was 10.23. Furthermore, An. jamesii's blood feeding rate (21.7% versus 85%), oocyst positivity rate (60% versus 90.6%), and sporozoite positivity (45.16% versus 83%) were significantly lower (P <0.05, unpaired t test) than those of An. stephensi, suggesting a recent adaptation to support Plasmodium sporogony.

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., (© 2024. The Author(s).)

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax . The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections., Competing Interests: The authors declare no conflict of interest.