Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

New insight into avian malaria vectors in New Zealand.

BACKGROUND: Mosquitoes (Culicidae) are vectors for most malaria parasites of the Plasmodium species and are required for Plasmodium spp. to complete their life cycle. Despite having 16 species of mosquitoes and the detection of many Plasmodium species in birds, little is known about the role of different mosquito species in the avian malaria life cycle in New Zealand. METHODS: In this study, we used nested polymerase chain reaction (PCR) and real-time PCR to determine Plasmodium spp. prevalence and diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across ten sites on the North Island of New Zealand during 2012-2014. The mosquitoes were pooled by species and location collected, and the thorax and abdomens were examined separately for Plasmodium spp. DNA. Akaike information criterion (AIC) modeling was used to test whether location, year of sampling, and mosquito species were significant predictors of minimum infection rates (MIR). RESULTS: We collected 788 unengorged mosquitoes of six species, both native and introduced. The most frequently caught mosquito species were the introduced Aedes notoscriptus and the native Culex pervigilans. Plasmodium sp DNA was detected in 37% of matched thorax and abdomen pools. When considered separately, 33% of abdomen and 23% of thorax pools tested positive by nested PCR. The MIR of the positive thorax pools from introduced mosquito species was 1.79% for Ae. notoscriptus and 0% for Cx. quinquefasciatus, while the MIR for the positive thorax pools of native mosquito species was 4.9% for Cx. pervigilans and 0% for Opifex fuscus. For the overall MIR, site and mosquito species were significant predictors of Plasmodium overall MIR. Aedes notoscriptus and Cx. pervigilans were positive for malaria DNA in the thorax samples, indicating that they may play a role as avian malaria vectors. Four different Plasmodium lineages (SYAT05, LINN1, GRW6, and a new lineage of P (Haemamoeba) sp. AENOT11) were identified in the pooled samples. CONCLUSIONS: This is the first detection of avian Plasmodium DNA extracted from thoraxes of native Culex and introduced Aedes mosquito species in New Zealand and therefore the first study providing an indication of potential vectors in this country.

Malaria transmission heterogeneity in different eco-epidemiological areas of western Kenya: a region-wide observational and risk classification study for adaptive intervention planning.

BACKGROUND: Understanding of malaria ecology is a prerequisite for designing locally adapted control strategies in resource-limited settings. The aim of this study was to utilize the spatial heterogeneity in malaria transmission for the designing of adaptive interventions. METHODS: Field collections of clinical malaria incidence, asymptomatic Plasmodium infection, and malaria vector data were conducted from 108 randomly selected clusters which covered different landscape settings including irrigated farming, seasonal flooding area, lowland dryland farming, and highlands in western Kenya. Spatial heterogeneity of malaria was analyzed and classified into different eco-epidemiological zones. RESULTS: There was strong heterogeneity and detected hot/cold spots in clinical malaria incidence, Plasmodium prevalence, and vector abundance. The study area was classified into four zones based on clinical malaria incidence, parasite prevalence, vector density, and altitude. The two irrigated zones have either the highest malaria incidence, parasite prevalence, or the highest malaria vector density; the highlands have the lowest vector density and parasite prevalence; and the dryland and flooding area have the average clinical malaria incidence, parasite prevalence and vector density. Different zones have different vector species, species compositions and predominant species. Both indoor and outdoor transmission may have contributed to the malaria transmission in the area. Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus s.s., and Anopheles leesoni had similar human blood index and malaria parasite sporozoite rate. CONCLUSION: The multi-transmission-indicator-based eco-epidemiological zone classifications will be helpful for making decisions on locally adapted malaria interventions.

Impact of climate change on temperature variations and extrinsic incubation period of malaria parasites in Chennai, India: implications for its disease transmission potential.

BACKGROUND: The global temperature has significantly risen in the past century. Studies have indicated that higher temperature intensifies malaria transmission in tropical and temperate countries. Temperature fluctuations will have a potential impact on parasite development in the vector Anopheles mosquito. METHODS: Year-long microclimate temperatures were recorded from a malaria-endemic area, Chennai, India, from September 2021 to August 2022. HOBO data loggers were placed in different vector resting sites including indoor and outdoor roof types. Downloaded temperatures were categorised by season, and the mean temperature was compared with data from the same study area recorded from November 2012 to October 2013. The extrinsic incubation period for Plasmodium falciparum and P. vivax was calculated from longitudinal temperatures recorded during both periods. Vector surveillance was also carried out in the area during the summer season. RESULTS: In general, temperature and daily temperature range (DTR) have increased significantly compared to the 2012-2013 data, especially the DTR of indoor asbestos structures, from 4.30 â„ƒ to 12.62 â„ƒ in 2021-2022, unlike the marginal increase observed in thatched and concrete structures. Likewise, the average DTR of outdoor asbestos structures increased from 5.02 â„ƒ (2012-2013) to 8.76 â„ƒ (2021-2022) although the increase was marginal in thatched structures and, surprisingly, showed no such changes in concrete structures. The key finding of the extrinsic incubation period (EIP) is that a decreasing trend was observed in 2021-2022 compared to 2012-2013, mainly in indoor asbestos structures from 7.01 to 6.35 days, which negatively correlated with the current observation of an increase in temperature. Vector surveillance undertaken in the summer season revealed the presence of Anopheles breeding in various habitats. Anopheles stephensi could be collected using CDC light traps along with other mosquito species. CONCLUSION: The microclimate temperature has increased significantly over the years, and mosquitoes are gradually adapting to this rising temperature. Temperature negatively correlates with the extrinsic incubation period of the parasite. As the temperature increases, the development of the parasite in An. stephensi will be faster because of a decrease in EIP, thus requiring relatively fewer days, posing a risk for disease transmission and a hindrance to malaria elimination efforts.

Rapid classification of epidemiologically relevant age categories of the malaria vector, Anopheles funestus.

BACKGROUND: Accurately determining the age and survival probabilities of adult mosquitoes is crucial for understanding parasite transmission, evaluating the effectiveness of control interventions and assessing disease risk in communities. This study was aimed at demonstrating the rapid identification of epidemiologically relevant age categories of Anopheles funestus, a major Afro-tropical malaria vector, through the innovative combination of infrared spectroscopy and machine learning, instead of the cumbersome practice of dissecting mosquito ovaries to estimate age based on parity status. METHODS: Anopheles funestus larvae were collected in rural south-eastern Tanzania and reared in an insectary. Emerging adult females were sorted by age (1-16 days old) and preserved using silica gel. Polymerase chain reaction (PCR) confirmation was conducted using DNA extracted from mosquito legs to verify the presence of An. funestus and to eliminate undesired mosquitoes. Mid-infrared spectra were obtained by scanning the heads and thoraces of the mosquitoes using an attenuated total reflection-Fourier transform infrared (ATR-FT-IR) spectrometer. The spectra (N = 2084) were divided into two epidemiologically relevant age groups: 1-9 days (young, non-infectious) and 10-16 days (old, potentially infectious). The dimensionality of the spectra was reduced using principal component analysis, and then a set of machine learning and multi-layer perceptron (MLP) models were trained using the spectra to predict the mosquito age categories. RESULTS: The best-performing model, XGBoost, achieved overall accuracy of 87%, with classification accuracy of 89% for young and 84% for old An. funestus. When the most important spectral features influencing the model performance were selected to train a new model, the overall accuracy increased slightly to 89%. The MLP model, utilizing the significant spectral features, achieved higher classification accuracy of 95% and 94% for the young and old An. funestus, respectively. After dimensionality reduction, the MLP achieved 93% accuracy for both age categories. CONCLUSIONS: This study shows how machine learning can quickly classify epidemiologically relevant age groups of An. funestus based on their mid-infrared spectra. Having been previously applied to An. gambiae, An. arabiensis and An. coluzzii, this demonstration on An. funestus underscores the potential of this low-cost, reagent-free technique for widespread use on all the major Afro-tropical malaria vectors. Future research should demonstrate how such machine-derived age classifications in field-collected mosquitoes correlate with malaria in human populations.

Case Report: Plasmodium vivax Sporozoite Melanization in the Midgut and Salivary Gland of the Malaria Vector Anopheles darlingi.

Anopheles darlingi is the primary malaria vector in the Amazon region and is highly susceptible to both Plasmodium vivax and Plasmodium falciparum parasites. Although anopheline mosquitoes may develop melanotic encapsulation in response to Plasmodium parasites, there is no record of An. darlingi exhibiting a melanization response to P. vivax, the main malaria parasite in the Americas. Here, we report the occurrence of P. vivax sporozoite melanization in An. darlingi mosquitoes.

The impact of mosquito sampling strategies on molecular xenomonitoring prevalence for filariasis: a systematic review.

Objective: To explore the impact of mosquito collection methods, sampling intensity and target genus on molecular xenomonitoring detection of parasites causing lymphatic filariasis. Methods: We systematically searched five databases for studies that used two or more collection strategies for sampling wild mosquitoes, and employed molecular methods to assess the molecular xenomonitoring prevalence of parasites responsible for lymphatic filariasis. We performed generic inverse variance meta-analyses and explored sources of heterogeneity using subgroup analyses. We assessed methodological quality and certainty of evidence. Findings: We identified 25 eligible studies, with 172 083 mosquitoes analysed. We observed significantly higher molecular xenomonitoring prevalence with collection methods that target bloodfed mosquitoes compared to methods that target unfed mosquitoes (prevalence ratio: 3.53; 95% confidence interval, CI: 1.52-8.24), but no significant difference compared with gravid collection methods (prevalence ratio: 1.54; 95% CI: 0.46-5.16). Regarding genus, we observed significantly higher molecular xenomonitoring prevalence for anopheline mosquitoes compared to culicine mosquitoes in areas where Anopheles species are the primary vector (prevalence ratio: 6.91; 95% CI: 1.73-27.52). One study provided evidence that reducing the number of sampling sites did not significantly affect molecular xenomonitoring prevalence. Evidence of differences in molecular xenomonitoring prevalence between sampling strategies was considered to be of low certainty, due partly to inherent limitations of observational studies that were not explicitly designed for these comparisons. Conclusion: The choice of sampling strategy can significantly affect molecular xenomonitoring results. Further research is needed to inform the optimum strategy in light of logistical constraints and epidemiological contexts.

Avian Plasmodium in invasive and native mosquitoes from southern Spain.

BACKGROUND: The emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife. METHODS: The presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus. RESULTS: Avian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions. CONCLUSIONS: Unlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required.

Global prevalence of Plasmodium infection in wild birds: A systematic review and meta-analysis.

Avian malaria is a vector-borne parasitic disease caused by Plasmodium infection transmitted to birds by mosquitoes. The aim of this systematic review was to analyze the global prevalence of malaria and risk factors associated with infection in wild birds. A systematic search of the databases CNKI, WanFang, VIP, PubMed, and ScienceDirect was performed from database inception to 24 February 2023. The search identified 3181 retrieved articles, of which 52 articles met predetermined inclusion criteria. Meta-analysis was performed using the random-effects model. The estimated pooled global prevalence of Plasmodium infection in wild birds was 16%. Sub-group analysis showed that the highest prevalence was associated with adult birds, migrant birds, North America, tropical rainforest climate, birds captured by mist nets, detection of infection by microscopy, medium quality studies, and studies published after 2016. Our study highlights the need for more understanding of Plasmodium prevalence in wild birds and identifying risk factors associated with infection to inform future infection control measures.

Entomological surveillance reveals transmission of malaria but not lymphatic filariasis in two communities in North-West Nigeria.

Malaria and lymphatic filariasis (LF) are two serious public health challenges in sub-Saharan Africa, and both diseases are transmitted by Anopheles mosquitoes. Successful control of both diseases requires detailed information on transmission dynamics; thus, this study investigated malaria and LF transmission indices in two (2) communities (Jidawa and Kargo) in North-West Nigeria. Anopheles mosquitoes were sampled from twenty-five (25) randomly selected houses from each of the two communities using pyrethrum spray collection (PSC). The samples were identified morphologically and molecularly characterised using polymerase chain reaction (PCR). Human biting rate (HBR), indoor resting density (IRD), sporozoite rate (SR) and entomological inoculation rate (EIR) were calculated using standard formulae. The thorax region of the collected samples were dissected and smeared; then, Plasmodium and Wuchereria bancrofti parasites were identified using microscopy. A total of 2417 Anopheles mosquitoes were collected, and all were identified morphologically as An. gambiae s.l. Further molecular identification of sibling species revealed that An. gambiae and An. arabiensis were the only sibling species present. A total of 818 Anopheles mosquitoes were screened for Plasmodium and Wuchereria bancrofti parasites. A total of 180 samples were positive for Plasmodium parasites (Jidawa = 151; Kargo = 29), and none was positive for W. bancrofti (0%). Result of entomological indices for malaria transmission showed that indoor resting density was higher in Jidawa (10 mosquitoes/room/night) while human biting rate (2.07 bites/person/night), sporozoite rate (29.3%) and entomological inoculation rate (0.61) were higher in Kargo. In total, 35.2% of the samples were blood-fed while 67.4% were parous. There is active transmission of malaria in the two communities but not LF, suggesting the effectiveness of mass drug administration for LF. Concerted efforts should be focused on malaria control as transmission of the disease persists.

Evolutionary modelling indicates that mosquito metabolism shapes the life-history strategies of Plasmodium parasites.

Within-host survival and between-host transmission are key life-history traits of single-celled malaria parasites. Understanding the evolutionary forces that shape these traits is crucial to predict malaria epidemiology, drug resistance, and virulence. However, very little is known about how Plasmodium parasites adapt to their mosquito vectors. Here, we examine the evolution of the time Plasmodium parasites require to develop within the vector (extrinsic incubation period) with an individual-based model of malaria transmission that includes mosquito metabolism. Specifically, we model the metabolic cascade of resource allocation induced by blood-feeding, as well as the influence of multiple blood meals on parasite development. Our model predicts that successful vector-to-human transmission events are rare, and are caused by long-lived mosquitoes. Importantly, our results show that the life-history strategies of malaria parasites depend on the mosquito's metabolic status. In our model, additional resources provided by multiple blood meals lead to selection for parasites with slow or intermediate developmental time. These results challenge the current assumption that evolution favors fast developing parasites to maximize their chances to complete their within-mosquito life cycle. We propose that the long sporogonic cycle observed for Plasmodium is not a constraint but rather an adaptation to increase transmission potential.

Unravelling the mosquito-haemosporidian parasite-bird host network in the southwestern Iberian Peninsula: insights into malaria infections, mosquito community and feeding preferences.

BAKGROUND: Vector-borne diseases affecting humans, wildlife and livestock have significantly increased their incidence and distribution in the last decades. Because the interaction among vectors-parasite-vertebrate hosts plays a key role driving vector-borne disease transmission, the analyses of the diversity and structure of vector-parasite networks and host-feeding preference may help to assess disease risk. Also, the study of seasonal variations in the structure and composition of vector and parasite communities may elucidate the current patterns of parasite persistence and spread as well as facilitate prediction of how climate variations may impact vector-borne disease transmission. Avian malaria and related haemosporidian parasites constitute an exceptional model to understand the ecology and evolution of vector-borne diseases. However, the characterization of vector-haemosporidian parasite-bird host assemblages is largely unknown in many regions. METHODS: Here, we analyzed 5859 female mosquitoes captured from May to November in five localities from southwestern Spain to explore the composition and seasonal variation of the vector-parasite-vertebrate host network. RESULTS: We showed a gradual increase in mosquito abundance, peaking in July. A total of 16 different haemosporidian lineages were found infecting 13 mosquito species. Of these assemblages, more than 70% of these vector-parasite associations have not been described in previous studies. Moreover, three Haemoproteus lineages were reported for the first time in this study. The prevalence of avian malaria infections in mosquitoes varied significantly across the months, reaching a maximum in November. Mosquito blood-feeding preference was higher for mammals (62.5%), whereas 37.5% of vectors fed on birds, suggesting opportunistic feeding behavior. CONCLUSION: These outcomes improve our understanding of disease transmission risk and help tovector control strategies.

Changes in contributions of different Anopheles vector species to malaria transmission in east and southern Africa from 2000 to 2022.

BACKGROUND: Malaria transmission in Africa is facilitated by multiple species of Anopheles mosquitoes. These vectors have different behaviors and vectorial capacities and are affected differently by vector control interventions, such as insecticide-treated nets and indoor residual spraying. This review aimed to assess changes in the contribution of different vector species to malaria transmission in east and southern Africa over 20 years of widespread insecticide-based vector control. METHODS: We searched PubMed, Global Health, and Web of Science online databases for articles published between January 2000 and April 2023 that provided species-specific sporozoite rates for different malaria vectors in east and southern Africa. We extracted data on study characteristics, biting rates, sporozoite infection proportions, and entomological inoculation rates (EIR). Using EIR data, the proportional contribution of each species to malaria transmission was estimated. RESULTS: Studies conducted between 2000 and 2010 identified the Anopheles gambiae complex as the primary malaria vector, while studies conducted from 2011 to 2021 indicated the dominance of Anopheles funestus. From 2000 to 2010, in 57% of sites, An. gambiae demonstrated higher parasite infection prevalence than other Anopheles species. Anopheles gambiae also accounted for over 50% of EIR in 76% of the study sites. Conversely, from 2011 to 2021, An. funestus dominated with higher infection rates than other Anopheles in 58% of sites and a majority EIR contribution in 63% of sites. This trend coincided with a decline in overall EIR and the proportion of sporozoite-infected An. gambiae. The main vectors in the An. gambiae complex in the region were Anopheles arabiensis and An. gambiae sensu stricto (s.s.), while the important member of the An. funestus group was An. funestus s.s. CONCLUSION: The contribution of different vector species in malaria transmission has changed over the past 20 years. As the role of An. gambiae has declined, An. funestus now appears to be dominant in most settings in east and southern Africa. Other secondary vector species may play minor roles in specific localities. To improve malaria control in the region, vector control should be optimized to match these entomological trends, considering the different ecologies and behaviors of the dominant vector species.

Systematic review of sporozoite infection rate of Anopheles mosquitoes in Ethiopia, 2001-2021.

BACKGROUND: Adult mosquitoes of the genus Anopheles are important vectors of Plasmodium parasites, causative agents of malaria. The aim of this review was to synthesize the overall and species-specific proportion of Anopheles species infected with sporozoites and their geographical distribution in the last 2 decades (2001-2021). METHODS: A comprehensive search was conducted using databases (PubMed, Google Scholar, Science Direct, Scopus, African Journals OnLine) and manual Google search between January 1 and February 15, 2022. Original articles describing work conducted in Ethiopia, published in English and reporting infection status, were included in the review. All the required data were extracted using a standardized data extraction form, imported to SPSS-24, and analyzed accordingly. The quality of each original study was assessed using a quality assessment tool adapted from the Joanna Briggs Institute critical appraisal checklist. This study was registered on PROSPERO (International Prospective Register of Systematic Reviews; registration no. CRD42022299078). RESULTS: A search for published articles produced a total of 3086 articles, of which 34 met the inclusion criteria. Data on mosquito surveillance revealed that a total of 129,410 anophelines comprising 25 species were captured, of which 48,365 comprising 21 species were tested for sporozoites. Anopheles arabiensis was the dominant species followed by An. pharoensis and An. coustani complex. The overall proportion infected with sporozoites over 21 years was 0.87%. Individual proportions included Anopheles arabiensis (1.09), An. pharoensis (0.79), An. coustani complex (0.13), An. funestus (2.71), An. demeilloni (0.31), An. stephensi (0.70), and An. cinereus (0.73). Plasmodium falciparum sporozoites accounted 79.2% of Plasmodium species. Mixed infection of Plasmodium vivax and P. falciparum was only reported from one An. arabiensis sample. CONCLUSIONS: Anopheles arebiensis was the dominant malaria vector over the years, with the highest sporozoite infection proportion of 2.85% and an average of 0.90% over the years. Other species contributing to malaria transmission in the area were An. pharoensis, An. coustani complex, An. funestus, An. stephensi, and An. coustani. The emergence of new vector species, in particular An. stephensi, is particularly concerning and should be investigated further.

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance.

BACKGROUND: The impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos' propensity to transmit Plasmodium spp. METHODS: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes. RESULTS: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1-5.2), p = 0.03; IRR = 2.5, 95% CI (1.2-5.8), p = 0.01 respectively). CONCLUSION: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance.

Avian Plasmodium spp. and Haemoproteus spp. parasites in mosquitoes in Germany.

BACKGROUND: Although haemosporidian parasites may cause considerable health and economic problems in aviaries, there is limited understanding of the vectors transmitting them. Mosquito-borne Plasmodium species are responsible for the deaths of numerous exotic (= immunologically naïve) birds in zoos every year, while native birds are adapted to the parasites and largely protected by an effective immune response. METHODS: Mosquitoes were collected in bird/animal parks, wetlands and private gardens in various regions of Germany from 2020 to 2022. Females were pooled with up to 10 specimens according to taxon, location and date. Extracted DNA was screened for avian Haemosporida-specific mitochondrial rDNA using real-time polymerase chain reaction (PCR). Positive samples were amplified by a Plasmodium/Haemoproteus-specific nested PCR targeting the partial cytochrome b gene, followed by sequencing of the PCR product for species identification. Sequences were checked against GenBank and MalAvi databases. RESULTS: PCR of 2633 pools with 8834 female mosquitoes signalled infection with Plasmodium in 46 pools and with Haemoproteus in one pool. Further amplification and sequencing demonstrated the occurrence of Haemoproteus majoris lineage PARUS1 (n = 1) as well as several Plasmodium species and lineages, including Plasmodium relictum SGS1 (n = 16) and GRW11 (n = 1), P. matutinum LINN1 (n = 13), P. vaughani SYAT05 (n = 10), P. circumflexum TURDUS01 (n = 3), P. cathemerium PADOM02 (n = 1) and Plasmodium sp. SYBOR02 (n = 1) and PLOPRI01 (n = 1). The infections were detected in Culex pipiens sensu lato (n = 40), Culiseta morsitans/fumipennis (n = 6) and Aedes cinereus/geminus (n = 1). CONCLUSIONS: Although the overall Plasmodium minimum infection rate (5.2) appears to be low, the results demonstrated not only the ongoing circulation of Plasmodium parasites in the German mosquito population, but also the occurrence of eight distinct Plasmodium lineages, with three of them (PADOM02, SYBOR02, PLOPRI01) being detected in Germany for the first time. This study highlights the importance of conducting mosquito-borne pathogen surveillance studies simultaneously targeting vectors and vertebrate hosts, as certain species may be detected more readily in their vectors than in their vertebrate hosts, and vice versa.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa.

Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case-control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April-July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito.

Spatial analyses of Plasmodium knowlesi vectors with reference to control interventions in Malaysia.

BACKGROUND: Malaria parasites such as Plasmodium knowlesi, P. inui, and P. cynomolgi are spread from macaques to humans through the Leucosphyrus Group of Anopheles mosquitoes. It is crucial to know the distribution of these vectors to implement effective control measures for malaria elimination. Plasmodium knowlesi is the most predominant zoonotic malaria parasite infecting humans in Malaysia. METHODS: Vector data from various sources were used to create distribution maps from 1957 to 2021. A predictive statistical model utilizing logistic regression was developed using significant environmental factors. Interpolation maps were created using the inverse distance weighted (IDW) method and overlaid with the corresponding environmental variables. RESULTS: Based on the IDW analysis, high vector abundances were found in the southwestern part of Sarawak, the northern region of Pahang and the northwestern part of Sabah. However, most parts of Johor, Sabah, Perlis, Penang, Kelantan and Terengganu had low vector abundance. The accuracy test indicated that the model predicted sampling and non-sampling areas with 75.3% overall accuracy. The selected environmental variables were entered into the regression model based on their significant values. In addition to the presence of water bodies, elevation, temperature, forest loss and forest cover were included in the final model since these were significantly correlated. Anopheles mosquitoes were mainly distributed in Peninsular Malaysia (Titiwangsa range, central and northern parts), Sabah (Kudat, West Coast, Interior and Tawau division) and Sarawak (Kapit, Miri, and Limbang). The predicted Anopheles mosquito density was lower in the southern part of Peninsular Malaysia, the Sandakan Division of Sabah and the western region of Sarawak. CONCLUSION: The study offers insight into the distribution of the Leucosphyrus Group of Anopheles mosquitoes in Malaysia. Additionally, the accompanying predictive vector map correlates well with cases of P. knowlesi malaria. This research is crucial in informing and supporting future efforts by healthcare professionals to develop effective malaria control interventions.

Genomic advances in the study of the mosquito vector during avian malaria infection.

Invertebrate host­parasite associations are one of the keystones in order to understand vector-borne diseases. The study of these specific interactions provides information not only about how the vector is affected by the parasite at the gene-expression level, but might also reveal mosquito strategies for blocking the transmission of the parasites. A very well-known vector for human malaria is Anopheles gambiae. This mosquito species has been the main focus for genomics studies determining essential key genes and pathways over the course of a malaria infection. However, to-date there is an important knowledge gap concerning other non-mammophilic mosquito species, for example some species from the Culex genera which may transmit avian malaria but also zoonotic pathogens such as West Nile virus. From an evolutionary perspective, these 2 mosquito genera diverged 170 million years ago, hence allowing studies in both species determining evolutionary conserved genes essential during malaria infections, which in turn might help to find key genes for blocking malaria cycle inside the mosquito. Here, we extensively review the current knowledge on key genes and pathways expressed in Anopheles over the course of malaria infections and highlight the importance of conducting genomic investigations for detecting pathways in Culex mosquitoes linked to infection of avian malaria. By pooling this information, we underline the need to increase genomic studies in mosquito­parasite associations, such as the one in Culex­Plasmodium, that can provide a better understanding of the infection dynamics in wildlife and reduce the negative impact on ecosystems.