Parasites & Vectors

Treatment of livestock with endectocides such as ivermectin is viewed as a complementary vector control approach to address residual transmission of malaria. However, efficacy of this treatment may vary between animal species. Hence, our purpose was to investigate the effects of ivermectin treatments of common livestock species on life history traits of the opportunistic malaria vector Anopheles coluzzii. Sheep, goats and pigs were treated using injectable veterinary ivermectin formulation at the species-specific doses (recommended dose for all species and high dose in pig). Mosquito batches were exposed to treated and control (not injected) animals at different days after treatment. Daily mosquito mortality was recorded and fecundity assessed through the count of gravid females and the number of eggs they developed. The recommended dose of ivermectin induced a significant decrease in mosquito survival for up to 7 days after injection (DAI), with a decrease of 89.7%, 66.7%, and 48.4% in treated pigs, goats and sheep, respectively, compared to control animals. In treated pigs, the triple therapeutic dose decreased mosquito survival of 68.97% relatively to controls up to 14 DAI. The average number in gravid females Anopheles that survived after feeding on treated animals were reduced when blood-meals were taken on sheep (2.57% and 42.03% at 2 and 7 DAI), or on goats (decrease of the 28.28% and 73.64% respectively at 2 and 7 DAI). This study shows that ivermectin treatments to animals negatively impacts An. coluzzii life history traits and could reduce vector densities in areas where livestock live near humans. However, due to short-term efficacy of single dose treatments, repeated treatments and potentially increased dosages would be required to span the transmission season. The use of long-acting ivermectin formulations is discussed as a mean for extending efficacy while remaining cost effective.

BackgroundAedes vittatus, an emerging invasive species and arboviral vector with high ecological plasticity and dispersal capacity, is expanding in the Caribbean, being detected in the Dominican Republic, Cuba, and Jamaica and recently in Yucatan, Mexico. ObjectivesThis study aimed to provide evidence of the establishment of Ae. vittatus in Haiti. MethodsLarval surveys were done across 12 localities around Cap-Haitien, in the North-East department of Haiti, the specimens were identified morphologically and confirmed by COI DNA barcoding. FindingsWe report, for the first time, the detection of the invasive vector Aedes (Fredwardsius) vittatus in Haiti, confirmed morphologically and molecularly, and being established in half of the municipalities sampled. Phylogenetic analyses group Haitian specimens with Caribbean and American populations, closely related to Mexico, indicating regional expansion. Main conclusionGiven its role as a vector of chikungunya, dengue, Zika and yellow fever, strengthened entomological surveillance and early detection in North America and the Caribbean are urgently needed.

Filarial nematodes are an important group of parasites that impact public, veterinary, and wildlife health globally. In order to understand these impacts and minimize their effects, scientists use molecular xenomonitoring techniques to understand their distribution and track elimination efforts. However, these molecular techniques can have narrow diagnostic capacity due to their species-specific approach which limits our understanding of important co-endemic filarial nematodes. Next-generation sequencing offers the ability to detect multiple species of coinfecting filarial nematodes and thus improve are ability to monitor, treat, and eliminate these pathogens. In this paper, we have developed a deep amplicon sequencing approach using filarial nematode primers targeting the cytochrome oxidase c subunit 1 (coxI) gene. To replicate molecular xenomonitoring conditions, third stage larvae (L3) of three species of filarioid nematodes (Brugia malayi, Brugia pahangi, Dirofilaria immitis) were spiked in different proportions to pools comprising various amounts of female Aedes aegypti mosquitoes (0, 10, 50, 100). Each pool was subjected to DNA extraction and Oxford Nanopore Technologies (ONT) deep amplicon sequencing protocols. Two sets of demultiplexing pipelines were utilized to optimize this novel approach, each reaching, 92.71% and 97.92% accuracy in identification of species composition across mock pools. However, in heterogenous pools, filarial species D. immitis exhibited an overrepresentation of reads and B. pahangi an underrepresentation of reads. We discuss reasons for recount biases and how this new molecular xenomonitoring tool could be implemented to serve public health, veterinary medicine, and scientific advancement. Note: Supplementary data associated with this article Author summaryHuman and animal diseases caused by filarial nematodes affect millions of people worldwide, particularly in low-income countries. These parasites are transmitted by blood-feeding arthropod vectors, such as mosquitoes and black flies. Thus, a major sector of public health research focuses on how to monitor, treat, and eliminate these harmful pathogens. An effective way is to capture these arthropod vectors and molecularly test these for filarial DNA in large sample pools. However, these pools can comprise multiple filarial species which targeted genetic analysis can miss. This proof-of-concept study seeks to circumvent these issues by using new next-generation sequencing approaches to capture the wider filarial diversity that may be contained in a single vector pool. We believe this tool could largely be beneficial to governments and organizations seeking to eliminate these filarial nematodes and become certified as a region free of certain devastating filarial species. Furthermore, we know very little about filarial diversity and this could be an integral tool to define their geographic distribution and future emerging threats to both human and animal health.

Malaria is transmitted when Anopheline mosquitoes ingest Plasmodium parasites during blood-feeding. Artificial feeding assays allow mosquitoes to take up blood from membrane feeders, and are widely used to study malaria transmission. These assays require large quantities of mosquitoes; insectaries optimize their rearing procedures to generate high yields of permissive, homogeneous mosquito populations. Rearing of Anopheline stephensi mosquitoes was protocolized at the Radboudumc in the 1980s, yet infection outcomes remain heterogeneous. This study explores possible improvements in mosquito rearing to improve homogeneity of the resulting mosquito populations. It compares the current mass-rearing standard with an adapted alternative approach from another institute that optimizes larval density per tray and applies a diet that has previously been reported. Differences between procedures were assessed by measuring mosquito size by proxy of wing-length and imbibed blood meal volume. To assess receptiveness to P. falciparum infection mosquitoes were fed cultured gametocytes. We observed a slight decrease in mosquito size when applying the alternative rearing procedure, but generated equally parasite-receptive P. falciparum mosquitoes compared to the standard procedure. We conclude that both rearing protocols can be used to generate susceptible mosquitoes for conducting malaria research.

Anopheles stephensi is an invasive malaria vector in Africa. To determine the status of the mosquito in Yemen, An. stephensi vector surveillance and molecular confirmation was conducted in Al Hudaydah Governorate in 2021 and 2022. Mosquito larvae were collected in suspected man-made breeding habitats in Ah Dahi and Zabid city in 2021 and 2022, respectively. Mosquitoes morphologically identified as An. stephensi underwent molecular confirmation through PCR assays, sequencing, and phylogenetic analysis of the cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 locus (ITS2). Analysis confirmed An. stephensi identification for the majority of samples (39/41), with two COI haplotypes detected: one newly reported haplotype and one haplotype common to Northeast Ethiopia and Somaliland. No clustering with An. stephensi from the Arabian Peninsula was observed. These findings provide preliminary insight into the diversity of An. stephensi in Yemen and the connection between An. stephensi in Yemen and East Africa.

Pyrethroid insecticides are widely used to control mosquitoes that transmit diseases such as West Nile virus (WNV) to humans. A single nucleotide polymorphism (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene of Culex mosquitoes confers knockdown resistance to pyrethroids. PCR-based assays that detect these SNPs in Culex species are currently available for Culex pipiens Linnaeus and Culex quinquefasciatus Say. RNAseq was employed to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. We utilized the cDNA sequence to develop a quantitative reverse transcriptase PCR assay that detects the L1014F mutation in the kdr of Vgsc. Because this locus is conserved, the assay successfully detected the SNPs in multiple Culex spp. vectors of WNV in the United States. The resulting Culex RTkdr assay was validated using quantitative PCR, CDC bottle bioassays, and sequencing of PCR products. Using sequencing, we determined the accuracy of the Culex RTkdr assay was 99%. Pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and co-occured with agriculture. We anticipate that public health and vector control agencies may utilize the Culex RTkdr assay to map the distribution of pyrethroid resistance in Culex species to more efficiently control mosquitoes and the diseases they transmit.

BACKGROUNDAngola ranks among the five countries with the highest malaria burden globally. The Ministry of Health in Angola has consistently partnered with international donors to sustain entomological surveillance and vector control strategies in a context of high malaria burden. METHODSVector surveillance was carried out in Luanda, Benguela, Namibe and Cuanza Sul provinces from 2016-2022. Collected adult mosquitoes were tested to assess the presence of Plasmodium parasites and determine blood sources. Larvae collections provided live material to test insecticide susceptibility in local Anopheles populations. Taxonomic determination of mosquitoes was based on external morphology and confirmed with molecular assays. The presence of Anopheles azevedoi was confirmed through morphology and genetic sequences, and errors in the original species determination were detected, discussed and corrected. OBJECTIVESThe study aimed to update the geographical range of Anopheles azevedoi in Angola and monitor the species susceptibility to public health insecticides. FINDINGS and MAIN CONCLUSIONSWe report on populations of Anopheles azevedoi occurring along the western coast of Angola, a highly abundant species with anthropophilic behavior in urban areas. Anopheles azevedoi is widely resistant to pyrethroids and DDT but fully susceptible to chlorfenapyr. We contribute with COI and ITS-2 barcoding sequences for future species identification and explain the reasons for which this species has been for long misidentified in Angola.

Pyrethroids are the most widely used insecticides to control vector borne diseases including malaria. Physiological resistance mechanisms to these insecticides have been well described, whereas those for behavioral resistance remain overlooked. Field data suggest the presence of spatial sensory detection by Anopheles mosquitoes of the pyrethroid molecules used in insecticide-based control tools, such as long-lasting insecticide nets or insecticide residual spraying, opening the way to the emergence of a wide range of behavioral adaptations among malaria vectors. However, the spatial sensory detection of these molecules is controversial and needs to be demonstrated. The goal of this study was to behaviorally characterize the non-contact detection of three of the most common pyrethroids used for malaria vector control: permethrin, deltamethrin an -cypermethrin. To reach this goal, we recorded the behavior (takeoff response) of Anopheles gambiae pyrethroid-sensitive and resistant laboratory strains, as well as field collected mosquitoes from the Gambiae complex, when exposed to the headspace of bottles containing different doses of the insecticides at 25 and 35{degrees}C, in order to represent a range of laboratory and field temperatures. We found the proportion of laboratory susceptible and resistant female mosquitoes that took off was, in all treatments, dose and the temperature dependent. Sensitive mosquitoes were significantly more prone to take off only in the presence of -cypermethrin, whereas sensitive and resistant mosquitoes showed similar responses to permethrin and deltamethrin. Field-collected mosquitoes of the Gambiae complex were also responsive to permethrin, independently of the species identity (An. gambiae, An. coluzzi and An. arabiensis) or their genotypes for the kdr mutation, known to confer resistance to pyrethroids. The observed ability of Anopheles spp. mosquitoes to detect insecticides without contact could favor the evolution of behavioral modifications that may allow them to avoid or reduce the adverse effect of insecticides and thus, the development of behavioral resistance.

BACKGROUNDThe Sierra Nevada de Santa Marta rainforest has diverse fauna due to its position in northern Colombia, a Caribbean region with predominantly tropical, dry, and rainforest ecosystems in which there is a high diversity of mosquito species that may act as arbovirus vectors. OBJECTIVESThe present study reports the molecular characterization of select mosquito species in this rainforest. METHODSManual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. The COI marker was used for species confirmation, and phylogenetic analysis was performed, using the neighbor-joining method, with the Kimura-2-Parameters model. FINDINGSAedes serratus, Psorophora ferox, Johnbelkinia ulopus, Sabethes cyaneus, Wyeomyia aporonoma, Wyeomyia pseudopecten, Wyeomyia ulocoma and Wyeomyia luteoventralis were identified and intra-species variation >2% for most species. MAIN CONCLUSIONSWe report the first records on the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed for identification at the species level, and the corroboration by means of classical taxonomy suggested complementarity of both methods, which may be employed when morphological or molecular data are poor or not available. The genetic and morphological characterization of jungle mosquito populations will help to understand their biology.

Malaria remains a major threat during humanitarian crises, necessitating targeted vector control strategies informed by local vector dynamics. Between May and July 2023, we conducted larval surveys in refugee settlements across Dadaab, Kakuma, and Kalobeyei (Kenya), collecting Anopheles larvae. Genotyping of 728 specimens revealed spatial variations in species composition. Overall, Anopheles arabiensis was the dominant species (59%, n=426), followed by Anopheles coluzzii (35%, n=252), and Anopheles rufipes (1%, n=7). In Dadaab, An. arabiensis was overwhelmingly dominant (94%, n=352/374). In contrast, the Kakuma/Kalobeyei complex was characterized by the co-occurrence of An. coluzzii (72%, n=252/350) and An. arabiensis (22%, n=74/350), with An. rufipes exclusively found in Kalobeyei (7%, n=6/89) (Figure 2B). Notably, no members of the Anopheles funestus group or Anopheles stephensi were detected. However, approximately 5% of the larvae across the sites could not be resolved molecularly. High frequencies of the L1014F kdr mutation, a pyrethroid resistance marker, were detected in An. coluzzii (Kakuma: 50%; Kalobeyei: 63%) and An. arabiensis (Kakuma: 10%; Kalobeyei: 30%) populations in Turkana County. Interestingly, no kdr mutations were observed in the An. arabiensis population from Dadaab. These findings highlight significant spatial diversity in vector species composition and resistance profiles, with An. coluzzii emerging as a dominant, pyrethroid-resistant vector in the Kakuma/Kalobeyei complex. The results underscore the urgent need for targeted interventions, including resistance monitoring and alternative insecticide-based strategies, to mitigate malaria transmission risks in fragile, humanitarian settings. Further studies are warranted to address unidentified larval species and seasonal transmission dynamics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/682237v1_fig2.gif" ALT="Figure 2"> View larger version (12K): org.highwire.dtl.DTLVardef@1f3788forg.highwire.dtl.DTLVardef@1701f58org.highwire.dtl.DTLVardef@19d566org.highwire.dtl.DTLVardef@95716_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Species composition of Anopheles mosquitoes in Kakuma, Kalobeyei, and Dadaab refugee camps, Kenya. (A) Overall relative abundance of Anopheles species identified by PCR and ITS2 sequencing. (B) Spatial distribution and abundance of the predominant species, An. coluzzii and An. arabiensis, across individual sampling sites. C_FIG

The landing patterns of mosquitoes are critical for disease prevention given their roles as vectors of numerous pathogens, especially given documented variability in their patterns across different geographies and contexts. In Ecuador, two of the most important vectors associated with human arboviruses infection, Aedes aegypti and Culex quinquefasciatus, are abundant. Ascertaining the patterns of these mosquitoes biting behaviors (and through this, their potential to transmit pathogens) is essential to better tailor public health interventions and thereby prevent the spread of arboviral diseases. From June 2021 to July 2022, we performed human landing collections of mosquitoes in two sites within Northern Ecuador, capturing mosquitoes in protected and unprotected settings, across the days span. We captured 18,280 mosquitoes, 95.51% were Cx. quinquefasciatus and 1.8% were Ae. aegypti. The results of this study reinforce traditional diel landing patterns and crepuscular patterns for Aedes aegypti and Culex quinquefasciatus, respectively. We emphasize the importance of elucidating landing dynamics in varied and unique contexts, as established patterns and new dynamics are not necessarily consistent across geographies. Finally, we describe differences in protected versus unprotected collection events, with vector species being more likely to land on protected collectors, of importance for future landing catch studies in disease-endemic areas.

Despite a decade of indoor residual spraying, malaria remains holoendemic in Nchelenge District in northern Zambia. This study examined the hypothesis that the intransigence of malaria in Nchelenge District is due in part to early evening and outdoor foraging of local anopheline species. Three replicate collections were performed at 24 households using CDC light traps over 6 weeks during the dry season of 2019. Traps were set indoors, in animal pens, and in outdoor gathering spaces from 16:00-22:00, and overnight indoors in all households during the third replicate. Twelve anopheline species were collected in early-evening traps. The major vector, Anopheles funestus, dominated collections (n = 1,865, 68.2%) and were captured primarily indoors (n = 1,370, 73.4%). Specimens with detectable human blood and Plasmodium falciparum positive specimens were found in all three trap types, indicating that infected anopheline foraging occurred before 22:00. EIR estimates for early-evening anophelines varied spatially and by species and were highest among indoor caught An. funestus at inland locations (18.74 bites/person/6months). There was little difference in anopheline abundance, blooded rates, and sporozoite infection rate when evening and overnight traps were compared, indicating there may be similar anopheline activity before and after 22:00. Overall, this study adds to the growing body of evidence that malaria transmission is not limited to late-night, indoor settings in Nchelenge District, Zambia.

BackgroundAccurate estimation of mosquito age is critical for malaria surveillance, as only older female Anopheles mosquitoes can transmit the parasite. Traditional age-grading methods, include the Polovodova technique in which dilatations on the ovarian pedicel to tally completed gonotrophic cycles (GC) are counted. Despite their widespread use, these techniques remain poorly validated under realistic operational conditions, with limited evidence on their precision and accuracy across different parity rates, GC, and species. In this study, the accuracy and precision of the Polovodova method was evaluated across multiple age-graders and species under both laboratory and field conditions. MethodsA blinded validation study was conducted using laboratory-reared Anopheles arabiensis and Anopheles funestus (N > 3,600) spanning four gonotrophic cycles, and field-collected mosquitoes from Ulanga district, Tanzania (N = 600). Three blinded researchers performed dissections and readings, while a fourth unblinded researcher served as the reference reader (R0) to obtain ground truth (for parity) or assumed truth (for GC) for laboratory mosquitoes. Accuracy and precision for parity and gonotrophic cycle classification were assessed using pairwise inter-rater comparisons, percent agreement, Fleiss kappa and Cohens kappa O_SCPCAP(C_SCPCAPO_SCPLOWKC_SCPLOWO_SCPCAP)C_SCPCAP statistics. For field mosquitoes, which had no age-referenced group, only precision was assessed. FindingsThe Polovodova method accurately classified parity in both species, correctly identifying 98-100% of nulliparous and 94-96% of parous mosquitoes compared with the reference dataset. Inter-rater reliability for parity was almost perfect in An. arabiensis and An. funestus (k = 0.81-0.83), with 82-96% pairwise agreement. GC classification was highly accurate for early stages (GC0-GC2: 85-100%) but declined at later stages GC4(34-46%), reflecting systematic underestimation of mosquito age. Overall GC reliability was moderate (k = 0.56-0.59). Field classifications showed almost perfect agreement (k = 0.87-0.88), supporting operational applicability. ConclusionsThe Polovodova method provides robust estimates of parity and early gonotrophic-cycle status in Anopheles mosquitoes, but late-cycle classifications are more prone to reader disagreement and systematic underestimation. Operational use should therefore rely on trained multiple-reader workflows, including adjudication of discordant or late-cycle specimens, rather than single-reader classification. These findings provide empirical benchmarks for quality-assured Polovodova age-grading in malaria vector surveillance and intervention evaluation.

BackgroundAnopheles mosquitoes vector pathogens responsible for more than 600,000 human deaths annually. Ecological studies of these insects are important to guide effective vector-control campaigns and to understand their broader ecological consequences. Molecular ecology methods, particularly qPCR, provide a valuable tool in such studies. By detecting trace DNA of a taxon of interest within mixed or environmental samples, qPCR can facilitate identification of prey taxa of interest in the diets of consumers. However, no protocol for the detection of An. gambiae complex mosquitoes in dietary samples has been available. MethodsWe introduce a new set of qPCR primers (Agam_CO1_F1 and Agam_CO1_R1) and a probe-based assay for detection of Anopheles gambiae-complex mosquitoes, even with short reads common in dietary and environmental samples. The primers were tested in vitro for their specificity and sensitivity, and in silico using Primer-BLAST to assess potential off-target amplification. ResultsThe qPCR primers amplified An. gambiae DNA even at low starting concentrations (5 copies {micro}l-1). The primers did not amplify any non-target DNA in either the in vitro or in silico tests, but consistently amplified An. gambiae complex DNA. The primers can therefore provide reliable tests for the presence or absence of An. gambiae complex in dietary or eDNA samples. ConclusionsThe new qPCR primers should allow advances in research into mosquito ecology by allowing detection of even trace amounts of An. gambiae DNA in dietary and environmental samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/707393v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1fcce3corg.highwire.dtl.DTLVardef@47e5f5org.highwire.dtl.DTLVardef@4a7063org.highwire.dtl.DTLVardef@1188d60_HPS_FORMAT_FIGEXP M_FIG C_FIG

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable ~2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

The Anopheles symbiont, Microsporidia MB, is maternally inherited and has a strong malaria transmission-blocking phenotype in Anopheles arabiensis. Microsporidia MB is also vertically transmitted, sexually transmitted and avirulent. These characteristics are expected to promote its spread through mosquito populations, enhancing the potential of Microsporidia MB as a candidate for the development of a symbiont-mediated malaria transmission blocking strategy. We found that the patterns of Microsporidia MB localisation over the development of An. arabiensis indicate accumulation in tissues linked to its transmission, specifically the male and female gonadal tissues. Transovarial vertical transmission of Microsporidia MB occurs in the female An. arabiensis ovary when Microsporidia MB becomes localised to the cytoplasm of the developing oocyte. In male An. arabiensis, Microsporidia MB is localised in the testis and vas deferens. Notably, a high intensity of Microsporidia MB can also be observed in the An. arabiensis adult but not larval gut. The levels of Microsporidia MB found in the female ovary are linked to the progression of oogenesis, increasing after blood feeding initiates the development of eggs. The levels of Microsporiodia MB in the male and female gonadal and gut tissue do not increase as mosquitoes age. Altogether, the high specificity of Microsporidia MB tissue localisation patterns and changes in infection prevalence and intensity suggest adaptation to maximise transmission and avirulence in Anopheles arabiensis. ImportanceMicrosporidia MB is a symbiont with strong malaria transmission-blocking phenotype in Anopheles arabiensis. It spreads in mosquito populations through mother-to-offspring and sexual transmission. The ability of Microsporidia MB to block Plasmodium transmission together with its ability to spread within Anopheles populations and its avirulence to the host makes it a very attractive candidate for developing a key strategy to stop malaria transmissions. Here, we report the basis of Microsporidia MB transmission. We find that Microsporidia MB accumulates in Anopheles arabiensis tissues linked to its sexual and vertical transmission. Its prevalence and intensity in the tissues over the mosquito life cycle suggest adaptation to maximise transmission and avirulence in Anopheles arabiensis. These findings provide the foundation for understanding the factors that affect Microsporidia MB transmission efficiency. This will contribute to the establishment of strategies to maximize Microsporidia MB transmission for Anopheles mosquito population replacement and malaria transmission blocking.

Anopheles stephensi, an efficient Asian malaria vector, recently spread into the Horn of Africa and may increase malaria receptivity in African urban areas. We assessed occurrence, genetic complexity, blood meal source and infection status of An. stephensi in Awash Sebat Kilo town, Ethiopia. We used membrane feeding assays to assess competence of local An. stephensi to P. vivax and P. falciparum isolates from clinical patients. 75.3% of the examined waterbodies were infested with An. stephensi developmental stages that were genetically closely related to isolates from Djibouti and Pakistan. Both P. vivax and P. falciparum were detected in wild-caught adult An. stephensi. Local An. stephensi was more receptive to P. vivax compared to a colony of An. arabiensis. We conclude that An. stephensi is an established vector in this part of Ethiopia, highly permissive for local P. vivax and P. falciparum isolates and presents an important new challenge for malaria control. Summary of the articleAn. stephensi, a metropolitan malaria vector that recently expanded to the Horn of African, was highly susceptible to local P. falciparum and P. vivax isolates from Ethiopia and may increase malariogenic potential of rapidly expanding urban settings in Africa.

BackgroundWolbachia pipientis is an endosymbiont bacteria that induce cytoplasmic incompatibility and inhibit arboviral replication in mosquitoes. This study aimed at estimating the prevalence and genetic diversity of Wolbachia in different mosquito (Diptera: Culicidae) species from Cape Verde. MethodsMosquitoes were collected in six islands of Cape Verde using dippers/pipettes, BG-sentinel(R) traps, CDC light traps, and dorsal aspirators. Samples were identified to species using morphological keys and PCR-based molecular assays. Wolbachia was detected by amplifying a fragment of the surface protein gene (wsp). Multilocus sequence typing (MLST) was performed with five housekeeping genes (coxA, gatB, ftsZ, hcpA and fbpA) and the wsp hypervariable region (HVR) for strain identification. Identification of wPip groups (wPip-I to wPip-V) was performed using PCR-RFLP assay on the ankyrin-domain gene pk1. ResultsNine mosquito species were collected, including the major vectors Aedes aegypti, Anopheles arabiensis, Culex pipiens s.s. and Culex quinquefasciatus. Wolbachia was detected in Cx. pipiens s.s. (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens/quinquefasciatus hybrids (100%) and Culex tigripes (100%). Results from MLST and wsp hypervariable region typing showed that Wolbachia from Cx. pipiens s.l. belong to Sequence Type 9, wPip clade and supergroup B Wolbachia. Phylogenetic analyses indicate that Wolbachia isolated from Cx. tigripes belongs to Supergroup B but integrates a distinct clade from wPip with no attributed MLST profile. PCR-RFLP revealed wPip-II, wPip-III and wPip-IV groups in Culex pipiens s.l. wPip-IV was the dominant group, while wPip-II and wPip-III were restricted to Maio and Fogo islands, respectively. ConclusionOur study showed a high prevalence and diversity of Wolbachia in Cx pipiens s.l. from Cape Verde islands and, to the best of our knowledge, is the first to detect Wolbachia in Cx. tigripes, being represented in this species by a previously undescribed MLST Sequence Type.

Barrier bednets (BBnets), regular bednets with a vertical insecticidal panel to target mosquitoes above the bednet roof, where activity is highest, have the potential to improve existing Insecticidal Treated Bednets (ITNs), by reducing quantity of insecticide required per net, reducing the toxic risks to those using the net, thus increasing the range of insecticides to choose from. We evaluated performance of different BBnet variants based on the PermaNet 3 (i.e., P3 BBnets with pyrethroid and piperonyl butoxide (PBO) on the roof or barrier; pyrethroid alone on the side walls) in room-scale bioassays, simultaneously video-recorded to track mosquitoes. Experimental results showed the longitudinal P3 barrier (P3L) to be highly effective: P3+P3L were consistently though not significantly more effective than the reference P3 bednet while performance of Ut+P3L was comparable to the reference P3. Comparing contact duration at the treated sections of each variant, the Ut+P3L accumulated 1273 contacts with 1374 seconds duration, all on the barrier, greatly exceeding the 792 seconds duration, from 8049 contacts, accumulated across the entire surface of the PermaNet 3 reference bednet. The BBnets potential to augment existing bednets and enhance their performance is considered.

Oviposition site selection is critical for mosquito population dynamics. Gravid mosquitoes rely on chemical cues to identify suitable breeding habitats. However, the sensory mechanisms governing this behavior in Anopheles stephensi remain poorly understood. Here, we examined the role of indole, a microbial volatile associated with aquatic environments, in oviposition site choice and assessed the involvement of sensory organs in its detection. In two-choice oviposition assays, water conditioned with first-instar larvae attracted gravid females (OAI = 0.56), whereas water from fourth-instar larvae was repellent (OAI = -0.20), consistent with avoidance of suboptimal, resource-depleted habitats. Indole elicited strong oviposition attraction across a broad concentration range (0.1-50 {micro}M), with no clear dose-response relationship. Surgical ablation of antennae and maxillary palps did not abolish indole-mediated preference but significantly reduced behavioral variability, suggesting that these structures modulate, rather than solely mediate, indole detection. Reanalysis of transcriptomes of antennae, maxillary palps, and legs in An. gambiae and An. coluzzii, along with quantitative RT-PCR in An. stephensi, revealed the expression of chemosensory genes (including Obp1, Obp13, Obp25, Obp71, Or2, and Or10) in the legs, indicating a potential role for leg chemosensation in oviposition decisions. These findings underscore the complexity of chemoreception and chemoperception in mosquito habitat assessment.