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.

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.

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.

Three molecularly undescribed filarial species were co-detected, while screening animals for Brugia malayi, the agent of lymphatic filariasis. Single microfilariae (Mf) isolated from blood samples of crab-eating macaques (Macaca fascicularis) from Belitung, Indonesia, and from pet dogs and cats in Sabah, Malaysia, were analyzed. Among 163 macaques, 33 (20.2%) were positive for large Mf (mean length 498.9 {micro}m) similar to Dirofilaria ( Belitung I). One macaque was infected with small Mf (mean length 150.4 {micro}m) ( Belitung II), with a high density of 17,150 Mf/mL. In two cats co-infected with B. malayi, Mf of a Dirofilaria species ( Sabah) with an average length of 299.1 {micro}m were detected. Morphometric analysis of Mf showed distinct differences between these three species and other Mf described in the area. Whole genome amplification and genome sequencing of 24 individual Mf enabled phylogenetic analysis of mitochondrial genomes, and analysis of specific mitochondrial and nuclear barcode regions. The three Mf groups formed distinct clusters and did not match any currently available reference sequence. Cluster Belitung I from macaques formed a sister group to all other Dirofilaria. Cluster Belitung II included bird filariae and primate filariae of the genus Mansonella as close relatives. The cluster Sabah formed a monophyletic group with the zoonotic species D. asiatica and Dirofilaria sp. Thailand. DNA of Wolbachia endobacteria was detected in Mf of Belitung I and Sabah, but not in Belitung II. These findings highlight the limited understanding of filarial diversity in macaques and cats in Asia and underscore the need for a more comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites. Author summaryFilarial worms are parasitic nematodes that infect humans and animals and are often transmitted by the same vector mosquito. We identified three molecularly undescribed filarial species while investigating animals as reservoirs for the agent of lymphatic filariasis, Brugia malayi on Belitung Island, Indonesia, and in Sabah, Malaysia. Blood samples were collected from Indonesian macaques and Malaysian pet cats. Out of 163 macaques, 20.2% tested positive for exceptionally large microfilariae (Mf) of an unclassified Dirofilaria-like species (Belitung I). Another filarial species ( Belitung II) with very small Mf, but with a remarkably high density of 17,150 Mf/mL was detected in one macaque. Two cats harbored medium sized Mf of a Dirofilaria species (Sabah). Genetic analysis revealed unique phylogenetic clusters that did not match any reference sequence. Dirofilaria sp. Sabah was closely related to the zoonotic D. asiatica complex, whereas Belitung I clustered as a sister group to Dirofilaria. Belitung II Mf clustered next to but not within the Mansonella spp. cluster. DNA of Wolbachia endobacteria was only detected in Mf of Belitung I and Sabah. These findings highlight the limited understanding of filarial diversity in animals and underscore the need for a comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites.

Long lasting insecticidal nets (LLINs) are a proven tool to reduce malaria transmission, but in Africa efficacy is being reduced by pyrethroid resistance in the major vectors. A cluster randomized trial in Muleba district, Tanzania demonstrated that permethrin LLINs co-treated with piperonyl butoxide (PBO), a synergist that can block pyrethroid-metabolizing enzymes in the mosquito, had much greater efficacy than pyrethroid-only nets. Insecticide resistance profiles and underlying mechanisms were investigated in Anopheles gambiae and An. funestus from Muleba during the trial. Diagnostic dose bioassays using permethrin, together with intensity assays, suggest pyrethroid resistance that is both strong and very common, but not extreme. Transcriptomic analysis found multiple P450 genes over expressed including CYP6M2, CYP6Z3, CYP6P3, CYP6P4, CYP6AA1 and CYP9K1 in An. gambiae and CYP6N1, CYP6M7, CYP6M1 and CYP6Z3 in An. funestus. Indeed, very similar suites of P450 enzymes commonly associated with resistant populations elsewhere in Africa were detected as over expressed suggesting a convergence of mechanisms across Sub-Saharan African malaria vectors. The findings give insight into factors that may correlate with pyrethroid PBO LLIN success, broadly supporting model predictions, but revision to guidelines previously issued by the World Health Organization is warranted.

Anopheles stephensi, an invasive malaria vector in Africa, has the potential to impact the landscape of malaria on the continent, threatening to put an additional 126 million people per year at risk of malaria, largely in peri-urban/urban areas. To accelerate the early detection and rapid response to An. stephensi and ensure no gains made in malaria control and elimination are lost, it is critical to confirm the presence of the species and the geographic extent of its spread to inform control. However, morphological identification may be misinterpreted if specimens are damaged and existing molecular species confirmation assays require specialized laboratory equipment and training and may be challenging to interpret, requiring additional sequencing confirmation. A colorimetric rapid loop-mediated isothermal amplification (LAMP) assay for molecular An. stephensi species identification was developed and optimized. The colorimetric assay requires only a heat source and reagents and can be used with or without DNA extraction resulting in positive color change in 30-35 minutes. To determine analytical sensitivity, a 1:10 dilution series of the DNA extract was conducted showing 100% assay sensitivity down to 0.003 nanograms. To determine specificity, three different An. stephensi laboratory strains (STE2, SDA 500, UCI), 8 other Anopheles mosquito species, and Aedes aegypti were compared, and the results indicated 100% specificity across these species. To determine use without the need for DNA extraction, samples evaluated included a single mosquito leg, whole adult or larval mosquitoes, and pooled DNA extract from several mosquito species. A total of 1687 individual reactions were tested during optimization and all LAMP assay results were compared against the conventional PCR assay and confirmed through Sanger sequencing. To validate the optimized assay on wild caught specimens, DNA extracted from 12 wild caught, sequence-confirmed An. stephensi from Marsabit, Kenya, were tested and the colorimetric assay was accurate in identifying all of the specimens as An. stephensi. The assay described presents an opportunity to accelerate An. stephensi molecular identification in new and existing locations in Africa, within its endemic range, and globally. These findings present a simple, rapid, unique alternative to existing PCR and sequencing-based An. stephensi species identification and confirmation strategies. With additional field validation studies, molecular screening tools like the colorimetric LAMP-based An. stephensi species identification (CLASS) assay fill an important gap of rapid confirmation of this invasive vector and presents an ideal opportunity to better understand the spread of the species in Africa and other recently invaded areas, thus accelerating a response to mitigate its long-term impacts on malaria on the continent.

Aedes albopictus is a cosmopolitan mosquito species capable of transmitting arboviral diseases such as dengue, chikungunya, and Zika. To control this and similar species, public and private entities often rely on pyrethroid insecticides. Insecticide resistance status and physiological traits, such as body size, may contribute to local patterns of abundance, which is important for planning vector control. In this study, we genetically screened Ae. albopictus collected from June to August, 2017, in Mecklenburg County, North Carolina, for mutations conferring pyrethroid resistance, and examined spatiotemporal patterns of specimen size, as measured by wing length. We hypothesized that size variation would be associated with factors found to influence abundance in similar populations of Ae. albopictus, and could therefore serve as a proxy measure. The genetic screening results indicated that known pyrethroid resistance alleles in two kdr regions are not present in this population. We detected no significant associations between wing length and socioeconomic and landscape factors, but mosquitoes collected in June had significantly longer wing length than in July or August. The lack of resistance indicators suggest that this population has not developed insecticide resistance via voltage-gated sodium channel mutations. The greater wing lengths in June are likely driven by meteorological patterns, suggesting that short-term weather cues may modulate morphological characteristics that, in turn, affect local fecundity and virus transmission potential.