The Anopheles coluzzii range extends into Kenya: detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

BACKGROUND: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. The current study sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well as describe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzii populations. METHODS: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing (WGS) data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Comparisons were made with An. coluzzii cohorts from West and Central Africa. RESULTS: This study reports the detection of An. coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points from which samples were analyzed and its presence confirmed through taxonomic analysis. Additionally, there was a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies up to 64%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. CONCLUSIONS: These findings emphasize the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

The Anopheles coluzzii range extends into Kenya: Detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

Background: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. In the current study, we sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well asdescribe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzi populations. Methods: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Results: This study reports the detection of Anopheles coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points sampled and its presence confirmed through taxonomic analysis. Additionally, we found a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies of ~60%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. Conclusions: These findings emphasise the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

Unraveling the genomic complexity of sylvatic mosquitoes in changing Neotropical environments.

Sylvatic New World mosquitoes (e.g. Old-growth Forest species) can transmit viruses among non-human primates. This could be a continuous source of viral cycling and spillover events from animals to humans, particularly in changing environments. However, most species of Neotropical sylvatic mosquitoes (genera Aedes, Haemagogus, and Sabethes), which include vector and non-vector species, currently lack genomic resources because there is no reliable and accurate approach for creating de novo reference genomes for these insects. This is a major knowledge gap in the biology of these mosquitoes, restricting our ability to predict and mitigate the emergence and spread of novel arboviruses in Neotropical regions. We discuss recent advances and potential solutions for generating hybrid de novo assemblies from vector and non-vector species using pools of consanguineous offspring. We also discussed research opportunities likely to emerge from these genomic resources.

The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmission.

Monitoring the invasion process of the Asian tiger mosquito Aedes albopictus and its interaction with the contender Aedes aegypti, is critical to prevent and control the arthropod-borne viruses (i.e., Arboviruses) they transmit to humans. Generally, the superior ecological competitor Ae. albopictus displaces Ae. aegypti from most geographic areas, with the combining factors of biology and environment influencing the competitive outcome. Nonetheless, detailed studies asserting displacement come largely from sub-tropical areas, with relatively less effort being made in tropical environments, including no comprehensive research about Aedes biological interactions in Mesoamerica. Here, we examine contemporary and historical mosquito surveillance data to assess the role of shifting abiotic conditions in shaping the spatiotemporal distribution of competing Aedes species in the Republic of Panama. In accordance with prior studies, we show that Ae. albopictus has displaced Ae. aegypti under suboptimal wet tropical climate conditions and more vegetated environments within the southwestern Azuero Peninsula. Conversely, in the eastern Azuero Peninsula, Ae. aegypti persists with Ae. albopictus under optimal niche conditions in a dry and more seasonal tropical climate. While species displacement was stable over the course of two years, the presence of both species generally appears to fluctuate in tandem in areas of coexistence. Aedes albopictus was always more frequently found and abundant regardless of location and climatic season. The heterogenous environmental conditions of Panama shape the competitive outcome and micro-geographic distribution of Aedes mosquitoes, with potential consequences for the transmission dynamics of urban and sylvatic zoonotic diseases. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10530-021-02482-y).

The genomic signal of local environmental adaptation in Aedes aegypti mosquitoes.

Local adaptation is important when predicting arthropod-borne disease risk because of its impacts on vector population fitness and persistence. However, the extent that vector populations are adapted to the environment generally remains unknown. Despite low population structure and high gene flow in Aedes aegypti mosquitoes across Panama, excepting the province of Bocas del Toro, we identified 128 candidate SNPs, clustered within 17 genes, which show a strong genomic signal of local environmental adaptation. This putatively adaptive variation occurred across fine geographical scales with the composition and frequency of candidate adaptive loci differing between populations in wet tropical environments along the Caribbean coast and dry tropical conditions typical of the Pacific coast. Temperature and vegetation were important predictors of adaptive genomic variation in Ae. aegypti with several potential areas of local adaptation identified. Our study lays the foundations of future work to understand whether environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and whether this could either aid or hinder efforts of population control.

Proteomic fingerprinting of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.

Correction: Habitat disturbance and the organization of bacterial communities in Neotropical hematophagous arthropods.

[This corrects the article DOI: 10.1371/journal.pone.0222145.].

Habitat disturbance and the organization of bacterial communities in Neotropical hematophagous arthropods.

The microbiome plays a key role in the biology, ecology and evolution of arthropod vectors of human pathogens. Vector-bacterial interactions could alter disease transmission dynamics through modulating pathogen replication and/or vector fitness. Nonetheless, our understanding of the factors shaping the bacterial community in arthropod vectors is incomplete. Using large-scale 16S amplicon sequencing, we examine how habitat disturbance structures the bacterial assemblages of field-collected whole-body hematophagous arthropods that vector human pathogens including mosquitoes (Culicidae), sand flies (Psychodidae), biting midges (Ceratopogonidae) and hard ticks (Ixodidae). We found that all comparisons of the bacterial community among species yielded statistically significant differences, but a difference was not observed between adults and nymphs of the hard tick, Haemaphysalis juxtakochi. While Culicoides species had the most distinct bacterial community among dipterans, tick species were composed of entirely different bacterial OTU's. We observed differences in the proportions of some bacterial types between pristine and disturbed habitats for Coquillettidia mosquitoes, Culex mosquitoes, and Lutzomyia sand flies, but their associations differed within and among arthropod assemblages. In contrast, habitat quality was a poor predictor of differences in bacterial classes for Culicoides biting midges and hard tick species. In general, similarities in the bacterial communities among hematophagous arthropods could be explained by their phylogenetic relatedness, although intraspecific variation seems influenced by habitat disturbance.

Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.

High infestation of invasive Aedes mosquitoes in used tires along the local transport network of Panama.

BACKGROUND: The long-distance dispersal of the invasive disease vectors Aedes aegypti and Aedes albopictus has introduced arthropod-borne viruses into new geographical regions, causing a significant medical and economic burden. The used-tire industry is an effective means of Aedes dispersal, yet studies to determine Aedes occurrence and the factors influencing their distribution along local transport networks are lacking. To assess infestation along the primary transport network of Panama we documented all existing garages that trade used tires on the highway and surveyed a subset for Ae. aegypti and Ae. albopictus. We also assess the ability of a mass spectrometry approach to classify mosquito eggs by comparing our findings to those based on traditional larval surveillance. RESULTS: Both Aedes species had a high infestation rate in garages trading used tires along the highways, providing a conduit for rapid dispersal across Panama. However, generalized linear models revealed that the presence of Ae. aegypti is associated with an increase in road density by a log-odds of 0.44 (0.73 ± 0.16; P = 0.002), while the presence of Ae. albopictus is associated with a decrease in road density by a log-odds of 0.36 (0.09 ± 0.63; P = 0.008). Identification of mosquito eggs by mass spectrometry depicted similar occurrence patterns for both Aedes species as that obtained with traditional rearing methods. CONCLUSIONS: Garages trading used tires along highways should be targeted for the surveillance and control of Aedes-mosquitoes and the diseases they transmit. The identification of mosquito eggs using mass spectrometry allows for the rapid evaluation of Aedes presence, affording time and cost advantages over traditional vector surveillance; this is of importance for disease risk assessment.

Maternal invasion history of Aedes aegypti and Aedes albopictus into the Isthmus of Panama: Implications for the control of emergent viral disease agents.

Despite an increase in dengue outbreaks and the arrival of chikungunya and Zika disease in Panama, studies on the demographic history of the invasive Aedes mosquitoes that are the principle vectors of these diseases are still lacking in this region. Here, we assess the genetic diversity of these mosquitoes in order to decipher their invasion histories into the Isthmus of Panama. DNA sequences from the mitochondrial cytochrome C oxidase I gene obtained from 30 localities in 10 provinces confirmed the presence of more than one mitochondrial haplogroup (i.e., maternal lineage) in each species. The invasion of Aedes albopictus was likely from temperate European countries, as the most frequent and widespread haplogroup in Panama harbored variants that are uncommon elsewhere in the Americas. Two infrequent and geographically restricted Ae. albopictus haplotypes appear to have subsequently invaded Panama from neighboring Costa Rica and the USA, respectively. In addition, we recovered two deeply divergent mitochondrial clades in Panamanian Aedes aegypti. The geographic origins of these clades is unknown, given that divergence in the mitochondrial genome is probably due to ancient population processes within the native range of Ae. aegypti, rather than due to its global expansion out of Africa. However, Panamanian Ae. aegypti mitochondrial sequences within the first clade were closely related to others from Colombia, Bolivia, Brazil, Mexico and the USA, suggesting two separate invasions from Western Hemisphere source populations. The pattern of increased genetic diversity in Aedes mosquitoes in Panama is likely facilitated by the numerous land and water inter-connections across the country, which allows them to enter via sea- and land-transportation from Europe, North, Central and South America. Our results here should be considered in disease mitigation programs if emergent arboviruses are to be effectively diminished in Panama through vector suppression.

The extracellular matrix protein betaIG-H3 is expressed at myotendinous junctions and supports muscle cell adhesion.

Molecules of the extracellular matrix (ECM) play important roles in the development and maintenance of myotendinous junctions (MTJs), specialized regions of muscle to bone union. In this report we provide evidence that skeletal muscle cells synthesize the collagen- and fibronectin-binding ECM protein betaIG-H3 and that betaIG-H3 is localized to MTJs. In situ hybridization experiments revealed that during E16.5-E18.5 of murine development, betaIG-H3 RNA transcripts were expressed where developing skeletal muscle fibers contact primordial cartilage and bone. Immunohistochemical analysis verified that the betaIG-H3 protein itself localized distinctively at MTJs, and ultrastructural analysis suggested that betaIG-H3 associates with extracellular fibers and the surface of cells. In vitro, recombinant betaIG-H3 functioned as an adhesion substratum for skeletal muscle cells. Adhesion was significantly reduced by anti-integrin alpha7 and beta1 antibodies, suggesting that betaIG-H3 binds to skeletal muscle cells via alpha7beta1 integrin. Localization of betaIG-H3 to the termini of skeletal muscle fibers and the binding of betaIG-H3 to cells and to molecules of the ECM suggests that betaIG-H3 may play an organizational and structural role in developing MTJs, linking skeletal muscle to components of the ECM.

Comparison of "selective" opiate receptor antagonists on the isolated mouse vas deferens.

The selectivity and relative potencies of opiate receptor antagonists were compared on the mouse vas deferens preparation. ICI-174864 was found to be a highly selective antagonist at delta opiate receptors equal in potency to naltrexone in blocking the actions of delta agonists. Although less potent than naltrexone, beta-funaltrexamine (beta-FNA) and Mr-1452, like naltrexone, were less selective in that they blocked the actions of mu, delta and kappa agonists. The relative potencies of beta-FNA and Mr-1452 in antagonizing the three types of agonists also were similar to naltrexone.