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We performed nanopore-based metagenomic screening on 885 ticks collected from 6 locations in Mongolia and divided the results into 68 samples: 23 individual samples and 45 pools of 2–12 tick samples each. We detected bacterial and parasitic pathogens Anaplasma ovis, Babesia microti, Coxiella burnetii, Borrelia miyamotoi, Francisella tularensis subsp. holarctica and novicida, Spiroplasma ixodetis, Theileria equi, and Rickettsia spp., including R. raoultii, R. slovaca, and R. canadensis. We identified the viral pathogens Crimean-Congo hemorrhagic fever virus (2.9%), recently described Alongshan virus (ALSV) (2.9%), and Beiji nairovirus (5.8%). We assembled ALSV genomes, and maximum-likelihood analyses revealed clustering with viruses reported in humans and ticks from China. For ALSV, we identified surface glycoprotein markers associated with isolates from Asia viruses hosted by Ixodes persulcatus ticks. We also detected 20 virus species of unknown public health impact, including a near-complete Yanggou tick virus genome. Our findings demonstrate that nanopore sequencing can aid in detecting endemic and emerging tickborne pathogens.

IntroductionCoxiella burnetii (C. burnetii)-infected livestock and wildlife have been epidemiologically linked to human Q fever outbreaks. Despite this growing zoonotic threat, knowledge of coxiellosis in wild animals remains limited, and studies to understand their epidemiologic role are needed. In C. burnetii-endemic areas, ticks have been reported to harbor and spread C. burnetii and may serve as indicators of risk of infection in wild animal habitats. Therefore, the aim of this study was to compare molecular techniques for detecting C. burnetii DNA in ticks.MethodsIn total, 169 ticks from wild animals and cattle in wildlife conservancies in northern Kenya were screened for C. burnetii DNA using a conventional PCR (cPCR) and two field-friendly techniques: Biomeme’s C. burnetii qPCR Go-strips (Biomeme) and a new C. burnetii PCR high-resolution melt (PCR-HRM) analysis assay. Results were evaluated, in the absence of a gold standard test, using Bayesian latent class analysis (BLCA) to characterize the proportion of C. burnetii positive ticks and estimate sensitivity (Se) and specificity (Sp) of the three tests.ResultsThe final BLCA model included main effects and estimated that PCR-HRM had the highest Se (86%; 95% credible interval: 56–99%), followed by the Biomeme (Se = 57%; 95% credible interval: 34–90%), with the estimated Se of the cPCR being the lowest (24%, 95% credible interval: 10–47%). Specificity estimates for all three assays ranged from 94 to 98%. Based on the model, an estimated 16% of ticks had C. burnetii DNA present.DiscussionThese results reflect the endemicity of C. burnetii in northern Kenya and show the promise of the PCR-HRM assay for C. burnetii surveillance in ticks. Further studies using ticks and wild animal samples will enhance understanding of the epidemiological role of ticks in Q fever.

Abstract Background Surveillance data documenting tick and tick-borne disease (TBD) prevalence is needed to develop risk assessments and implement control strategies. Despite extensive research in Africa, there is no standardized, comprehensive review. Methods Here we tackle this knowledge gap, by producing a comprehensive review of research articles on ticks and TBD between 1901 and 2020 in Chad, Djibouti, Ethiopia, Kenya, Tanzania, and Uganda. Over 8356 English language articles were recovered. Our search strategy included 19 related MeSH terms. Articles were reviewed, and 331 met inclusion criteria. Articles containing mappable data were compiled into a standardized data schema, georeferenced, and uploaded to VectorMap. Results Tick and pathogen matrixes were created, providing information on vector distributions and tick–pathogen associations within the six selected African countries. Conclusions These results provide a digital, mappable database of current and historical tick and TBD distributions across six countries in Africa, which can inform specific risk modeling, determine surveillance gaps, and guide future surveillance priorities. Graphical Abstract

The threat posed by emerging infectious diseases is a major concern for global public health, animal health and food security, and the role of birds in transmission is increasingly under scrutiny. Each year, millions of mass-reared game-farm birds are released into the wild, presenting a unique and a poorly understood risk to wild and susceptible bird populations, and to human health. In particular, the shedding of enteric pathogens through excrement into bodies of water at shared migratory stop-over sites, and breeding and wintering grounds, could facilitate multi-species long-distance pathogen dispersal and infection of high numbers of naive endemic birds annually. The Mallard (Anas platyrhynchos) is the most abundant of all duck species, migratory across much of its range, and an important game species for pen-rearing and release. Major recent population declines along the US Atlantic coast has been attributed to game-farm and wild mallard interbreeding and the introduction maladaptive traits into wild populations. However, pathogen transmission and zoonosis among game-farms Mallard may also impact these populations, as well as wildlife and human health. Here, we screened 16 game-farm Mallard from Wisconsin, United States, for enteric viral pathogens using metatranscriptomic data. Four families of viral pathogens were identified – Picobirnaviridae (Genogroup I), Caliciviridae (Duck Nacovirus), Picornaviridae (Duck Aalivirus) and Sedoreoviridae (Duck Rotavirus G). To our knowledge, this is the first report of Aalivirus in the Americas, and the first report of Calicivirus outside domestic chicken and turkey flocks in the United States. Our findings highlight the risk of viral pathogen spillover from peri-domestically reared game birds to naive wild bird populations.

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum.

Abstract We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Abstract Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188–250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

IntroductionWe evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays.MethodsForty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach.ResultsEleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted segment read counts was observed between PCR-positive and PCR-negative samples. NS further enabled the initial description of Quaranjavirus sequences in ticks, where human and avian pathogenicity of particular isolates had been previously documented.DiscussionNS was observed to surpass broad-range and nested amplification in detection and to generate sufficient genome-wide data for investigating virus diversity. It can be employed for monitoring pathogens in tick vectors or human/animal clinical samples in hot-spot regions for examining zoonotic spillover.

Abstract Background Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex. Methods Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees. Results Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species—Anopheles janconnae and An. albitarsis G—which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits. Conclusion The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J. Graphical Abstract

Abstract With over 3500 mosquito species described, accurate species identification of the few implicated in disease transmission is critical to mosquito borne disease mitigation. Yet this task is hindered by limited global taxonomic expertise and specimen damage consistent across common capture methods. Convolutional neural networks (CNNs) are promising with limited sets of species, but image database requirements restrict practical implementation. Using an image database of 2696 specimens from 67 mosquito species, we address the practical open-set problem with a detection algorithm for novel species. Closed-set classification of 16 known species achieved 97.04 ± 0.87% accuracy independently, and 89.07 ± 5.58% when cascaded with novelty detection. Closed-set classification of 39 species produces a macro F1-score of 86.07 ± 1.81%. This demonstrates an accurate, scalable, and practical computer vision solution to identify wild-caught mosquitoes for implementation in biosurveillance and targeted vector control programs, without the need for extensive image database development for each new target region.

Focusing on the utility of ticks as xenosurveillance sentinels to expose circulating pathogens in Kenyan drylands, host-feeding ticks collected from wild ungulates [buffaloes, elephants, giraffes, hartebeest, impala, rhinoceros (black and white), zebras (Grévy’s and plains)], carnivores (leopards, lions, spotted hyenas, wild dogs), as well as regular domestic and Boran cattle were screened for pathogens using metagenomics. A total of 75 host-feeding ticks [Rhipicephalus (97.3%) and Amblyomma (2.7%)] collected from 15 vertebrate taxa were sequenced in 46 pools. Fifty-six pathogenic bacterial species were detected in 35 pools analyzed for pathogens and relative abundances of major phyla. The most frequently observed species was Escherichia coli (62.8%), followed by Proteus mirabilis (48.5%) and Coxiella burnetii (45.7%). Francisella tularemia and Jingmen tick virus (JMTV) were detected in 14.2 and 13% of the pools, respectively, in ticks collected from wild animals and cattle. This is one of the first reports of JMTV in Kenya, and phylogenetic reconstruction revealed significant divergence from previously known isolates and related viruses. Eight fungal species with human pathogenicity were detected in 5 pools (10.8%). The vector-borne filarial pathogens (Brugia malayi, Dirofilaria immitis, Loa loa), protozoa (Plasmodium spp., Trypanosoma cruzi), and environmental and water-/food-borne pathogens (Entamoeba histolytica, Encephalitozoon intestinalis, Naegleria fowleri, Schistosoma spp., Toxoplasma gondii, and Trichinella spiralis) were detected. Documented viruses included human mastadenovirus C, Epstein-Barr virus and bovine herpesvirus 5, Trinbago virus, and Guarapuava tymovirus-like virus 1. Our findings confirmed that host-feeding ticks are an efficient sentinel for xenosurveillance and demonstrate clear potential for wildlife-livestock-human pathogen transfer in the Kenyan landscape.

Abstract Background The need to develop new products and novel approaches for malaria vector control is recognized as a global health priority. One approach to meeting this need has been the development of new products for indoor residual spraying (IRS) with novel active ingredients for public health. While initial results showing the impact of several of these next-generation IRS products have been encouraging, questions remain about how to best deploy them for maximum impact. To help address these questions, a 2-year cluster-randomized controlled trial to measure the impact of IRS with a microencapsulated formulation of pirimiphos-methyl (PM) in an area with high ownership of long-lasting insecticidal nets (LLINs) was conducted in a high-transmission district of central Mozambique with pyrethroid resistant vectors. Presented here are the results of the vector surveillance component of the trial. Methods The 2 year, two-armed trial was conducted in Mopeia District, Zambezia Province, Mozambique. In ten sentinel villages, five that received IRS with PM in October–November 2016 and again in October–November 2017 and five that received no IRS, indoor light trap collections and paired indoor-outdoor human landing collections catches (HLCs) were conducted monthly from September 2016 through October 2018. A universal coverage campaign in June 2017, just prior to the second spray round, distributed 131,540 standard alpha-cypermethrin LLINs across all study villages and increased overall net usage rates in children under 5 years old to over 90%. Results The primary malaria vector during the trial was Anopheles funestus sensu lato (s.l.), and standard World Health Organization (WHO) tube tests with this population indicated variable but increasing resistance to pyrethroids (including alpha-cypermethrin, from > 85% mortality in 2017 to 7% mortality in 2018) and uniform susceptibility to PM (100% mortality in both years). Over the entire duration of the study, IRS reduced An. funestus s.l. densities by 48% (CI95 33–59%; p

Abstract Long-distance migration of insects impacts food security, public health, and conservation–issues that are especially significant in Africa. Windborne migration is a key strategy enabling exploitation of ephemeral havens such as the Sahel, however, its knowledge remains sparse. In this first cross-season investigation (3 years) of the aerial fauna over Africa, we sampled insects flying 40–290 m above ground in Mali, using nets mounted on tethered helium-filled balloons. Nearly half a million insects were caught, representing at least 100 families from thirteen orders. Control nets confirmed that the insects were captured at altitude. Thirteen ecologically and phylogenetically diverse species were studied in detail. Migration of all species peaked during the wet season every year across localities, suggesting regular migrations. Species differed in flight altitude, seasonality, and associated weather conditions. All taxa exhibited frequent flights on southerly winds, accounting for the recolonization of the Sahel from southern source populations. “Return” southward movement occurred in most taxa. Estimates of the seasonal number of migrants per species crossing Mali at latitude 14°N were in the trillions, and the nightly distances traversed reached hundreds of kilometers. The magnitude and diversity of windborne insect migration highlight its importance and impacts on Sahelian and neighboring ecosystems.

Arboviral mosquito vectors are key targets for the surveillance and control of vector-borne diseases worldwide. In recent years, changes to the global distributions of these species have been a major research focus, aimed at predicting outbreaks of arboviral diseases. In this study, we analyzed a global scenario of climate change under regional rivalry to predict changes to these species’ distributions over the next century. Using occurrence data from VectorMap and environmental variables (temperature and precipitation) from WorldClim v. 2.1, we first built fundamental niche models for both species with the boosted regression tree modelling approach. A scenario of climate change on their fundamental niche was then analyzed. The shared socioeconomic pathway scenario 3 (regional rivalry) and the global climate model Geophysical Fluid Dynamics Laboratory Earth System Model v. 4.1 (GFDL-ESM4.1; gfdl.noaa.gov) were utilized for all analyses, in the following time periods: 2021–2040, 2041–2060, 2061–2080, and 2081–2100. Outcomes from these analyses showed that future climate change will affect Ae. aegypti and Ae. albopictus distributions in different ways across the globe. The Northern Hemisphere will have extended Ae. aegypti and Ae. albopictus distributions in future climate change scenarios, whereas the Southern Hemisphere will have the opposite outcomes. Europe will become more suitable for both species and their related vector-borne diseases. Loss of suitability in the Brazilian Amazon region further indicated that this tropical rainforest biome will have lower levels of precipitation to support these species in the future. Our models provide possible future scenarios to help identify locations for resource allocation and surveillance efforts before a significant threat to human health emerges.

Background and Methods: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. Results: Viruses belonging to the Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. Conclusions: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Abstract Background Currently, knowledge regarding the phlebotomine sand fly (Diptera: Psychodidae) fauna of Turkey is restricted to regions with endemic leishmaniasis. However, rapidly changing environmental and social conditions highlight concerns on the possible future expansion of sand fly-borne diseases in Turkey, promoting risk assessment through biosurveillance activities in non-endemic regions. Traditional morphological approaches are complicated by extensive cryptic speciation in sand flies, thus integrated studies utilizing DNA markers are becoming increasingly important for correct sand fly identification. This study contributes to the knowledge of the sand fly fauna in understudied regions of Turkey, and provides an extensive DNA barcode reference library of expertly identified Turkish sand fly species for the first time. Methods Fly sampling was conducted at 101 locations from 29 provinces, covering all three biogeographical regions of Turkey. Specimens were morphologically identified using available keys. Cytochrome c oxidase I (cox1) barcode sequences were analyzed both for morphologically distinct species and those specimens with cryptic identity. A taxon identity tree was obtained using Neighbor Joining (NJ) analysis. Species boundaries among closely related taxa evaluated using ABGD, Maximum Likelihood (ML) and haplotype network analyses. Sand fly richness of all three biogeographical regions were compared using nonparametric species richness estimators. Results A total of 729 barcode sequences (including representatives of all previously reported subgenera) were obtained from a total of 9642 sand fly specimens collected in Turkey. Specimens belonging to the same species or species complex clustered together in the NJ tree, regardless of their geographical origin. The species delimitation methods revealed the existence of 33 MOTUs, increasing the previously reported 28 recorded sand fly species by 17.8%. The richest sand fly diversity was determined in Anatolia, followed by the Mediterranean, and then the Black Sea regions of the country. Conclusions A comprehensive cox1 reference library is provided for the sand fly species of Turkey, including the proposed novel taxa discovered herein. Our results have epidemiological significance exposing extensive distributions of proven and suspected sand fly vectors in Turkey, including those areas currently regarded as non-endemic for sand fly-borne disease.

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling-a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)-mosquitoes and Triatominae (Hemiptera: Reduviidae)-kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.

BackgroundThe recent reports of Aedes aegypti and Ae. albopictus populations in Turkey, in parallel with the territorial expansion identified in several surrounding countries, have raised concerns about the establishment and re-establishment of these invasive Aedes mosquitoes in Turkey. This cross-sectional study was performed to detect Aedes aegypti and Ae. albopictus in regions of recent incursions, and screen for viral pathogens known to be transmitted elsewhere by these species.MethodologyMosquitoes were collected at several locations in Artvin, Rize and Trabzon provinces of the Black Sea region during 2016-2017, identified morphologically, pooled and analyzed via generic or specific nucleic acid amplification assays. Viruses in positive pools were identified by product sequencing, cell culture inoculation and next generation sequencing (NGS) in selected specimens.Principal findingsThe study group comprised 791 specimens. Aedes albopictus was the most abundant species in all locations (89.6%), followed by Ae. aegypti (7.8%) and Culex pipiens (2.5%). Mosquitoes were screened for viruses in 65 pools where fifteen (23.1%) were reactive. The infecting strains was identified as West Nile virus (WNV) in 5 pools (7.7%) with Ae. albopictus or Cx. pipiens mosquitoes. The obtained WNV sequences phylogenetically grouped with local and global lineage 1 clade 1a viruses. In 4 (6.2%) and 6 (9.2%) pools, respectively, cell fusing agent virus (CFAV) and Aedes flavivirus (AEFV) sequences were characterized. NGS provided a near-complete AEFV genome in a pool of Ae. albopictus. The strain is provisionally called "AEFV-Turkey", and functional analysis of the genome revealed several conserved motifs and regions associated with virus replication. Merida-like virus Turkey (MERDLVT), a recently-described novel rhabdovirus, was also co-detected in a Cx. pipiens pool also positive for WNV.Conclusions/significanceInvasive Aedes mosquitoes are established in certain locations of northeastern Turkey. Herein we conclusively show the role of these species in WNV circulation in the region. Biosurveillance is imperative to monitor the spread of these species further into Asia Minor and to detect possible introduction of pathogens.

Abstract Background Pseudoskusea, Rusticoidus and Protomacleaya were well-recognized, morphologically distinct subgenera within the genus Aedes prior to a series of taxonomic changes over the past 15 years by Reinert, Harbach and Kitching, when they were recognized as subgenera of the genus Ochlerotatus. In our recent effort to stabilize the Tribe Aedini, we synonymized these subgenera and associated species back into the genus Aedes, but incorrectly assigned them as putative informal groups, instead of reinstating them to subgenera. Conclusion Here we formally elevate three traditionally recognized subgenera (Pseudoskusea, Rusticoidus and Protomacleaya) within the genus Aedes.

Abstract Background Malaria remains a significant public health issue in South America. Future climate change may influence the distribution of the disease, which is dependent on the distribution of those Anopheles mosquitoes competent to transmit Plasmodium falciparum. Herein, predictive niche models of the habitat suitability for P. falciparum, the current primary vector Anopheles darlingi and nine other known and/or potential vector species of the Neotropical Albitarsis Complex, were used to document the current situation and project future scenarios under climate changes in South America in 2070. Methods To build each ecological niche model, we employed topography, climate and biome, and the currently defined distribution of P. falciparum, An. darlingi and nine species comprising the Albitarsis Complex in South America. Current and future (i.e., 2070) distributions were forecast by projecting the fitted ecological niche model onto the current environmental situation and two scenarios of simulated climate change. Statistical analyses were performed between the parasite and each vector in both the present and future scenarios to address potential vector roles in the dynamics of malaria transmission. Results Current distributions of malaria vector species were associated with that of P. falciparum, confirming their role in transmission, especially An. darlingi, An. marajoara and An. deaneorum. Projected climate changes included higher temperatures, lower water availability and biome modifications. Regardless of future scenarios considered, the geographic distribution of P. falciparum was exacerbated in 2070 South America, with the distribution of the pathogen covering 35-46 % of the continent. As the current primary vector An. darlingi showed low tolerance for drier environments, the projected climate change would significantly reduce suitable habitat, impacting both its distribution and abundance. Conversely, climate generalist members of the Albitarsis Complex showed significant spatial and temporal expansion potential in 2070, and we conclude these species will become more important in the dynamics of malaria transmission in South America. Conclusions Our data suggest that climate and landscape effects will elevate the importance of members of the Albitarsis Complex in malaria transmission in South America in 2070, highlighting the need for further studies addressing the bionomics, ecology and behaviours of the species comprising the Albitarsis Complex.

We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. We subsequently screened 7223 ticks, representing 15 species in five genera, collected from various regions in Anatolia and eastern Thrace, Turkey. Moreover, we tested specimens from various patient cohorts (n = 103), and canine (n = 60), bovine (n = 20) and avian specimens (n = 65). JMTV nucleic acids were detected in 3.9% of the tick pools, including those from several tick species from the genera Rhipicephalus and Haemaphysalis, and Hyalomma marginatum, the main vector of CCHFV in Turkey. Phylogenetic analysis supported two separate clades, independent of host or location, suggesting ubiquitous distribution in ticks. JMTV was not recovered from any human, animal or bird specimens tested. Near-complete viral genomes were sequenced from the prototype isolate and from three infected tick pools. Genome topology and functional organization were identical to the members of Jingmen group viruses. Phylogenetic reconstruction of individual viral genome segments and functional elements further supported the close relationship of the strains from Kosovo. We further identified probable recombination events in the JMTV genome, involving closely-related strains from Anatolia or China.

We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kırklareli, and Tekirdağ (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Şanlıurfa (Southeastern Anatolia) provinces from 2013−2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequences in three pools. A comprehensive analysis revealed three distinct, monophyletic virus genotypes, comprised of previously-described viruses from Anatolia and the Balkans, with unique fingerprints in conserved amino acid motifs in viral replicase. A novel tick phlebovirus group was discovered circulating in the Balkans and Turkey, with at least three genotypes or species. No evidence for replication in vertebrates or infections in clinical cases could be demonstrated.

Two snapshot surveys to establish the diversity and ecological preferences of mosquitoes (Diptera: Culicidae) in the terra firme primary rain forest surrounding the Tiputini Biodiversity Station in the UNESCO Yasuní Biosphere Reserve of eastern Amazonian Ecuador were carried out in November 1998 and May 1999. The mosquito fauna of this region is poorly known; the focus of this study was to obtain high quality link-reared specimens that could be used to unequivocally confirm species level diversity through integrated systematic study of all life stages and DNA sequences. A total of 2,284 specimens were preserved; 1,671 specimens were link-reared with associated immature exuviae, all but 108 of which are slide mounted. This study identified 68 unique taxa belonging to 17 genera and 27 subgenera. Of these, 12 are new to science and 37 comprise new country records. DNA barcodes [658-bp of the mtDNA cytochrome c oxidase ( COI ) I gene] are presented for 58 individuals representing 20 species and nine genera. DNA barcoding proved useful in uncovering and confirming new species and we advocate an integrated systematics approach to biodiversity studies in future. Associated bionomics of all species collected are discussed. An updated systematic checklist of the mosquitoes of Ecuador (n = 179) is presented for the first time in 60 years.

INTRODUCTION:Yellow fever continues to be a problem in sub-Saharan Africa with repeated epidemics occurring. The mosquito Aedes bromeliae is a major vector of yellow fever, but it cannot be readily differentiated from its non-vector zoophilic sister species Ae. lilii using morphological characters. Genetic differences have been reported between anthropophilic Ae. bromeliae and zoophilic Ae. lilii and between forest and domestic populations. However, due to the application of different molecular markers and non-overlapping populations employed in previous studies, interpretation of species delimitation is unclear. METHODOLOGY/PRINCIPLE FINDINGS:DNA sequences were generated from specimens of Ae. simpsoni s.l. from the Republic of Benin, Tanzania and Uganda for two nuclear genes apolipophorin 2 (apoLp2) and cytochrome p450 (CYPJ92), the ribosomal internal transcribed spacer region (ITS) and the mitochondrial cytochrome c oxidase (COI) barcoding region. Nuclear genes apoLp2 and CYPJ92 were unable to differentiate between species Ae. bromeliae and Ae. lilii due to ancestral lineage sorting, while ITS sequence data provided clear topological separation on a phylogeny. The standard COI barcoding region was shown to be subject to species introgression and unable to clearly distinguish the two taxa. Here we present a reliable direct PCR-based method for differentiation of the vector species Ae. bromeliae from its isomorphic, sympatric and non-biomedically important sister taxon, Ae. lilii, based on the ITS region. Using molecular species verification, we describe novel immature habitats for Ae. lilii and report both sympatric and allopatric populations. Whereas only Ae. lilii is found in the Republic of Benin and only Ae. bromeliae in Tanzania, both species are sympatric in Uganda. CONCLUSIONS/SIGNIFICANCE:Our accurate identification method will allow informed distribution and detailed ecological studies that will facilitate assessment of arboviral disease risk and development of future targeted vector control.

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

The tribe Aedini (Family Culicidae) contains approximately one-quarter of the known species of mosquitoes, including vectors of deadly or debilitating disease agents. This tribe contains the genus Aedes, which is one of the three most familiar genera of mosquitoes. During the past decade, Aedini has been the focus of a series of extensive morphology-based phylogenetic studies published by Reinert, Harbach, and Kitching (RH&K). Those authors created 74 new, elevated or resurrected genera from what had been the single genus Aedes, almost tripling the number of genera in the entire family Culicidae. The proposed classification is based on subjective assessments of the "number and nature of the characters that support the branches" subtending particular monophyletic groups in the results of cladistic analyses of a large set of morphological characters of representative species. To gauge the stability of RH&K's generic groupings we reanalyzed their data with unweighted parsimony jackknife and maximum-parsimony analyses, with and without ordering 14 of the characters as in RH&K. We found that their phylogeny was largely weakly supported and their taxonomic rankings failed priority and other useful taxon-naming criteria. Consequently, we propose simplified aedine generic designations that 1) restore a classification system that is useful for the operational community; 2) enhance the ability of taxonomists to accurately place new species into genera; 3) maintain the progress toward a natural classification based on monophyletic groups of species; and 4) correct the current classification system that is subject to instability as new species are described and existing species more thoroughly defined. We do not challenge the phylogenetic hypotheses generated by the above-mentioned series of morphological studies. However, we reduce the ranks of the genera and subgenera of RH&K to subgenera or informal species groups, respectively, to preserve stability as new data become available.

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.