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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.

Triatomines are infamous as vectors of the parasite Trypanosoma cruzi, the causative agent of Chagas disease. However, climate-driven range expansion and urbanization adaptation of triatomine populations, coupled with their highly diverse feeding strategies (vertebrate haematophagy, kleptohaematophagy, and coprophagy), and has elevated interest in triatomines as potential arboviral vectors. Information on the triatomine virome is scant, with prior records including only eight insect-specific viruses: Triatoma virus (TrV) and Rhodnius prolixus viruses 1-7. Here, we leverage publicly available transcriptome datasets to assess viral diversity in 122 wild and colony kissing bugs representing eight species from six countries. In total, six viruses were detected (including Rhodnius prolixus viruses 4-6), and TrV was detected in almost half of all screened triatomines. This is the first report of TrV in Triatoma brasiliensis and in members of the genus Mepraia (M. gajardoi, M. spinolai, and M. parapatrica), and this effort has vastly expanded the publicly available genomic resources of TrV, adding 39 genome sequences to the single genome sequence currently available in the GenBank database. Furthermore, two additional viruses-Meccus longipennis virus 1 and Drosophila melanogaster Nora virus-are herein reported for the first time from kissing bugs. Meccus longipennis virus 1 was detected in Triatoma infestans from Argentina, Brazil, Chile, and Peru, and Drosophila melanogaster Nora virus was found in T. infestans from Argentina. Our results illustrate the advantage and utility of low-cost transcriptome data mining for the discovery of known and novel arboviruses in triatomines and other potential insect vectors., (© 2024. The Author(s).)

Members of the Orthonairovirus genus (family Nairoviridae) include many tick-borne viruses of significant human and animal health impact, with several recently-documented pathogenic viruses lacking sufficient epidemiological information. We screened 215 adult ticks of seven species collected in Bulgaria, Georgia, Latvia and Poland for orthonairoviruses, followed by nanopore sequencing (NS) for genome characterization. Initial generic amplification revealed Sulina virus (SULV, Orthonairovirus sulinaense), for which an updated amplification assay was used, revealing an overall prevalence of 2.7% in Ixodes ricinus ticks from Latvia. Three complete and additional partial SULV genomes were generated, that consistently formed a separate, distinct clade with further intragroup divergence in the maximum likelihood analyses. Comparisons with previously described viruses from Romania exhibited similar genome topologies, albeit with divergent motifs and cleavage sites on the glycoprotein precursor. Preliminary evidence of recombination involving the S segment was documented, in addition to variations in predicted viral glycoproteins. Generic screening further identified Tacheng tick virus 1 (TCTV1, Orthonairovirus tachengense), with documented human infections, in Dermacentor reticulatus ticks from Poland, with a prevalence of 0.9%. Subsequent NS and assembly provided the first complete TCTV1 genome outside of China, where it was originally described. Phylogenetic analysis of virus genome segments revealed TCTV1-Poland as a discrete taxon within the TCTV1 cluster in the Orthonairovirus genus, representing a geographically segregated clade. Comparable genome topology with TCTV1 from China was observed, aside from minor variations in the M segment. Similar to SULV, TCTV1 exhibited several mismatches on previously described screening primer binding sites, likely to prevent amplification. These findings indicate presence of novel TCTV1 and SULV clades in Eastern Europe, confirming the expansion of orthonairoviruses with pathogenic potential., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Coxiella 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., Methods: In 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., Results: The 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., Discussion: These 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kamau, Witte, Goosen, Mutinda, Villinger, Getange, Khogali, von Fricken, Fèvre, Zimmerman, Linton and Miller.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bourke, Dusek, Ergunay, Linton and Drovetski.)

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., Competing Interests: Declaration of Competing Interest None, (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: This manuscript was prepared whilst KE and BPB held a National Research Council (NRC) Research Associateship Awards at the Walter Reed Biosystematics Unit (WRBU), through the Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, USA.

Mosquito-borne diseases (MBDs) threaten public health and food security globally. We provide the first biogeographic description of the African mosquito fauna (677 species) and the 151 mosquito-borne pathogens (MBPs) they transmit. While mosquito species richness agrees with expectations based on Africa's land surface, African arboviruses and mammalian plasmodia are more speciose than expected. Species assemblages of mosquitoes and MBPs similarly separate sub-Saharan Africa from North Africa, and those in West and Central Africa from eastern and southern Africa. Similarities between mosquitoes and MBPs in diversity and range size suggest that mosquitoes are key in delimiting the range of MBPs. With approximately 25% endemicity, approximately 50% occupying one to three countries and less than 5% occupying greater than 25 countries, the ranges of mosquitoes and MBPs are surprisingly small, suggesting that most MBPs are transmitted by a single mosquito species. Exceptionally widespread mosquito species feed on people and livestock, and most are high-altitude-windborne migrants. Likewise, widespread MBPs are transmitted among people or livestock by widespread mosquitoes, suggesting that adapting to people or livestock and to widespread mosquito species promote range expansion in MBPs. Range size may predict range expansion and emergence risk. We highlight key knowledge gaps that impede prediction and mitigation of future emergence of local and global MBDs.

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., (© 2023. The Author(s).)

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., (© 2023. Springer Nature Limited.)

The genus Dirphys Howard 1914 syn. n. is synonymized with Encarsia Förster, and treated as a species-group of Encarsia , referred to henceforth as the Encarsia mexicana species-group. The monophyly of Encarsia is discussed in relation to Dirphys . The new synonymy is based on phylogenetic analyses of the nuclear ribosomal 28S-D2 gene region (43 taxa, 510 bp). The Encarsia mexicana species-group is recovered as strongly monophyletic within Encarsia. All species of the Encarsia mexicana species-group are revised. The group includes six previously described species, and fourteen newly described species. All species are described (or redescribed) and illustrated. Detailed distributional data, and, where available, plant associate and host records are provided for all species. Encarsia myartsevae Kresslein and Polaszek nom. nov. is here proposed as a replacement name for Encarsia mexicana Myartseva, now preoccupied by Encarsia mexicana (Howard). A dichotomous identification key, supplemented by an online multiple-entry key, is provided for all species.

Introduction: We evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays., Methods: Forty 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., Results: Eleven 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., Discussion: NS 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ergunay, Dincer, Justi, Bourke, Nelson, Liao, Timurkan, Oguz, Sahindokuyucu, Gokcecik, Reinbold-Wasson, Jiang, Achee, Grieco and Linton.)

The Anopheles subgenus Kerteszia is a poorly understood group of mosquitoes that includes several species of medical importance. Although there are currently twelve recognized species in the subgenus, previous studies have shown that this is likely to be an underestimate of species diversity. Here, we undertake a baseline study of species delimitation using the barcode region of the mtDNA COI gene to explore species diversity among a geographically and taxonomically diverse range of Kerteszia specimens. Beginning with 10 of 12 morphologically identified Kerteszia species spanning eight countries, species delimitation analyses indicated a high degree of cryptic diversity. Overall, our analyses found support for at least 28 species clusters within the subgenus Kerteszia . The most diverse taxon was Anopheles neivai , a known malaria vector, with eight species clusters. Five other species taxa showed strong signatures of species complex structure, among them Anopheles bellator , which is also considered a malaria vector. There was some evidence for species structure within An. homunculus , although the results were equivocal across delimitation analyses. The current study, therefore, suggests that species diversity within the subgenus Kerteszia has been grossly underestimated. Further work will be required to build on this molecular characterization of species diversity and will rely on genomic level approaches and additional morphological data to test these species hypotheses.

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.

Recent studies have reported Anopheles mosquitoes captured at high-altitude (40-290 m above ground) in the Sahel. Here, we describe this migration modality across genera and species of African Culicidae and examine its implications for disease transmission and control. As well as Anopheles , six other genera- Culex, Aedes, Mansonia, Mimomyia, Lutzia , and Eretmapodites comprised 90% of the 2,340 mosquitoes captured at altitude. Of the 50 molecularly confirmed species ( N = 2,107), 33 species represented by multiple specimens were conservatively considered high-altitude windborne migrants, suggesting it is a common migration modality in mosquitoes (31-47% of the known species in Mali), and especially in Culex (45-59%). Overall species abundance varied between 2 and 710 specimens/species (in Ae. vittatus and Cx. perexiguus , respectively). At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one blood meal on a vertebrate host prior to their departure. Most taxa were more common at higher sampling altitudes, indicating that total abundance and diversity are underestimated. High-altitude flight activity was concentrated between June and November coinciding with availability of surface waters and peak disease transmission by mosquitoes. These hallmarks of windborne mosquito migration bolster their role as carriers of mosquito-borne pathogens (MBPs). Screening 921 mosquitoes using pan- Plasmodium assays revealed that thoracic infection rate in these high-altitude migrants was 2.4%, providing a proof of concept that vertebrate pathogens are transported by windborne mosquitoes at altitude. Fourteen of the 33 windborne mosquito species had been reported as vectors to 25 MBPs in West Africa, which represent 32% of the MBPs known in that region and include those that inflict the heaviest burden on human and animal health, such as malaria, yellow fever, dengue, and Rift Valley fever. We highlight five arboviruses that are most likely affected by windborne mosquitoes in West Africa: Rift Valley fever, O'nyong'nyong, Ngari, Pangola, and Ndumu. We conclude that the study of windborne spread of diseases by migrating insects and the development of surveillance to map the sources, routes, and destinations of vectors and pathogens is key to understand, predict, and mitigate existing and new threats of public health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yaro, Linton, Dao, Diallo, Sanogo, Samake, Ousmane, Kouam, Krajacich, Faiman, Bamou, Woo, Chapman, Reynolds and Lehmann.)

Data on the prevalence and distribution of ticks and tick-borne diseases in Belize are lacking. Ticks (n = 564) collected from dogs, horses, and vegetation in two villages in Stann Creek District in southeastern Belize in 2018, were molecularly identified and screened for tick-borne nonviral human pathogens. The identity of 417 ticks was molecularly confirmed by DNA barcoding as Rhipicephalus sanguineus (Latreille) (66.43%), Amblyomma ovale Koch (15.59%), Dermacentor nitens Neumann (11.51%), Amblyomma sp. ADB0528 (3.6%), and the remainder being small records (2.87%) of Amblyomma coelebs Neumann, Amblyomma imitator Kohls, Amblyomma tapirellum Dunn, Amblyomma auricularium Conil, and Amblyomma maculatum Koch. Individual tick extracts were screened for the presence of Rickettsia spp., Babesia spp., Babesia microti, Borrelia spp., Ehrlichia spp., and Anaplasma spp. using available conventional polymerase chain reaction (PCR) assays. Rickettsia parkeri strain Atlantic Rainforest was identified in five specimens of A. ovale, and one other unidentified tick, all collected from dogs. Another unidentified tick-also collected from a dog-tested positive for an undefined but previously detected Ehrlichia sp. With the exception of D. nitens, all eight other tick species identified in this study were collected on dogs, suggesting that dogs could be usefully employed as sentinel animals for tick surveillance in Belize., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

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., Competing Interests: MM was employed by Kenya Wildlife Services Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ergunay, Mutinda, Bourke, Justi, Caicedo-Quiroga, Kamau, Mutura, Akunda, Cook, Gakuya, Omondi, Murray, Zimmerman and Linton.)

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., (© 2021. The Author(s).)

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.

Increasingly intimate associations between human society and the natural environment are driving the emergence of novel pathogens, with devastating consequences for humans and animals alike. Prior to emergence, these pathogens exist within complex ecological systems that are characterized by trophic interactions between parasites, their hosts and the environment. Predicting how disturbance to these ecological systems places people and animals at risk from emerging pathogens-and the best ways to manage this-remains a significant challenge. Predictive systems ecology models are powerful tools for the reconstruction of ecosystem function but have yet to be considered for modelling infectious disease. Part of this stems from a mistaken tendency to forget about the role that pathogens play in structuring the abundance and interactions of the free-living species favoured by systems ecologists. Here, we explore how developing and applying these more complete systems ecology models at a landscape scale would greatly enhance our understanding of the reciprocal interactions between parasites, pathogens and the environment, placing zoonoses in an ecological context, while identifying key variables and simplifying assumptions that underly pathogen host switching and animal-to-human spillover risk. As well as transforming our understanding of disease ecology, this would also allow us to better direct resources in preparation for future pandemics.

The Neotropical Albitarsis Group is a complex assemblage of essentially isomorphic species which currently comprises eight recognized species-five formally described (Anopheles albitarsis Lynch-Arribalzaga, An. deaneorum Rosa-Freitas, An. janconnae Wilkerson and Sallum, An. marajoara Galvao and Damasceno, An. oryzalimnetes Wilkerson and Motoki) and three molecularly assigned (An. albitarsis F, G & I)-and one mitochondrial lineage (An. albitarsis H). To further explore species recognition within this important group, 658 base pairs of the mitochondrial DNA cytochrome oxidase subunit I (COI) were analyzed from 988 specimens from South America. We conducted statistical parsimony network analysis, generated estimates of haplotype, nucleotide, genetic differentiation, divergence time, and tested the effect of isolation by distance (IBD). Ten clusters were identified, which confirmed the validity of the eight previously determined species, and confirmed the specific status of the previous mitochondrial lineage An. albitarsis H. High levels of diversity were highlighted in two samples from Pará (= An. albitarsis J), which needs further exploration through additional sampling, but which may indicate another cryptic species. The highest intra-specific nucleotide diversity was observed in An. deaneorum, and the lowest in An. marajoara. Significant correlation between genetic and geographical distance was observed only in An. oryzalimnetes and An. albitarsis F. Divergence time within the Albitarsis Group was estimated at 0.58-2.25 Mya, during the Pleistocene. The COI barcode region was considered an effective marker for species recognition within the Albitarsis Group and a network approach was an analytical method to discriminate among species of this group., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Anopheles tessellatus is a potentially important vector found across South, East and Southeast Asia. While it was formerly considered a formidable vector of human Plasmodium and filarial parasites in the Maldives, and of lesser importance as a vector of human Plasmodium in Sri Lanka and parts of Indonesia, it is currently of little or unknown health importance in many other parts of its range. This study describes the genetic diversity and evolutionary relationships among An. tessellatus populations in nine Asian countries at the COI gene using maximum-likelihood and Bayesian phylogenetic inference tree and cluster-based species delimitation approaches. These analyses reveal exceptional levels of genetic diversity in An. tessellatus populations across its known range, and identify up to six putative species in the newly determined Tessellatus Complex. The existence of such cryptic diversity has potentially important consequences for vector management and disease control. Differences in the ecologies and life histories among these species may have considerable impact on vectorial capacity and may go some way towards explaining why An. tessellatus s.l. has such varying degrees of public health importance across its range., (Copyright © 2020. Published by Elsevier B.V.)

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., Competing Interests: The authors have declared that no competing interests exist.

Following vaccination with the live attenuated, recombinant vesicular stomatitis virus Indiana serotype Ebola virus (rVSV-EBOV) vaccine, persons may exhibit a transient vaccine-associated viremia. To investigate the potential for Old World sand flies to transmit this vaccine following feeding on a viremic person, we fed laboratory-reared Phlebotomus papatasi an artificial blood meal containing 7.2 log10 plaque-forming units of rVSV-EBOV. Replication or dissemination was not detected in the body or legs of any P. papatasi collected at seven (n = 75) or 15 (n = 75) days post-feed. These results indicate a low potential for rVSV-EBOV to replicate and disseminate in P. papatasi, a species whose geographic distribution ranges from Morocco to southwest Asia and as far north as southern Europe.

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% (CI 95 33-59%; p < 0.001) in indoor light traps and by 74% (CI 95 38-90%; p = 0.010) during indoor and outdoor HLC, though in each study year reductions in vector density were consistently greatest in those months immediately following the IRS campaigns and waned over time. Overall there was no strong preference for An. funestus to feed indoors or outdoors, and these biting behaviours did not differ significantly across study arms: observed indoor-outdoor biting ratios were 1.10 (CI 95 1.00-1.21) in no-IRS villages and 0.88 (CI 95 0.67-1.15) in IRS villages. The impact of IRS was consistent in reducing HLC exposures both indoors (75% reduction: CI 95 47-88%; p = 0. < 0.001) and outdoors (68% reduction: CI 95 22-87%; p = 0.012). While substantially fewer Anopheles gambiae s.l. were collected during the study, trends show a similar impact of IRS on this key vector group as well, with a 33% (CI 95 7-53%; p = 0.019) reduction in mosquitoes collected in light traps and a non-statistically significant 39% reduction (p = 0.249) in HLC landing rates., Conclusion: IRS with PM used in addition to pyrethroid-only LLINs substantially reduced human exposures to malaria vectors during both years of the cluster-randomized controlled trial in Mopeia-a high-burden district where the primary vector, An. funestus s.l., was equally likely to feed indoors or outdoors and demonstrated increasing resistance to pyrethroids. Findings suggest that IRS with PM can provide effective vector control, including in some settings where pyrethroid-only ITNs are widely used. Trial registration clinicaltrials.gov , NCT02910934. Registered 22 September 2016, https://www.clinicaltrials.gov/ct2/show/NCT02910934.

Routine biosurveillance efforts at the Naval Station Guantanamo Bay, Cuba, on 18 June 2019, detected two unusual mosquitos in a CO 2 -baited CDC light trap. Morphological and molecular analysis confirmed the presence of Aedes (Fredwardsius) vittatus (Bigot, 1861) - the first record of the Old World dengue, chikungunya, Zika and yellow fever virus vector into the Americas - and provides evidence for its establishment in Cuba. Newly submitted GenBank sequences from Dominican Republic further evidence its establishment in the Caribbean, and a median-joining network analysis using mitochondrial COI gene sequences clearly supports multiple introductions of Ae. vittatus into the Caribbean from the Indian subcontinent. It was determined that many Ae. vittatus COI barcode sequences in GenBank are currently misidentified as Aedes (Fredwardsius) cogilli Edwards, 1922., (Copyright © 2020. Published by Elsevier B.V.)

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.

We conducted orthonairovirus RNA screening of 7043 tick specimens-representing 16 species-collected from various regions of Anatolia. In 602 pools, Crimean-Congo hemorrhagic fever virus (CCHFV) Europe 1 and 2 lineages were detected in seven pools (1.1 %) comprising Hyalomma marginatum, Hyalomma scupense, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks. In pools of Hyalomma aegyptium, we detected Tamdy virus (TAMV) and an unclassified nairovirus sequence. Next-generation sequencing revealed complete coding regions of three CCHFV Europe 2 (AP92-like) viruses, TAMV and the novel orthonairovirus, tentatively named herein as Meram virus. We further performed in silico functional analysis of all available CCHFV Europe 2, TAMV, Meram and related virus genomes. The CCHFV Europe 2 viruses possessed several conserved motifs, including those with OTU-like cysteine protease activity. Probable recombinations were identified in L genome segments of CCHFV and TAMV. Through phylogeny reconstruction using individual genome segments, Meram virus emerged as a distinct virus among species within the Orthonairovirus genus. It further exhibited conserved motifs associated with RNA binding, encapsidation, signal peptidase cleavage, post-translational modification, RNA-dependent RNA polymerase and OTU-like activities. Bole tick virus 3 was also detected in two pools with CCHFV reactivity. Hereby, we describe a novel tick-associated orthonairovirus, in a CCHFV-endemic region with confirmed TAMV activity. Human and animal health impact of these viruses need to be addressed., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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.

Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in areas where extreme reductions have been sustained, such as South Africa 1,2 . Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8 months of the year have suggested that some species of Anopheles mosquito use long-distance migration 3 . Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290 m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235 anopheline mosquitoes that were captured during 617 nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all of the mosquitoes that we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens are probably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300 km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000 Anopheles gambiae sensu stricto, 6 million A. coluzzii and 44 million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.

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.

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.

Background: The 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., Methodology: Mosquitoes 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 Findings: The 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/significance: Invasive 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., Competing Interests: The authors have declared that no competing interests exist.

The study of demographic processes involved in species diversification and evolution ultimately provides explanations for the complex distribution of biodiversity on earth, indicates regions important for the maintenance and generation of biodiversity, and identifies biological units important for conservation or medical consequence. African and forest biota have both received relatively little attention with regard to understanding their diversification, although one possible mechanism is that this has been driven by historical climate change. To investigate this, we implemented a standard population genetics approach along with Approximate Bayesian Computation, using sequence data from two exon-primed intron-crossing (EPIC) nuclear loci and mitochondrial cytochrome oxidase subunit I, to investigate the evolutionary history of five medically important and inherently forest dependent mosquito species of the genus Aedes . By testing different demographic hypotheses, we show that Aedes bromeliae and Aedes lilii fit the same model of lineage diversification, admixture, expansion, and recent population structure previously inferred for Aedes aegypti . In addition, analyses of population structure show that Aedes africanus has undergone lineage diversification and expansion while Aedes hansfordi has been impacted by population expansion within Uganda. This congruence in evolutionary history is likely to relate to historical climate-driven habitat change within Africa during the late Pleistocene and Holocene epoch. We find differences in the population structure of mosquitoes from Tanzania and Uganda compared to Benin and Uganda which could relate to differences in the historical connectivity of forests across the continent. Our findings emphasize the importance of recent climate change in the evolution of African forest biota.

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n=3), Cx. pipiens s.l. (n=1) and Aedes caspius (n=1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s.l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Little is known about tick-borne rickettsial pathogens in Belize, Central America. We tested ixodid ticks for the presence of Rickettsia species in three of the six northern and western Belizean districts. Ticks were collected from domestic animals and tick drags over vegetation in 23 different villages in November 2014, February 2015, and May 2015. A total of 2,506 collected ticks were identified to the following species: Dermacentor nitens Neumann (46.69%), Rhipicephalus sanguineus (Latreille) (19.55%), Rhipicephalus microplus (Canestrini) (19.47%), Amblyomma cajennense complex (9.74%), Amblyomma maculatum Koch (3.47%), Amblyomma ovale Koch (0.68%), Ixodes nr affinis (0.16%), Amblyomma nr maculatum (0.12%), and Amblyomma nr oblongoguttatum (0.12%). Ticks were pooled according to species, life stage (larva, nymph, or adult), and location (n = 509) for DNA extraction and screened for genus Rickettsia by quantitative real-time polymerase chain reaction (qPCR). All 42 positive pools were found to be positive for spotted fever group (SFG) Rickettsia in pools of A. cajennense complex (n = 33), A. maculatum (n = 4), A. nr maculatum (n = 1), A. ovale (n = 1), R. sanguineus (n = 1), and I. nr affinis (n = 2). Rickettsia amblyommatis was identified from A. cajennense complex and A. nr maculatum. Rickettsia parkeri was found in A. maculatum, and Rickettsia sp. endosymbiont was detected in I. nr affinis. The presence of infected ticks suggests a risk of tick-borne rickettsioses to humans and animals in Belize. This knowledge can contribute to an effective tick management and disease control program benefiting residents and travelers., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.)

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Background: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates., Results: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as "Ochlerotatus caspius flavivirus Turkey", was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified., Conclusions: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses from mosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed a monophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Increasing globalization has promoted the spread of exotic species, including disease vectors. Understanding the evolutionary processes involved in such colonizations is both of intrinsic biological interest and important to predict and mitigate future disease risks. The Aedes aegypti mosquito is a major vector of dengue, chikungunya and Zika, the worldwide spread of which has been facilitated by Ae. aegypti's adaption to human-modified environments. Understanding the evolutionary processes involved in this invasion requires characterization of the genetic make-up of the source population(s). The application of approximate Bayesian computation (ABC) to sequence data from four nuclear and one mitochondrial marker revealed that African populations of Ae. aegypti best fit a demographic model of lineage diversification, historical admixture and recent population structuring. As ancestral Ae. aegypti were dependent on forests, this population history is consistent with the effects of forest fragmentation and expansion driven by Pleistocene climatic change. Alternatively, or additionally, historical human movement across the continent may have facilitated their recent spread and mixing. ABC analysis and haplotype networks support earlier inferences of a single out-of-Africa colonization event, while a cline of decreasing genetic diversity indicates that Ae. aegypti moved first from Africa to the Americas and then to Asia. ABC analysis was unable to verify this colonization route, possibly because the genetic signal of admixture obscures the true colonization pathway. By increasing genetic diversity and forming novel allelic combinations, divergence and historical admixture within Africa could have provided the adaptive potential needed for the successful worldwide spread of Ae. aegypti., (© 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

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.

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.

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.