Interindividual variation among Culex pipiens larvae in terms of thermal response.

This study aims to determine the phenological characteristics of thermal responses in the larvae of a Culex pipiens complex field population at the individual level under the influence of thermal regime of its habitat. The analysis is based on a structured population model quantifying the thermal responses of development time and survival under variable conditions and characterising the variety between the larvae (interindividual variety). During the study performed in Turkish Thrace on a monthly basis between May 2021 and June 2022, a total of 3744 larvae were reared as peer larval cohorts and 2330 larvae as siblings in artificial containers to be fully exposed to the natural thermal condition that was recorded hourly. The development process of larvae was monitored daily from egg to adult. As a result, a total of 4788 adult mosquitoes emerged, with a development period ranging from 8 to 52 days in the females and 7 to 50 days in the males, and the survival rate was found to range from 0% to 100%. Both parameters varied by month and individuals, and the variations manifested itself, particularly in the colder periods. The results indicate that the variation between the individuals in terms of thermal response in the larvae of C. pipiens, along with the thermal acclimation ability, appears to be fate determinant in resisting fluctuating thermal regimes, surviving in concurrent climate change and adapting to new conditions with modifications in the seasonal phenology, such as maintaining reproductive dynamics throughout the winter thanks to global warming.

Identification of Ochrobactrum as a bacteria with transstadial transmission and potential for application in paratransgenic control of leishmaniasis.

Leishmaniasis is a zoonotic vector-borne disease with worldwide distribution. All current approaches in leishmaniasis control or development of vaccines/cures showed only limited success. Recently, paratransgenesis has been marked as a promising strategy for leishmaniasis control. Thus, the investigations of the gut microbial content of sand flies have gained popularity. Gut microbial composition of the laboratory colony of Phlebotomus papatasi was investigated via microbial culturomics approach which refers to the combination of multiple culture conditions and different selective and/or enriched culture mediums, followed by 16S rDNA sequencing. Investigations were conducted on three offspring generations, with six samplings of immature stages (four larval samplings, one pre-pupa, one pupa) and samplings of adults before and after blood feeding. The aim was to determine if microbiome changes during the sand fly development and to identify bacteria with transstadial potential. The presence of 8 bacterial taxa (Bacillus sp., Terribacillus sp., Staphylococcus sp., Alcaligenes sp., Microbacterium sp., Leucobacter sp., Ochrobactrum sp. and Enterobacter sp.), 2 fungi (Fusarium sp. and Acremonium sp.) and 1 yeast (Candida sp.) were recorded. Gram-positive bacteria were more diverse, but gram-negative bacteria were more abundant. All taxa were recorded among immature stage samples, while only one bacterium was detected in adults. Microbial diversity among larval samples was stable, with a steady decrease in pre-pupa and pupa, resulting in the survival of only Ochrobactrum sp. in adults. Abundance of microbes was higher when larvae were actively feeding, with a gradual decrease after larvae stopped feeding and commenced pupation. Ochrobactrum sp. is the bacteria with transstadial potential, worthy of future in-depth analysis for the application in paratransgenic approach for the control of Leishmania sp.

VectorNet: collaborative mapping of arthropod disease vectors in Europe and surrounding areas since 2010.

BackgroundArthropod vectors such as ticks, mosquitoes, sandflies and biting midges are of public and veterinary health significance because of the pathogens they can transmit. Understanding their distributions is a key means of assessing risk. VectorNet maps their distribution in the EU and surrounding areas.AimWe aim to describe the methodology underlying VectorNet maps, encourage standardisation and evaluate output.Methods: Vector distribution and surveillance activity data have been collected since 2010 from a combination of literature searches, field-survey data by entomologist volunteers via a network facilitated for each participating country and expert validation. Data were collated by VectorNet members and extensively validated during data entry and mapping processes.ResultsAs of 2021, the VectorNet archive consisted of ca 475,000 records relating to > 330 species. Maps for 42 species are routinely produced online at subnational administrative unit resolution. On VectorNet maps, there are relatively few areas where surveillance has been recorded but there are no distribution data. Comparison with other continental databases, namely the Global Biodiversity Information Facility and VectorBase show that VectorNet has 5-10 times as many records overall, although three species are better represented in the other databases. In addition, VectorNet maps show where species are absent. VectorNet's impact as assessed by citations (ca 60 per year) and web statistics (58,000 views) is substantial and its maps are widely used as reference material by professionals and the public.ConclusionVectorNet maps are the pre-eminent source of rigorously validated arthropod vector maps for Europe and its surrounding areas.

Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Seasonal dynamics of a population of Phlebotomus (Larroussius) perfiliewi Parrot, 1930 (Diptera: Psychodidae) in North-Eastern Romania.

Sand flies were collected in a location from Romania in order to estimate their abundance and seasonal variation in correlation with environmental and anthropic factors. From May to October 2017, eight premises with different animal species were sampled for sand flies in a household from Fundatura village, Vaslui County, in North-Eastern Romania. Animal-related data, shelter-related data, and climatic parameters were recorded. All (n = 150) collected sand flies were Phlebotomus perfiliewi. A mono-modal type of abundance trend has been recorded (a single peak at the beginning of August). The first day of capture was in mid-July. The total number of females during the peak season was significantly higher than the total number of males. The highest percentage of males was recorded at the beginning and at the end of the sand fly activity. Only the traps placed in the poultry enclosure built from clay and wood were positive. A strong positive correlation was recorded between the total number of collected sand flies and the minimum and the maximum temperature. The analysis of the climatic data shows that the first presence of sand flies was registered only after the average minimum temperature for the previous 7 days was above 15 °C.

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

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.

Isolation, genetic characterization, and seroprevalence of Adana virus, a novel phlebovirus belonging to the Salehabad virus complex, in Turkey.

UNLABELLED: A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies. IMPORTANCE: Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece.

Influence of Bloodmeal Source on Reproductive Output of the Potential West Nile Vector, Culex theileri (Diptera: Culicidae).

Culex theileri Theobald (Diptera: Culicidae) has a wide Afrotropical, southern Palaearctic, northern Oriental, and European distribution. It is mainly considered as a mammophilic mosquito and also feeds on birds and serves as a vector for various zoonotic diseases including West Nile virus. Despite its broad distribution and evidence indicating that Cx. theileri is a competent vector of human and domestic animal pathogens, basic biological and ecological features of this species have not been well investigated. We evaluated the impact of bloodmeal source (human, chicken, cow, and a double bloodmeal such as human and cow or chicken and cow and mixed bloodmeals [cow, chicken, and human] via artificial feeding) on fecundity, hatching rates, developmental times, and viability from egg to adult for laboratory colonized Cx. theileri. Fecundity in mosquitoes that took a chicken bloodmeal, a double bloodmeal and mixed bloodmeals was significantly higher than in females fed on a single cow or single human blood. This is the first study about the bloodmeal sources effect on laboratory-reared Cx. theileri populations and these findings contribute to our understanding of the impact of bloodmeal source on reproduction in Cx. theileri. As it is known that Cx. theileri is a vector for West Nile virus, the potential impacts of bloodmeal source on virus transmission are discussed.

Differential expression of Phlebotomus tobbi Adler, Theodor & Lourie, 1930 (Diptera: Psychodidae) genes under different environmental conditions.

Phlebotomus tobbi is a widely distributed sand fly species in Turkey and is the proven vector of Leishmania infantum and several Phleboviruses. Information regarding the genetic basis of phenotypic plasticity is crucial for managing vector-borne diseases, as the changing environmental conditions have consequences for the survival of arthropods and the disease agents they transmit. However, limited data is available on the impacts of environmental conditions on the traits associated with sand fly survival, reproduction, and vectorial competence. The present study aimed to reveal the changes in the expression levels of three selected P. tobbi genes using laboratory-reared and wild-caught populations. A nervous system protein, Cacophony (PtCac), related to the life history traits of sand flies, and two sand fly salivary protein genes, PtSP32 and PtSP38, influence the infection of the vertebrate hosts, were assessed. Sand flies were maintained at 23 °C and 27 °C in the laboratory to evaluate the relationship between temperature and the expressed phenotypes. Field collections were carried out in three climatically distinct regions of Turkey to establish the regional differences in the gene expression levels of natural P. tobbi populations. In the laboratory, PtCac expression increased with the temperature. However, PtCac expression was negatively correlated with local temperature and humidity conditions. No differences were detected in the PtSP32 gene expression levels of both laboratory-reared and wild-caught females, but a negative correlation was observed with relative humidity in natural populations. Although the expression levels of PtSP38 did not differ among the females collected from distinct regions, a positive correlation was detected in the laboratory-reared colony. The findings indicated that changes in environmental conditions could drive the expression levels of P. tobbi genes, which influence population dynamics and the transmission risk of the disease.

Entomological surveillance and spatiotemporal risk assessment of sand fly-borne diseases in Cyprus.

Visceral and cutaneous leishmaniases are important public health concerns in Cyprus. Although the diseases, historically prevalent on the island, were nearly eradicated by 1996, an increase in frequency and geographical spread has recently been recorded. Upward trends in leishmaniasis prevalence have largely been attributed to environmental changes that amplify the abundance and activity of its vector, the phlebotomine sand flies. Here, we performed an extensive field study across the island to map the sand fly fauna and compared the presence and distribution of the species found with historical records. We mapped the habitat preferences of Phlebotomus papatasi and P. tobbi, two medically important species, and predicted the seasonal abundance of P. papatasi at unprecedented spatiotemporal resolution using a climate-sensitive population dynamics model driven by high-resolution meteorological forecasting. Our compendium holds a record of 18 species and the locations of a subset, including those of potential public and veterinary health concern. We confirmed that P. papatasi is widespread, especially in densely urbanized areas, and predicted that its abundance uniformly peaks across the island at the end of summer. We identified potential hotspots of P. papatasi activity even after this peak. Our results form a foundation to inform public health planning and contribute to the development of effective, efficient, and environmentally sensitive strategies to control sand fly populations and prevent sand fly-borne diseases.

Reconstructing the post-glacial spread of the sand fly Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) in Europe.

Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread.

Identification and characterization of single nucleotide polymorphisms (SNPs) in Culex theileri (Diptera: Culicidae).

Culex theileri Theobald (Diptera: Culicidae) is one of the most common mosquito species in northeastern Turkey and serves as a vector for various zoonotic diseases including West Nile virus. Although there have been some studies on the ecology of Cx. theileri, very little genetic data has been made available. We successfully sequenced 11 gene fragments from Cx. theileri specimens collected from the northeastern part of Turkey. On average, we found a Single nucleotide polymorphism every 45 bp. Transitions outnumbered transversions, at a ratio of 2:1. This is the first report of genetic polymorphisms in Cx. theileri and Single nucleotide polymorphism discovered from this study can be used to investigate population structure and gene-environmental interactions.

A dynamically structured matrix population model for insect life histories observed under variable environmental conditions.

Various environmental drivers influence life processes of insect vectors that transmit human disease. Life histories observed under experimental conditions can reveal such complex links; however, designing informative experiments for insects is challenging. Furthermore, inferences obtained under controlled conditions often extrapolate poorly to field conditions. Here, we introduce a pseudo-stage-structured population dynamics model to describe insect development as a renewal process with variable rates. The model permits representing realistic life stage durations under constant and variable environmental conditions. Using the model, we demonstrate how random environmental variations result in fluctuating development rates and affect stage duration. We apply the model to infer environmental dependencies from the life history observations of two common disease vectors, the southern (Culex quinquefasciatus) and northern (Culex pipiens) house mosquito. We identify photoperiod, in addition to temperature, as pivotal in regulating larva stage duration, and find that carefully timed life history observations under semi-field conditions accurately predict insect development throughout the year. The approach we describe augments existing methods of life table design and analysis, and contributes to the development of large-scale climate- and environment-driven population dynamics models for important disease vectors.

The Asian tiger mosquito Aedes albopictus (Skuse) in Kosovo: First record.

The Asian tiger mosquito, Aedes albopictus, is an invasive mosquito species that is considered a potential vector of about 22 arboviruses, among which dengue, chikungunya and Zika. Here we report the first record of Aedes albopictus in the territory of the Republic of Kosovo. The first finding, in July 2020, was driven by a photo of an adult mosquito published by a citizen in social media. The subsequent field investigation in July 2020 confirmed the presence of adult mosquitoes by human landing catch and collection of eggs in ovitraps at the village Zhur. Monitoring was performed for 7 weeks with ovitraps and BG-Sentinel adult traps at 36 sampling stations from 7 localities, in the Southern part of Kosovo, on the border with Albania. Fifty-two out of 81 ovitrap samples resulted positive for the presence of Ae. albopictus. A total of 2,711 eggs are collected in 22 out 36 stations and a total of 78 adults from 4 out 4 stations with BG Sentinel trap and 15 adults with handled electric aspirator. Our finding shows that the tiger mosquito is expanding its geographical range in the Balkans, southeastern Europe.

Assessment of expertise in morphological identification of mosquito species (Diptera, Culicidae) using photomicrographs.

Accurate identification of insect species is an indispensable and challenging requirement for every entomologist, particularly if the species is involved in disease outbreaks. The European MediLabSecure project designed an identification (ID) exercise available to any willing participant with the aim of assessing and improving knowledge in mosquito taxonomy. The exercise was based on high-definition photomicrographs of mosquitoes (26 adult females and 12 larvae) collected from the western Palaearctic. Sixty-five responses from Europe, North Africa and the Middle East were usable. The study demonstrated that the responders were better at identifying females (82% correct responses) than larvae (63%). When the responders reported that they were sure of the accuracy of their ID, the success rate of ID increased (92% for females and 88% for larvae). The top three tools used for ID were MosKeyTool (72% of responders), the ID key following Becker et al. [2010. Mosquitoes and their control, 2nd edn. Berlin: Springer] (38%), and the CD-ROM of Schaffner et al. [2001. Les moustiques d'Europe: logiciel d'identification et d'enseignement - The mosquitoes of Europe: an identification and training programme. Montpellier: IRD; EID] (32%), while other tools were used by less than 10% of responders. Responders reporting the identification of mosquitoes using the MosKeyTool were significantly better (80% correct responses) than non-MosKeyTool users (69%). Most responders (63%) used more than one ID tool. The feedback from responders in this study was positive, with the exercise being perceived as halfway between educational training and a fun quiz. It raised the importance of further expanding training in mosquito ID for better preparedness of mosquito surveillance and control programmes.

Title: Évaluation de l'expertise en identification morphologique des espèces de moustiques (Diptera, Culicidae) à l'aide de photomicrographies. Abstract: L'identification précise des espèces d'insectes est une exigence indispensable et difficile pour tout entomologiste, en particulier si l'espèce est impliquée dans des épidémies. Le projet européen MediLabSecure a conçu un exercice d'identification (ID) accessible à tout participant volontaire dans le but d'évaluer et d'améliorer les connaissances en taxonomie des moustiques. L'exercice était basé sur des photomicrographies haute définition de moustiques (26 femelles adultes et 12 larves) prélevées dans le Paléarctique occidental. Soixante-cinq réponses d'Europe, d'Afrique du Nord et du Moyen-Orient ont été utilisables. L'étude a démontré que les répondants étaient meilleurs pour identifier les femelles (82 % de réponses correctes) que les larves (63 %). Lorsque les répondants ont déclaré être sûrs de l'exactitude de leur ID, le taux de réussite de l'identification était meilleur (92 % pour les femelles et 88 % pour les larves). Les trois principaux outils utilisés pour les ID étaient MosKeyTool (72 % des répondants), la clé d'identification du livre de Becker et al. (38%) et le CD-ROM de Schaffner et al. (32 %), tandis que d'autres outils étaient utilisés par moins de 10 % des répondants. Les répondants déclarant identifier des moustiques à l'aide de MosKeyTool étaient significativement meilleurs (80 % de réponses correctes) que les non-utilisateurs de MosKeyTool (69 %). La plupart des répondants (63 %) ont utilisé plus d'un outil d'identification. Les commentaires des répondants de cette étude ont été positifs, l'exercice étant perçu comme à mi-chemin entre une formation pédagogique et un quiz amusant. Il a souligné l'importance d'étendre la formation complémentaire à l'identification des moustiques pour une meilleure préparation des programmes de surveillance et de contrôle des moustiques.

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe.

BACKGROUND: There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. METHODS: Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. RESULTS: Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004

Updated checklist of Culicoides Latreille (Diptera: Ceratopogonidae) of Turkey with ten new records.

We investigated the Culicoides fauna in Turkey during the years 2016-2019 in the process of entomological surveillance for arboviral diseases. The entomological survey was conducted at 104 sampling stations in 51 provinces in Turkey during four consecutive years. There were approximately 450,000 specimens and 59 identified species collected during the surveillance. Ten species were newly recorded for Turkey: C. chiopterus, C. grisescens, C. paradoxalis, C. santonicus, C. poperinghensis, C. sergenti, C. tbilisicus, C. comosioculatus, C. haranti, and C. univittatus. Identification of C. chiopterus and C. grisescens was confirmed using species-specific PCR and DNA sequencing. With our recent findings, previous data were critically reviewed and updated, and the number of Culicoides species has been increased to 71 for Turkey. The presence of C. chiopterus has particular importance due to its potential vector status for bluetongue virus (BTV) and Schmallenberg virus (SBV). This study presents result of the first large-scale integrated faunistic survey on Culicoides species in Turkey.

Sand fly fauna of South-Eastern Romania, with the description of Phlebotomus (Transphlebotomus) simonahalepae n. sp. (Diptera: Psychodidae).

BACKGROUND: An entomological study was conducted in the Canaraua Fetii Special Protection Area in the Dobrogea region, South-Eastern Romania. Four sand fly species were recorded at this location between 1968 and 1970: Phlebotomus neglectus, Ph. balcanicus, Ph. sergenti and Sergentomyia minuta. The most abundant sand fly species recorded at that time were Ph. balcanicus and Se. minuta. In the context of a countrywide study to update the sand fly species diversity, we surveyed the same area, recording also a previously unknown Ph. (Transphlebotomus) sp., for which we provide a formal description here. METHODS: Sand flies were collected between July and August in 2018 and 2019 in three sites from Canaraua Fetii, Dobrogea region, Romania. The general aspect of the landscape is of a canyon (vertical, narrow walls and deep valleys). Species identification was done using both morphological and molecular analyses. RESULTS: Out of 645 collected sand flies, 644 (99.8%) were morphologically identified as Ph. neglectus, while one female specimen (0.2%) was assigned to a previously unknown species, belonging to the subgenus Transphlebotomus. The morphological and molecular examination of this specimen showed that it is a previously unknown species which we formally describe here as Phlebotomus (Transphlebotomus) simonahalepae n. sp. Also, Ph. balcanicus, Ph. sergenti, and Se. minuta (previously recorded in this location) were not present. CONCLUSIONS: The study revealed for the first time the presence of sand flies of the subgenus Transphlebotomus in Romania. Moreover, a new species, Ph. simonahalepae n. sp., was described based on a female specimen, raising the number of species in this subgenus to six. In the investigated natural habitat, the predominant species was Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the predominant species in 1968-1970).

Phlebotomine sand fly survey in the Republic of Moldova: species composition, distribution and host preferences.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a first update on their occurrence, species composition and bloodmeal sources after 50 years. METHODS: During 5 seasons (2013-2017), 58 localities from 20 regions were surveyed for presence of sand flies using CDC light traps and manual aspirators. Species identification was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profiling). In engorged females, host blood was identified by three molecular techniques (RFLP, cytb sequencing and MALDI-TOF peptide mass mapping). Population structure of most abundant species was studied by cox1 haplotyping; phylogenetic analyses of ITS2 and cox1 genetic markers were used to resolve relationships of other detected species. RESULTS: In total, 793 sand flies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identified by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfiliewi and Phlebotomus sp. (Adlerius), the first being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only five haplotypes of cox1 detected. Distinct morphological and molecular characters of Phlebotomus sp. (Adlerius) specimens suggest the presence of a yet undescribed species. CONCLUSIONS: Our study revealed the presence of stable sand fly populations of three species in Moldova that represent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied.

Biting activity of mosquito species (Diptera: Culicidae) in the Turkey-Armenia border area, Ararat Valley, Turkey.

During nine consecutive nights in July 2007 (from 18:15-05:45 h), mosquitoes landing-biting on humans were collected outdoors and indoors at the Turkey-Armenia border. A total of 1005 females were collected consisting of nine species. The dominant species was Aedes dorsalis (Meigen) (47.5% of total catch), followed by Anopheles hyrcanus (Pallas) (22.9%), Culex theileri (Theobald) (9.3%),Ae. vexans (Meigen) (6.6%), Ae. caspius (Pallas) (4.9%),Anopheles maculipennis s.l. (Meigen) (3.1%), Culex territans (Walker) (2.8%), Coquillettidia richiardii (Ficalbi) (1.6%), and Cx. pipiens L. (1.5%). The biting rate outdoors (15.1 mosquitoes/human/h) was greater than indoors (3.4 mosquitoes/human/h). The landing-biting of Ae. dorsalis peaked at dusk (19:15-19:45 h) and dawn (04:15-04:45 h). Ae. vexans activity increased soon after dark (20:15-20:45 h) and reached a peak at dawn (04:15-04:45 h). Maximum biting activity of An. hyrcanus and Cx. theileri occurred during the first sampling interval after dusk (20:15-20:45 h). A large number of An. maculipennis s.l. adults were collected during the second half of the night. We believe that these findings will contribute to decisions on the timing of mosquito control in Ararat Valley.