A cross-sectional study on phlebotomine sand flies in relation to disease transmission in the Republic of Kosovo.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross-sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate-related factors into account. In addition, PCR-based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo.

The role of sand flies as vectors of viruses other than phleboviruses.

Sand flies (Diptera: Phlebotominae) are proven vectors of various pathogens of medical and veterinary importance. Although mostly known for their pivotal role in the transmission of parasitic protists of the genus Leishmania that cause leishmaniases, they are also proven or suspected vectors of many arboviruses, some of which threaten human and animal health, causing disorders such as human encephalitis (Chandipura virus) or serious diseases of domestic animals (vesicular stomatitis viruses). We reviewed the literature to summarize the current published information on viruses detected in or isolated from phlebotomine sand flies, excluding the family Phenuiviridae with the genus Phlebovirus, as these have been well investigated and up-to-date reviews are available. Sand fly-borne viruses from four other families (Rhabdoviridae, Flaviviridae, Reoviridae and Peribunyaviridae) and one unclassified group (Negevirus) are reviewed for the first time regarding their distribution in nature, host and vector specificity, and potential natural transmission cycles.

Factors Influencing Suicidal Tendencies of Patients with Diagnosis of Attempted Suicide in Medical History and Potential Prevention of Relapse Prevention.

OBJECTIVE: The authors researched the incidence of suicidal thoughts and related factors in 123 patients of the psychiatric ward of the Hospital of Ceské Budejovice with diagnosed attempted suicide in their medical history for the period from January 2013 ­ June 2015. METHODS: The research was carried out in two stages. At the beginning of the hospitalization, quantitative data collection was implemented using a semi-structured questionnaire, followed by qualitative research conducted with semi-structured phone conversation, based on previous patient's written consent. The research data were statistically processed to obtain information about the character of relations among individual characteristics. To quantify them, the Bayesian Network (BN) was constructed, and to identify relations among individual characteristics, the Hill-Climbing algorithm was used. Before deriving the network, variables were discretized. The network parameters were set based on a data matrix using the maximal plausibility method. RESULTS: The results of analysed set show that the probability of suicidal thoughts is high, achieving a value of 0.750 (0.781 for women and 0.724 for men). If the patient visits a contact centre for drug-addicted persons, the probability of suicidal thoughts decreases to 0.683. If the patient visits a psychotherapist, the values of 0.736 are achieved. If a daily care centre is visited, the estimated risk rises to 0.832 and the probability of the patient repetitively attempting suicide is 0.606. If the interviewed person regularly consumes alcohol, the probable relapse amounts to 0.616. But if the person consumes alcohol from time to time, the probability rises to 0.701. In case of abstinence, the probable relapse decreases to 0.565. CONCLUSIONS: The incidence of suicidal thoughts in observed patients was high, and the amount of risk was influenced by gender, by visiting follow-up care facilities, psychotherapy, and particularly by the frequency of alcohol consumption. Intermittent alcohol consumption is the highest-risk factor in connection with relapsing suicide. In case of psychiatric patients with attempted suicide in their medical history, all verified preventive and therapeutic procedures that can contribute to prevention of relapses should be used within follow-up professional care. Specific approach of the closest social environment, medical literacy of the population and state safety measures are important.

Comparative Genomics Uncovers the Evolutionary Dynamics of Detoxification and Insecticide Target Genes Across 11 Phlebotomine Sand Flies.

Sand flies infect more than 1 million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled, and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs), carboxyl/cholinesterases (CCEs), and ATP-binding cassette (ABC) transporters. Using this comparative approach, we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of (i) CYP4G genes involved in cuticular hydrocarbon biosynthesis, (ii) ABCB genes involved in xenobiotic toxicity, and (iii) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and voltage gated sodium channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Phlebotomine sand flies (Diptera: Psychodidae) of Bosnia and Herzegovina: distribution, ecology and environmental preferences.

Sand flies (Diptera: Phlebotominae) are the principal vectors for the protozoan parasites Leishmania spp. and for phleboviruses. The sand fly fauna on the Balkan Peninsula, including Bosnia and Herzegovina (BIH), is diverse and the circulation of Leishmania infantum as well as phleboviruses has been proven. However, recent data on the sand fly fauna in BIH are scarce. In this study, we surveyed understudied regions in central and northeastern BIH to update the sand fly distribution and gain insights into the ecological and environmental factors shaping their appearance. CDC light trapping was conducted in 2022 and 2023 and a combination of morphological and molecular methods (cytochrome oxidase I barcoding) was performed for species identifications. We mapped the currently known distribution, modelled climatic suitability patterns and performed environmental analyses by applying machine learning methods. In addition, we analyzed blood meals by host gene sequencing and MALDI-TOF peptide mass mapping and screened for Leishmania spp. DNA and Phlebovirus RNA. Altogether, 591 sand flies of four species were trapped, predominantly Phlebotomus neglectus (97 %), but also Ph. balcanicus, Ph. mascittii, and Ph. papatasi. Records of seven sand fly species known to be endemic were plotted onto distribution maps based on 101 datapoints, identifying Ph. neglectus as the overall predominant species. The environmental analyses of sand fly species indicated variation in altitudinal, thermal, and precipitation conditions across the sand fly-positive sites. Phlebotomus simici, Phlebotomus tobbi, and Sergentomyia minuta are typically found exclusively in Mediterranean and subtropical climate zones, whereas other species typically inhabit continental regions. The Inverse Distance Weighted (IDW) interpolation of sand fly species numbers and Shannon entropy values suggested the southeastern coastal region of BIH as a primary focus for sand fly occurrence. This finding was corroborated by modeled average climatic suitability patterns for sand flies, depicting four distinct meso-regions for sand fly occurrence. The results of the ensemble method highlight the importance of annual precipitation to distinguish between positive and negative sand fly trapping sites in BIH. In total, 55 blood meals of two sand fly species, Ph. neglectus and Ph. balcanicus, were analyzed and five host species identified. Our comprehensive assessment of ecological and environmental preferences of sand flies in BIH may support further entomological surveys and help to better understand and evaluate potential hot spots of disease transmission in the country.

Ecological setting of phlebotomine sand flies in the Republic of Kosovo.

Sand flies (Diptera, Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis, as well as phleboviruses. In the Balkans, the endemicity and spreading of sand fly-borne diseases are evident, particularly in the Republic of Kosovo, a country with a predominantly humid continental climate. To date, understanding the drivers behind the spatial structure and diversity patterns of sand fly communities in humid continental regions remains limited. Therefore, elucidating the geographical and ecological factors contributing to the presence of potential vector species in the country is crucial. We aimed to enhance our understanding of factors influencing sand fly occurrence in cool and wet wintering humid continental areas, which could serve as a model for other countries with similar climatic conditions. Therefore, we assessed the currently known sand fly fauna through detailed environmental analyses, including Voronoi tessellation patterns, entropy calculations, Principal Coordinate and Component Analyses, Hierarchical Clustering, Random Trees, and climatic suitability patterns. Notable differences in the ecological tolerance of the species were detected, and the most important climatic features limiting sand fly presence were wind speed and temperature seasonality. Sand flies were observed to prefer topographical environments with little roughness, and the modelled climatic suitability values indicated that, dominantly, the western plain regions of Kosovo harbour the most diverse sand fly fauna; and are the most threatened by sand fly-borne diseases. Phlebotomus neglectus and P. perfiliewi, both confirmed vectors for L. infantum and phleboviruses, were identified as two main species with vast distribution in Kosovo. Contrary to this, most other present species are relatively sparse and restricted to temperate rather than humid continental regions. Our findings reveal a diverse potential sand fly fauna in Kosovo, indicating the need for tailored strategies to address varying risks across the country's western and eastern regions in relation to leishmaniasis control amidst changing environmental conditions.

Population Genetic Structure of Phlebotomus sergenti (Diptera: Psychodidae) Collected in Four Regions of Morocco Based on the Analysis of Cyt b and EF-1α Genes.

Phlebotomus (Ph.) sergenti is the main vector of Leishmania (L.) tropica (Trypanosomatida: Trypanosomatidae), the causative agent of anthroponotic cutaneous leishmaniasis in Morocco. This species has an extended geographical distribution, wider than that of the parasite. The main objective of our study was to analyze the genetic diversity of Ph. sergenti collected in four foci in Morocco: Taza, Foum Jemâa, El Hanchane, and Ouarzazate. We studied a set of diversity and population structure indices by sequencing two markers; nuclear EF-1α and mitochondrial Cyt b from 175 individual sand flies. Our results showed a considerable degree of intraspecific polymorphism with a high number of haplotypes identified in both genes. Many polymorphic sites detected in the Cyt b sequences (SCyt b = 45) indicate that it is the most polymorphic marker showing a distinct distribution of haplotypes according to their geographical origin, whereas the EF-1α marker showed no geographical isolation. Analysis by Tajima's D and Fu's Fs tests revealed a possible recent expansion of the populations, especially with the EF-1α marker, showing significant values in Taza and Ouarzazate sequences. The present study revealed significant genetic diversity within Ph. sergenti populations in Morocco. The results warrant further research using a combination of more than two markers including mitochondrial and non-mitochondrial markers, which may provide more information to clarify the genetic status of Ph. sergenti.

Species identification of phlebotomine sandflies using deep learning and wing interferential pattern (WIP).

Sandflies (Diptera; Psychodidae) are medical and veterinary vectors that transmit diverse parasitic, viral, and bacterial pathogens. Their identification has always been challenging, particularly at the specific and sub-specific levels, because it relies on examining minute and mostly internal structures. Here, to circumvent such limitations, we have evaluated the accuracy and reliability of Wing Interferential Patterns (WIPs) generated on the surface of sandfly wings in conjunction with deep learning (DL) procedures to assign specimens at various taxonomic levels. Our dataset proves that the method can accurately identify sandflies over other dipteran insects at the family, genus, subgenus, and species level with an accuracy higher than 77.0%, regardless of the taxonomic level challenged. This approach does not require inspection of internal organs to address identification, does not rely on identification keys, and can be implemented under field or near-field conditions, showing promise for sandfly pro-active and passive entomological surveys in an era of scarcity in medical entomologists.

First Detection and Molecular Analysis of Leishmania infantum DNA in Sand Flies of Kosovo.

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum.

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.

Phlebotomine sand flies (Diptera: Psychodidae) of the Maghreb region: A systematic review of distribution, morphology, and role in the transmission of the pathogens.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various human and animal pathogens such as Bartonella bacilliformis, Phlebovirus, and parasitic protozoa of the genus Leishmania, causative agent of leishmaniases that account among most significant vector-borne diseases. The Maghreb countries Mauritania, Morocco, Algeria, Tunisia, and Libya occupy a vast area of North Africa and belong to most affected regions by these diseases. Locally varying climatic and ecological conditions support diverse sand fly fauna that includes many proven or suspected vectors. The aim of this review is to summarize often fragmented information and to provide an updated list of sand fly species of the Maghreb region with illustration of species-specific morphological features and maps of their reported distribution. MATERIALS AND METHODS: The literature search focused on scholar databases to review information on the sand fly species distribution and their role in the disease transmissions in Mauritania, Morocco, Algeria, Tunisia, and Libya, surveying sources from the period between 1900 and 2020. Reported distribution of each species was collated using Google Earth, and distribution maps were drawn using ArcGIS software. Morphological illustrations were compiled from various published sources. RESULTS AND CONCLUSIONS: In total, 32 species of the genera Phlebotomus (Ph.) and Sergentomyia (Se.) were reported in the Maghreb region (15 from Libya, 18 from Tunisia, 23 from Morocco, 24 from Algeria, and 9 from Mauritania). Phlebotomus mariae and Se. africana subsp. asiatica were recorded only in Morocco, Ph. mascitti, Se. hirtus, and Se. tiberiadis only in Algeria, whereas Ph. duboscqi, Se. dubia, Se. africana africana, Se. lesleyae, Se. magna, and Se. freetownensis were reported only from Mauritania. Our review has updated and summarized the geographic distribution of 26 species reported so far in Morocco, Algeria, Tunisia, and Libya, excluding Mauritania from a detailed analysis due to the unavailability of accurate distribution data. In addition, morphological differences important for species identification are summarized with particular attention to closely related species such as Ph. papatasi and Ph. bergeroti, Ph. chabaudi, and Ph. riouxi, and Se. christophersi and Se. clydei.

Phlebotomine Sand Flies in Southern Thailand: Entomological Survey, Identification of Blood Meals and Molecular Detection of Trypanosoma spp.

An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.

Identification of blood source preferences and Leishmania infection in sand flies (Diptera: Psychodidae) in north-eastern Algeria.

Leishmaniases are among the most neglected vector-borne diseases, infecting humans as well various animal hosts with clinical outcomes varying from cutaneous disorders to visceral and life-threatening disease. In Algeria, canine leishmaniasis (CanL) caused by Leishmania infantum is endemic mainly throughout the northern regions of the country with the Mediterranean climate that favours the occurrence of Larroussius sand flies, the vectors of the parasite. This study conducted in Bougaa and Kherrata, two regions located in north-eastern Algeria and endemic for CanL, focuses on: i) composition of sand fly fauna, ii) screening of Leishmania parasites and iii) the blood sources of engorged females. Entomological surveys were conducted between June and September 2019 using CDC light-traps in rural areas of both regions. Sand fly specimens were morphologically identified, females were screened for Leishmania DNA using kDNA and ITS1 primers, blood meals in engorged females were identified by peptide mass mapping (PMM)-based MALDI-TOF mass spectrometry and confirmed by DNA sequencing analysis. Overall, 1940 specimens (844 males, 1096 females) were collected, all belonging to the subgenus Larroussius: Phlebotomus perniciosus, (94.64%), Ph. perfiliewi (4.74%) and Ph. longicuspis (0.62%). No Leishmania DNA was detected in the evaluated pools (n = 106) (1096 females). PMM-based MALDI-TOF MS successfully identified a source of blood in 92% (141/154) of engorged females (135 Ph. perniciosus and 6 Ph. perfiliewi). All blood meals were taken from domestic cattle (Bos taurus) except for one originating from a dog (Canis lupus familiaris) and one from sheep (Ovis aries). Sequencing of host cytochrome B gene confirmed these identifications but showed lower success rate of 58% (29/50), demonstrating the high effectivity of peptide mass mapping (PMM)-based MALDI-TOF mass spectrometry for routine identification of blood meals of varying degree of digestion. Our findings represent first record of cattle and dog blood in sand flies in Algeria and striking feeding preference of local sand fly population at domestic sites of studied regions for cattle which may play an important role in parasite transmission. Further studies are needed to better understand potential contribution of cattle to ecology of sand flies and epidemiology of leishmaniasis in north-eastern Algeria.

Entomological study in an anthroponotic cutaneous leishmaniasis focus in Morocco: Fauna survey, Leishmania infection screening, molecular characterization and MALDI-TOF MS protein profiling of relevant Phlebotomus species.

In Morocco, leishmaniases are a major public health problem due to their genetic diversity and geographical distribution. Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted typically by bite of phlebotomine sand flies. This study identifies sand fly fauna in Ibaraghen village, province of Azilal, which is a focus of CL, by combination of morphological and molecular methods (sequencing of COI gene, MALDI-TOF MS protein profiling). Nested-kDNA PCR was used to detect and identify Leishmania species within potential vector species. 432 CDC light traps were placed at different heights above ground level at four capture sites during a whole year. Traps at 1.5 m above the ground yielded capture of sand flies almost double compared to above ground level (29.33%), while the collection reached 55.09% when the traps were placed 2.5 m above ground. A total of 2,830 sand flies were collected, 2,213 unfed specimens were morphologically identified, 990 males (44.73%) and 1,223 females (55.26%) of 13 species; ten Phlebotomus species and three Sergentomyia species. Six species were analysed by MALDI-TOF MS protein profiling (4 Phlebotomus and 2 Sergentomiya species), and their identification was confirmed by COI sequencing. 1,375 unfed females were screened for the presence of Leishmania by nested-kDNA PCR in pools, 11/30 pools of P. sergenti showing a single band of 750 bp corresponding to L. tropica. Our results confirm the role of P. sergenti as a proven vector in Azilal focus of cutaneous leishmaniasis; however, the relative abundance of other species known as vectors of Leishmania species emphasizes the risk of introduction of L. infantum and L. major in this province. For the first time in Morocco, a combined approach to identify sand flies by both morphology and molecular methods based on DNA barcoding and MALDI-TOF MS protein profiling was applied.

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.

Ecology, seasonality and host preferences of Austrian Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, populations.

BACKGROUND: Sand flies are principal vectors of the protozoan parasites Leishmania spp. and are widely distributed in all warmer regions of the world, including the Mediterranean parts of Europe. In Central European countries, the sand fly fauna is still under investigation. Phlebotomus mascittii, a suspected but unproven vector of Leishmania infantum, is regarded as the most widely distributed species in Europe. However, many aspects of its biology and ecology remain poorly known. The aim of this study was to provide new data on the biology and ecology of Ph. mascittii in Austria to better understand its current distribution and potential dispersal. METHODS: Sand flies were collected by CDC light traps at four localities in Austria for 11 (2018) and 15 weeks (2019) during the active sand fly season. Climatic parameters (temperature, relative humidity, barometric pressure and wind speed) were retrospectively obtained for the trapping periods. Sand flies were identified by a combined approach (morphology, DNA barcoding, MALDI-TOF protein profiling), and blood meals of engorged females were analysed by DNA sequencing and MALDI-TOF mass spectrometry. RESULTS: In total, 450 individuals of Ph. mascittii were caught. Activity was observed to start at the beginning of June and end at the end of August with peaks in mid-July at three locations and early August at one location. Increased activity was associated with relatively high temperatures and humidity. Also, more individuals were caught on nights with low barometric pressure. Analysis of five identified blood meals revealed chicken (Gallus gallus) and equine (Equus spp.) hosts. Sand fly abundance was generally associated with availability of hosts. CONCLUSION: This study reports unexpectedly high numbers of Ph. mascittii at selected Austrian localities and provides the first detailed analysis of its ecology to date. Temperature and humidity were shown to be good predictors for sand fly activity. Blood meal analyses support the assumption that Ph. mascittii feeds on mammals as well as birds. The study significantly contributes to understanding the ecology of this sand fly species in Central Europe and facilitates prospective entomological surveys.

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas.

BACKGROUND: Simulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC-MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC-MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase. CONCLUSIONS/SIGNIFICANCE: This study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.

Phlebotomus (Adlerius) simici NITZULESCU, 1931: first record in Austria and phylogenetic relationship with other Adlerius species.

BACKGROUND: Phlebotomine sand flies are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). Information on sand flies in Central Europe is scarce and, to date, in Austria, only Phlebotomus mascittii has been recorded. In 2018 and 2019, entomological surveys were conducted in Austria with the aim to further clarify sand fly distribution and species composition. RESULTS: In 2019, a Ph. simici specimen was trapped in Austria for the first time. Analyses of two commonly used marker genes, cytochrome c oxidase I (coxI) and cytochrome b (cytb), revealed high sequence identity with Ph. simici specimens from North Macedonia and Greece. Phylogenetic analyses showed high intraspecific distances within Ph. simici, thereby dividing this species into three lineages: one each from Europe, Turkey and Israel. Low interspecific distances between Ph. simici, Ph. brevis and an as yet unidentified Adlerius sp. from Turkey and Armenia highlight how challenging molecular identification within the Adlerius complex can be, even when standard marker genes are applied. CONCLUSION: To our knowledge, this study reports the first finding of Ph. simici in Austria, representing the northernmost recording of this species to date. Moreover, it reveals valuable insights into the phylogenetic relationships among species within the subgenus Adlerius. Phlebotomus simici is a suspected vector of L. infantum and therefore of medical and veterinary importance. Potential sand fly expansion in Central Europe due to climatic change and the increasing import of Leishmania-infected dogs from endemic areas support the need for further studies on sand fly distribution in Austria and Central Europe in general.

Sand fly fauna of Crete and the description of Phlebotomus (Adlerius) creticus n. sp. (Diptera: Psychodidae).

BACKGROUND: The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. METHODS: All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. RESULTS: Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. CONCLUSIONS: The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

An integrative approach to identify sand fly vectors of leishmaniasis in Ethiopia by morphological and molecular techniques.

BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.