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.

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.

Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey.

BACKGROUND: Sand flies (Diptera: Psychodidae) are medically important vectors of human and veterinary disease-causing agents. Among these, the genus Leishmania (Kinetoplastida: Trypanosomatidae), and phleboviruses are of utmost importance. Despite such significance, updated information about sand fly fauna is missing for Balkan countries where both sand flies and autochtonous leishmaniases are historically present and recently re-emerging. Therefore, a review of historical data on sand fly species composition and distribution in the region was followed by a large-scale entomological survey in eight Balkan countries to provide a recent update on local sand fly fauna. METHODS: The literature search involved the period 1910-2019. The entomological survey was conducted at 1189 sampling stations in eight countries (Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Montenegro, North Macedonia, Serbia and Slovenia), covering 49 settlements and 358 sampling sites between June and October in the years 2014 and 2016, accumulating 130 sampling days. We performed a total of 1189 trapping nights at these stations using two types of traps (light and CO2 attraction traps) in each location. Sampling was performed with a minimal duration of 6 (Montenegro) and a maximal of 47 days (Serbia) between 0-1000 m.a.s.l. Collected sand flies were morphologically identified. RESULTS: In total, 8490 sand fly specimens were collected. Morphological identification showed presence of 14 species belonging to genera Phlebotomus and Sergentomyia. Historical data were critically reviewed and updated with our recent findings. Six species were identified in Bosnia and Herzegovina (2 new records), 5 in Montenegro (2 new records), 5 in Croatia (2 new records), 9 in Bulgaria (5 new records), 11 in North Macedonia (1 new record), 10 in Serbia (no new records), 9 in Kosovo (3 new records) and 4 in Slovenia (no new records). CONCLUSIONS: This study presents results of the first integrated sand fly fauna survey of such scale for the Balkan region, providing first data on sand fly populations for four countries in the study area and presenting new species records for six countries and updated species lists for all surveyed countries. Our findings demonstrate presence of proven and suspected vectors of several Leishmania species.

A survey of sand flies (Diptera, Phlebotominae) along recurrent transit routes in Serbia.

Serbia was a country endemic for leishmaniasis with rich and abundant sand fly fauna during the middle of the 20th century. After 1968, the disease was considered as eradicated, and as a consequence, all research on vectors and pathogens was neglected. The recent detection of Leishmania infantum in sand flies and confirmed cases of leishmaniasis in humans and dogs indicated the risk of re-emergence in Serbia. Also, for millions of refugees/immigrants, Serbia is a transit route between East-Mediterranean and Middle-East countries, and Central/North Europe, and it is under constant risk of vector and disease introduction. Primary objectives of this research were to determine which sand fly species are present around transit routes, whether they include vectors of the Leishmania spp. moreover, is/are Leishmania spp. present in the vectors. Surveys were conducted at 55 locations, organised in four clusters, which covered main immigrant routes and shelters/camps as well as tourist/trade transit routes. In total, 367 sand fly specimens were collected. Nine species were identified: Phlebotomus papatasi, P. perfiliewi, P. tobbi, P. neglectus, P. sergenti, P. alexandri, P. simici, P. balcanicus and P. mascittii. Detection of P. alexandri represents the first record in Serbia. The diversity of sand fly species increased while the number of collected specimens per night decreased during the period of research neglection. Phlebotomus neglectus, a proven vector of L. infantum, was a predominant species in all surveyed clusters, and in 56,52% of locations, it was the only species present. Although all detected species are regarded either as proven or suspected vectors of Leishmania spp., screening of females for Leishmania presence resulted negative. Our study provides insight into the significant changes of sand fly fauna in Serbia during the end of XX and beginning of XXI century. Diverse sand fly fauna in Serbia suggests that the establishment of new leishmaniasis foci is possible.

Updates on the distribution and diversity of sand flies (Diptera: Psychodidae) in Romania.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodidae) are haematophagous insects that transmit the protozoan parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), the main causative agent of both zoonotic visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean basin. Eight species of sand flies have been previously recorded in Romania: Phlebotomus papatasi, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus longiductus, Phlebotomus balcanicus and Sergentomyia minuta. Three of them (P. perfiliewi, P. neglectus and P. balcanicus) were incriminated as vectors of L. infantum. Recent reports of autochthonous CanL in Romania require updates on sand fly distribution and diversity in this country. METHODS: Between 2013-2014 and 2016-2018, CDC light traps and mouth aspirators were used to collect sand flies in 132 locations from Romania, indoors and around various animal species shelters. Species identification of collected specimens was done using morphological keys, genetic tools and MALDI-TOF protein profiling. RESULTS: Sand flies were present in seven localities (5.3%): Eibenthal, Baia Noua, Gura Vaii (south-western Romania, Mehedinti County); Fundatura, Pâhnesti, Epureni (eastern Romania, Vaslui County); and Schitu (southern Romania, Giurgiu County). Of the total number of collected sand flies (n = 251), 209 (83.27%) were Phlebotomus neglectus, 39 (15.53%) P. perfiliewi, 1 (0.40%) P. papatasi, 1 (0.40%) P. balcanicus and 1 (0.40%) P. sergenti (sensu lato). CONCLUSIONS: We confirmed the presence of five sand fly species previously recorded in Romania. However, their updated distribution differs from historical data. The diversity of sand fly species in Romania and their presence in areas with Mediterranean climatic influences constitutes a threat for the reemergence of vector-borne diseases. In the context of CanL and VL reemergence in Romania, but also due to imported cases of the diseases in both humans and dogs, updates on vector distribution are imperative.

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.

Midgut Bacterial Diversity of Wild Populations of Phlebotomus (P.) papatasi, the Vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in Turkey.

Phlebotomine sand flies are hematophagous insects that harbor bacterial, viral and parasitic agents like Bartonella sp., Phleboviruses and Leishmania spp., respectively. There are few reports on bacterial microbiota of Phlebotomus (P.) papatasi but no data available for natural populations of Turkey, where leishmaniasis is endemic. Therefore, we aimed to investigate the midgut bacterial flora of different populations of P. papatasi. Sand flies were collected from different towns (Karaburun, Urla, Ayvacik and Basçayir) located in the western part of Turkey. Laboratory reared P. papatasi were included in the study as an insectarium population. After sterile washing steps, sand flies were dissected and guts were separated. Three pools, (males, unfed females and blood-fed females) were generated for each population. Prokaryotic 16 S rRNA gene was amplified and DGGE was performed. Fourteen different organisms belonging to two Phylum (Proteobactericea and Furmicutes) were identified according to sequence results in the studied pools. The presence of Wolbachia sp. was shown for the first time in the wild-caught sand fly populations of Turkey. This is the first report of gut bacterial flora of wild-caught P. papatasi collected in an endemic area for leishmaniasis in Turkey. Microbiome profiling of wild-caught sand flies will be of great help in the investigating of possible vector control candidates for paratransgenic control approach.

Wing morphology variations in a natural population of Phlebotomus tobbi Adler and Theodor 1930.

Cutaneous leishmaniasis (CL) is highly endemic in the Cukurova region, located on the crossroads of main refugee routes from the Middle East to Europe on the eastern Mediterranean part of Turkey. Our purpose was to investigate the phenotypic variation of Phlebotomus tobbi, the known vector of CL in the region, during one active season. Sand flies and microclimatic data were collected monthly from May to October, 2011, from five locations in six villages in the study area. A geometric morphometric approach was used to investigate wing morphology. Shape analyses revealed that males collected in May and June comprised one group, while specimens collected in August, September, and October formed a second group. Specimens from July were found to be distributed within these two groups. A similar distribution pattern was observed for females, but specimens from October were represented as the third district group. Significant size variation was detected for both sexes between months. Wing size and temperature were negatively correlated for females, but there was no temperature effect for males. Wing size of both sexes was increased in correlation to increasing relative humidity. Males were found to have smaller wings with increasing population density.

Sand fly and Leishmania spp. survey in Vojvodina (Serbia): first detection of Leishmania infantum DNA in sand flies and the first record of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908.

BACKGROUND: Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis started to emerge for the first time in Vojvodina Province (previously non-endemic region of Serbia). Reports of introduced, and later on autochthonous cases of leishmaniasis alerted the possibility of disease emergence. The aim of this study was to bridge more than a half a century wide gap in entomological surveillance of sand fly vectors in Vojvodina, as well as to verify the presence of the vector species that could support Leishmania spp. circulation. RESULTS: During the period 2013-2015, a total of 136 sand flies were collected from 48 of 80 surveyed locations. Four sand fly species of the genus Phlebotomus were detected: P. papatasi, P. perfiliewi, P. mascittii and P. neglectus. Detection of P. mascittii represents the first record of this species for the sand fly fauna in Vojvodina and in Serbia. All female specimens (n = 80) were tested for Leishmania spp. DNA, and three blood-fed P. papatasi specimens were positive (4%). One positive DNA sample was successfully amplified by ITS1 nPCR. The RFLP analysis of the resulting 350 bp fragment showed a typical pattern of L. infantum, and the ITS1 partial sequence blasted in GenBank confirmed 100% identity with L. infantum and L. donovani complex sequences. This result represents the first record of both Leishmania spp. and L. infantum DNA from sand flies in Vojvodina, and in Serbia. CONCLUSIONS: Presence of autochthonous canine leishmaniasis cases, records of Phlebotomus (Larroussius) species proven vectors of L. infantum (P. perfiliewi and P. neglectus) and detection of L. infantum DNA from wild caught (non-competent) vectors, prove that L. infantum is present in Vojvodina and indicates a probable circulation in the region.

Assessment of diagnostic doses for widely used synthetic pyrethroids (Deltamethrin & Permethrin) in an endemic focus of leishmaniasis in Turkey.

BACKGROUND: Leishmania is a group of parasitic flagellated protozoons, which are transmitted by female sand flies and produces health problems in humans and also in wild and domestic animals. So far, 25 Phlebotomus and 4 Sergentomyia species were recorded in Turkey including proven or possible vectors of Leishmania spp. As no single insecticide susceptibility test was conducted targeting the sand flies in Turkey, we aimed to determine the diagnostic dose against two commonly used synthetic pyrethroids (deltamethrin and permethrin) in a hyperendemic area for leishmaniasis. METHODS: Sand flies were collected from villages of Adana in 2-4 September 2013 using Centers for Disease Control and Prevention (CDC) light traps and transferred to the laboratory. The World Health Organisation tube test method was conducted using self-prepared filter papers with different concentrations. In order to determine the diagnostic dose, lethal doses (LD) were calculated by EPA Probit Analysis. Sand flies used in the experiments were dissected, mounted and identified. RESULTS: For the lowest (0.025 %) and highest dose of permethrin (0.5 %), the mortality rate was recorded as 52.6 % and 100 % by the end of 24-h period and the diagnostic dose was recorded as 0.36 %. The mortality rate for lowest (0.0025 %) and highest (0.05 %) doses of deltamethrin was recorded as 54.8 % and 100 %. The diagnostic dose of deltamethrin was determined as 0.9 %. CONCLUSION: An insecticide susceptibility study was conducted in Turkey for the first time and effective doses were determined by calculating the LDs. According to presented results, the wild population of sand flies collected from a hyper-endemic region of Adana Province is still susceptible to deltamethrin and permethrin.

Effects of environmental factors and storage conditions on the performance of Olyset® Plus against sand flies in WHO cone bioassays.

BACKGROUND: Leishmaniasis, visceral and cutaneous, is seen in Turkey and has both public and veterinary importance. So far, four Leishmania species and their vectors have been detected in Turkey. Vector control is essential in endemic areas and several personal protection methods are available including long-lasting insecticidal nets (LLINs). In this study, we aimed to measure the effects of usage and storage conditions on LLINs in a village-scale study. METHODS: Olyset(®) Plus bed nets were set up in different climatic conditions (rain, exposed to sunlight and humidity) and collected after 6 months. The effectiveness of bed nets were tested by WHO's cone test method using wild-caught sand flies. RESULTS: Bed nets, which were placed directly exposed to sunlight (A1, A2) showed lower (17.2%) knock down effect compared to bed nets placed indoors (A3, B1). Twenty-four hour mortality was 100% for the five study groups (A2, A3, B1, C1, C2) whereas group A1 was found to have a lower mortality rate (44.4%). CONCLUSION: Bed nets need to avoid direct exposure to sunlight. When used and stored in appropriate conditions (cool, well-ventilated place away from sunlight) they can be used as an effective vector control tool in endemic areas.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Evaluation of the efficacy of Olyset® Plus in a village-based cohort study in the Cukurova Plain, Turkey, in an area of hyperendemic cutaneous leishmaniasis.

The aim of this study was to measure the protective efficacy of Olyset(®) Plus, a new long-lasting factory-treated insecticidal net (LLIN) incorporated with 2% permethrin and 1% of the synergist piperonyl butoxide (PBO), against cutaneous leishmaniasis (CL) transmission under field conditions. A village-scale trial, promoting the use of LLIN by the local inhabitants of the study area was conducted as a pilot study in a new hyperendemic focus of CL caused by a Leishmania infantum/L. donovani hybrid parasite transmitted by proven vector species Phlebotomus tobbi in Cukurova Plain, Adana, Turkey, between May, 2013 and May, 2014. The study area comprised eight villages; two of them were selected as an intervention village with Olyset(®) Plus net (Kizillar) and a control village without net application (Malihidirli). Six villages with surrounding allopatric barriers were utilized as a buffer zone cluster between intervention and control villages. Monthly entomological surveys were performed in the intervention and control villages and Damyeri, representing the other six villages, to collect adults of Phlebotomus tobbi. Results showed a significant reduction in cutaneous leishmaniasis incidence in the intervention village from 4.78% to 0.37%. The protective efficacy rate of LLIN was 92.2%. In contrast, incidence rates increased in the control village from 3.67% to 4.69%. We also evaluated residual insecticide levels of used nets after six and 12 months of usage. It was determined that the nets had retained full insecticidal strength. These results highlight the value of real-world data on bed net effectiveness and longevity to guide decisions regarding sand fly control strategies. To the best of our knowledge, this is the first field study to evaluate Olyset(®) Plus efficacy in a hyperendemic cutaneous leishmaniasis area.