Șuleșco et al. Parasites Vectors (2021) 14:371 https://doi.org/10.1186/s13071-021-04858-4 Parasites & Vectors RESEARCH Open Access Phlebotomine sand fy survey in the Republic of Moldova: species composition, distribution and host preferences Tatiana Șuleșco1* , Ozge Erisoz Kasap2 , Petr Halada3 , Gizem Oğuz2, Dimian Rusnac1, Marketa Gresova4, Bulent Alten2 , Petr Volf4 and Vit Dvorak4 Abstract Background: Phlebotomine sand fies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a frst 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 fies using CDC light traps and manual aspirators. Species identifcation was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profling). In engorged females, host blood was identifed 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 fies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identifed by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfliewi and Phleboto- mus sp. (Adlerius), the frst being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only fve 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 fy populations of three species in Moldova that repre- sent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied. Keywords: Phlebotomus, Bloodmeal analysis, Haplotype network, MALDI-TOF MS protein profling Background transmission, which occurs mostly in the Mediterranean Phlebotomine sand fies (Diptera: Psychodidae) are vec- countries where L. infantum, transmitted by several spe- tors of several pathogens including parasitic protozoans cies of the subgenus Larroussius, is well established and of the genus Leishmania and phleboviruses and thus of where two other Leishmania species, L. donovani and L. great importance in human and veterinary medicine tropica, have recently re-emerged [2]. However, potential [35]. In Europe, they are incriminated in Leishmania expansion of sand fies due to climatic and environmen- tal changes into regions where they were not previously *Correspondence: [email protected]; [email protected] established is expected in the near future [36], posing a 1 Laboratory of Entomology, Institute of Zoology, Chisinau, Republic risk of Leishmania introduction into non-endemic areas of Moldova [47]. Recently, permanent sand fy populations were Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Șuleșco et al. Parasites Vectors (2021) 14:371 Page 2 of 17 recorded in several countries north of their traditional permanent sites in Ceadir-Lunga close to two poultry distribution [38, 43], and the knowledge of sand fy fauna houses between June 22 and September 26, 2015. Each in long-time understudied regions like ex-Yugoslavia site was sampled 2–3 times per week; number of per- countries [14] and Romania [5] was signifcantly updated, formed samplings was reduced to once per week in case further demonstrating the importance of entomological of rain. Collection by manual aspirators inside houses feld research at the edge of their occurrence. (n = 13) and animal shelters (n = 14) was conducted sev- Moldova is considered a country non-endemic for eral times per week between June 26 and September 14, leishmaniasis; there are no records of autochthonous 2015. Every time one house and/or one animal shelter human or canine cases [37]. In the past, presence of three was visited. In 2017, two CDC light traps operated every Phlebotomus species was reported: P. papatasi (Scopoli), week on the permanent sites between July 6 and Septem- P. perfliewi Parrot and P. chinensis Newstead [41, 42, 45]. ber 25. Additionally, sand fies were collected manually However, the historical studies provided scarce informa- inside a chicken coop and a house (July 30–31, 2017). tion about their abundance and geographical distribu- tion within the country, and there has been no update Morphological identifcation of sand fies regarding species composition, spatial distribution and Te collected insects were killed by freezing in dry ice bloodmeal preferences of sand fies in Moldova since and preserved in 70 or 96% molecular grade ethanol. then. After several decades, increasing biting nuisance For morphological identifcation, head and genitalia of reported by residents from southern Moldova since each specimen were dissected and mounted on slides 2011 initiated this study, which presents frst data to our using CMCP-10 high viscosity mounting medium (Poly- knowledge on sand fies in Moldova after 50 years. sciences, Hirschberg, Germany) or Berlese mounting medium, and the rest of the body was stored in ethanol Methods for molecular analyses. Species identifcation was based Sand fy sampling on decisive morphological characters using published A countrywide feld survey was conducted to assess the keys and descriptions [32, 40]. For specimens belonging presence of sand fies at 58 localities from 20 regions to the subgenus Adlerius, morphometric measurements between 2013 and 2017. Initially, feld surveys were of decisive characters on head and genitalia were done conducted at a rural locality in southern Moldova (July using a light microscope Olympus BX51 (Olympus Life 2013, August 2014 and June–September 2015) and an Science, Waltham, USA) with a camera system Olympus urban area in central Moldova (July–September 2013) D70. Morphological characters were measured using the using miniature Centre for Disease Control (CDC) light QuickPHOTO MICRO 3.0 software (Promicra, Prague, traps (John W. Hock Company, model 512, Gainesville, Czech Republic) and compared with previously pub- Florida, USA) and manual aspirators (Additional fle 1: lished values [3, 13]. Table S1). In 2015, two CDC light traps were operated on the same sites close to an animal shelter between June Molecular taxonomy of sand fies 22 and September 26, 2015. Cross-sectional entomologi- Te remaining body parts of sand fies were stored in 96% cal surveys were then conducted between 2016 and 2017. ethanol for DNA extraction that was done using High A total of 55 localities from 16 regions were surveyed in Pure PCR Template Preparation Kit (Roche Life Sci- 2016 and 10 localities from 9 regions in 2017 (Fig. 1a). ence, Penzberg, Germany). Species identity of selected Sampling was performed using CDC light traps (Trap- sand fy specimens was further assessed by amplifcation pola per Monitoraggio Zanzare, IMT Original 2002, of cytochrome oxidase I (cox1) of mtDNA region using Italy) baited with CO 2 (dry ice) and placed inside or out- LCO/HCO primer pair or amplifcation of the second side of the animal shelters (livestock sheds, hen houses, internal transcribed spacer 2 (ITS2) using the primer pair dog kennels) (Fig. 1b). Tey were used overnight in the JTS3/C1a following the protocols published by Folmer places protected from wind exposure. Entomological col- et al. [17] and Depaquit et al. [7], respectively. Relevant lections were complemented by mouth aspirators inside molecular markers were chosen for analyzed species the houses and animal shelters. Mouth aspirators were depending on the availability of reference sequences, also used within human dwellings and animal shelters. beside cox1, which is widely used as a universal DNA Each collecting site was numbered and mapped using a barcoding primer for sand fies [6]. ITS2 was used for P. global positioning system (GPS). Ceadir-Lunga, a rural perfliewi as it was deployed in the previous study of P. locality in southern Moldova, was selected for seasonal perfliewi complex [9]. PCR products were purifed using sand fy collections in 2015 and 2017 using CDC traps a QIAquick PCR Purifcation Kit (Qiagen, Hilden, Ger- and manual aspirators (WGS84 coordinates: 46.06549 many) and directly sequenced in both directions using N, 28.84219 E). Two CDC light traps operated on two the same primers used for DNA amplifcation. Sequences Șuleșco et al. Parasites Vectors (2021) 14:371 Page 3 of 17 a 1. Malaesti 26. Vorniceni 2. Parcani 27. Sadova 3. Bender 28. Andrusul de Sus 4. Gisca 29. Andrusul de Jos U 5. Suclea; Caragas 30. Rosu 46 45 K 6. Slobozia 31. Manta; Crihana Veche 44 R 7. Ciobruciu 32. Vadul lui Isac 43 8. Balabanesti 33. Colibasi 42 A 9. Corjova; Malaesti Noi 34. Slobozia Mare I 10. Dubasarii Vechi 35. Chisinau 47 11.