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. Slobozia Dusca 36. Milestii Mici N 12. Onitcani 37. Costesti 17 E 13. Boscana 38. Ciobanovca 18 14. Magdacesti; Ratus 39. Anenii Noi 19 15. Peresecina 40. Troita Noua 20 24 16. Ciopleni 41. Geamana 23 21 22 17. Braniste 42. Stoicani 18. Cobani 43. Alexandru cel Bun 19. Balatina; Padurea Domneasca 44. Soroca; Rubelnita 20. Moara Domneasca 45. Badiceni 15 21. Navirnet 46. Visoca; Darcauti 27 16 11 22. Albinetul Vechi 47. Floresti 26 14 25 13 12 10 23. Calinesti 48. Ceadir-Lunga 8 9 24. Chetris 49. Corten 35 R 1 25. Lozova; Codrii reserve 50. Taraclia O 36 39 38 2 5 37 40 M 41 3 6 A 4 7 N I A

48 49 28 29 50 30 U K R A I N E 31 32 33 34

b

Fig. 1 Sand fy collection localities and sites in the Republic of Moldova. a Map showing the sand fy collection locations. Settlements located close to each other marked under a same number (dots 5, 9, 14, 19, 25, 31, 44, 46 represent two localities). Black dots: positive sampling localities; white dots: negative sampling localities. b Sampling sites in rural localities of the study area Șuleșco et al. Parasites Vectors (2021) 14:371 Page 4 of 17

were edited and aligned using the BioEdit (v.7.0.9) [22] For species identifcation and cluster analysis, the protein alignment editor. profles were processed using MALDI Biotyper 3.1 and Statistical parsimony network analysis implemented compared with reference spectra of an in-house database in TCS method has been frequently used for reveal- constructed using protein spectra of 25 diferent sand ing the genetic divergence at the intraspecifc level. Tis fy species. For MSP dendrogram creation, an individual method has also been proven as a robust and practical main spectrum (MSP) was generated from each analyzed way for species delimitation in several taxa [23]. Using spectrum. a nucleotide data set, this method calculates the maxi- mum number of mutational steps to link the haplotypes Bloodmeal analysis in a parsimonious way with 95% probability. Tus, the Bloodmeals of engorged females, all identifed as P. DNA sequences of haplotypes belonging to the same papatasi and captured in 11 diferent villages mainly species are grouped in a single network, while the DNA by manual aspirators, were analyzed by three molecu- sequences of haplotypes representing diferent spe- lar approaches, using two DNA-based techniques and a cies are grouped in separate networks. Cox1 sequences mass spectrometry approach. For morphological species obtained for P. papatasi were compared with those avail- identifcation, specimens were dissected and mounted able in GenBank using BLAST algorithm to confrm spe- on slides, and for molecular techniques, abdomens con- cies identifcation. Haplotype network for 36 P. papatasi taining host blood were used. When morphological specimens collected at 20 diferent localities (Additional identifcation was not conclusive, species identifcation fle 2: Table S2) was constructed by PopArt [31, 30] using was further confrmed by MALDI-TOF MS protein pro- TCS method. Te position of collected P. perfliewi speci- fling using remaining thoraxes of analyzed specimens. mens within the species complex Phlebotomus perfliewi Additional fle 3: Table S3 summarizes details about all (s.l.) was assessed by sequencing analysis of ITS2 for six engorged females analyzed for identifcation of blood specimens and comparison with sequences available in origin, providing information about trapping meth- GenBank. Cox1 sequences obtained for Phlebotomus sp. ods, localization of the traps, potential hosts available (Adlerius) from Moldova and the sequences available for at the trapping sites and methods applied to identify the the other members of the subgenus in GenBank were bloodmeals. analyzed together to construct a neighbor joining tree Bloodmeals of engorged females collected in 2016, all based on the Kimura’s two parameter (K2P) substitu- identifed as P. papatasi, were analyzed by combination of tion model in MEGA v.6.0 [46]. Relationship between the RFLP assay by HaeIII and HinfI restriction enzymes and members of Adlerius subgenus was further evaluated by sequencing of cytochrome B (cyt b). In total, 100 blood- constructing parsimony networks using TCS method in fed females were analyzed, originating from 10 of the PopArt. surveyed localities. Tese females were collected mostly indoors, inside either human dwellings (91 specimens) or Identifcation of sand fies using MALDI‑TOF mass hen houses (5 specimens); four specimens were collected spectrometry by a trap placed outside an animal shelter. A 359-bp frag- Sample preparation and analysis by MALDI-TOF MS ment of vertebrate cyt b gene was amplifed using the protein profling followed a protocol optimized for sand modifed vertebrate-universal specifc primers cyt bb1/ fies [12]. In total, 20 specimens (10 collected in 2016, cyt bb2 [29, 34]. PCR amplifcation was performed as 10 collected in 2017) were analyzed. Toraxes of dis- described by González et al. [19], using water instead of sected specimens were homogenized by a manual Bio- DNA template as a negative control to exclude contami- Vortexer homogenizer (BioSpec, Bartlesville, USA) with nation by the reagents. Te PCR product was purifed by sterile disposable pestles in 10 μl of 25% formic acid QIAquick PCR Purifcation Kit (QIAGEN), and 15 µl was and briefy centrifuged at 10,000g for 15 s. Two micro- digested by 1 µl of the enzyme HaeIII or HinfI (New Eng- liters of the homogenate was mixed with 2 µl of freshly land Biolabs) at 37 °C for 20 min in 50 µl of total solution prepared MALDI matrix, which was an aqueous 60% according to a protocol of the producer. Digested product acetonitrile/0.3% TFA solution of sinapinic acid (30 mg/ was separated on 2% agarose gel and observed under UV ml, Bruker Daltonics, Bremen, Germany). One micro- light. For a proportion of randomly chosen specimens, liter of the mixture was then spotted on a steel MALDI the purifed amplicons were sequenced in both directions plate in duplicate. Protein mass spectra were measured using the same primers, cyt bb1/cyt bb2, to confrm the in a mass range of 4–25 kDa on an Ultrafex III MALDI- bloodmeal identifcation by RFLP. TOF spectrometer (Bruker Daltonics) as a sum of 2000 Bloodmeals of 25 chosen engorged females collected in laser shots (20 × 100 shots from diferent positions of the 2016 (8 specimens) and 2017 (17 specimens) at 4 locali- sample spot) and analyzed by FlexAnalysis 3.4 software. ties (Ceadir Lunga, Corjova, Malaesti Noi and Slobozia Șuleșco et al. Parasites Vectors (2021) 14:371 Page 5 of 17

Mare), all identifed by morphology as P. papatasi, were manual aspirators inside the buildings. Phlebotomus per- analyzed by peptide mass mapping of host-specifc hemo- fliewi was present in 13.8% (n = 8) of all sampled locali- globin peptides using MALDI-TOF mass spectrometry ties and 6.6% (n = 10) of all sites, located in six regions as described by Hlavackova et al. [24]. From dissected of the country. Tis species was sampled only by CDC specimens, thoraxes were stored for confrmation of spe- light traps with or without dry ice, placed inside or out- cies identifcation by MALDI-TOF MS protein profling, side the animal shelters with the potential animal hosts and abdomens were homogenized in 50 μl of distilled represented mainly by goats, poultry and dogs. Phleboto- water (Merck KGaA, Darmstadt, Germany) by BioVor- mus sp. (Adlerius) specimens were collected from 10.3% texer homogenizer (BioSpec); 10 μl of the homogenate (n = 6) of all inspected localities belonged to four regions. was then incubated with 10 μl of 50 mM N-ethylmorpho- Tis taxon was sampled mainly by CDC light traps; only line acetate bufer (pH 8.1; Sigma-Aldrich) and 100 ng of two females were captured by manual aspirator inside a trypsin (Promega) at 37˚C for 30 min. After the diges- hen house and one male was collected inside the house. tion, 0.5 μl of the mixture was deposited on a MALDI A total of 225 and 47 sand fies were sampled in 2015 plate in duplicate, air-dried and overlaid with 0.5 μl of and 2017, respectively, in the locality Ceadir-Lunga. In MALDI matrix (aqueous 50% acetonitrile/0.1% TFA total, 18 specimens of all three species were trapped using solution of α-cyano-4-hydroxycinnamic acid; 5 mg/ml; CDC traps between June 22 and September 26, 2015: P. Bruker Daltonics). Peptide mass mapping spectra were papatasi (4 males, 5 females), P. perfliewi (4 females) acquired on an Ultrafex III MALDI-TOF instrument in and Phlebotomus sp. (Adlerius) (5 females). First speci- the mass range of 700–4000 Da and calibrated externally mens were trapped in mid-July and last in mid-August. using a peptide standard PepMix II (Bruker Daltonics). Collection by manual aspirators inside houses and ani- At least two peptides per sample were selected for MS/ mal shelters between June 26 and September 14, 2015, MS sequencing using LIFT technology. MS/MS data yielded 207 sand fies (140 specimens in the houses and were searched against the SwissProt database subset of 67 specimens in animal shelters). Te highest number of vertebrate proteins using in-house MASCOT 2.1 search sand fies (n = 160) of two species was collected in July in engine (Matrix Science). hen houses: P. papatasi (49 males, 108 females) and Phle- botomus sp. (Adlerius) (1 male, 2 females). Indoor sand Results fy activity was markedly longer, frst specimens being Entomological survey collected at frst aspiration in June 24 and last in Septem- In total, 151 sites from 58 localities were sampled using ber 14 which was the last day of collections by aspirators. CDC traps and manual aspirators, of which 65 (43.0%) In 2017, a total of 18 sand fies were collected by two sites were positive for sand fies (Additional fle 1: CDC traps, operated on the permanent sites between July Table S1). A total of 793 sand fies (34.2% males) were 6 and September 25. Te majority of specimens (n = 10) collected and identifed between 2013 and 2017 from belonged to P. papatasi (3 males, 3 females), Phlebotomus 30 (51.7%) localities belonged to 12 regions of Moldova sp. (Adlerius) (3 females) and P. perfliewi (1 female) was (Fig. 1a). collected in July. Seven sand fy specimens of two species Overall, 66.9% (534/793) of specimens were collected [4 males of P. papatasi and 3 females of Phlebotomus sp. by manual aspirators from 31 sites belonging to 21 vil- (Adlerius)] were sampled in August and one specimen (P. lages, inside the houses (381 specimens, 26 sites) and papatasi male) in September 2017. Manual collection of animal shelters (153 specimens, 5 sites) (Fig. 1b). Tree sand fies inside a chicken coop and a house (July 30–31, sand fy species of the genus Phlebotomus were identifed 2017) provided additional 29 P. papatasi specimens (12 based on their morphological characters as described males, 17 females). Numbers of specimens trapped by later: P. papatasi, P. perfliewi and Phlebotomus sp. the CDC traps during the course of active sand fy sea- (Adlerius). Te most abundant species was P. papatasi son in 2015 and 2017, respectively, are shown on graphs (n = 754/793; 265 males, 489 females), followed by Phle- in Additional fle 6: Figure S2. botomus sp. (Adlerius) (n = 20; 3 males, 17 females) and Morphological species identifcation P. perfliewi (n = 19; 4 males, 15 females). P. papatasi was the most widely distributed species, present in 48.3% Specimens of the subgenera Phlebotomus and Lar- (n = 28) of all sampled localities and 41.7% (n = 65) of all roussius were all identified based on morphological collected sites from ten regions in Moldova. During the characters on their head (pharyngeal armature) and entomological surveys, the species was present in high genitalia (spermathecas of females, morphology of numbers inside the hen houses and human dwellings external terminalia of males) as Phlebotomus papa- located in central and southern Moldova. Overall, 71.9% tasi and P. perfiliewi, respectively. For Phlebotomus (n = 542) of P. papatasi specimens were collected by sp. (Adlerius) specimens, decisive morphological Șuleșco et al. Parasites Vectors (2021) 14:371 Page 6 of 17

characters were compared with previously published Species identifcation by molecular techniques values of described species within the subgenus, espe- DNA barcoding by cox1 of 36 randomly selected P. cially four species previously reported from Europe: papatasi specimens provided an alignment 602 bp long, Phlebotomus balcanicus, P. creticus, P. longiductus and showing presence of fve unique haplotypes at the sam- P. simici (Table 1). Three analyzed males had mark- pling localities. Obtained sequences of all specimens edly longer flagellomere 3 (A3) and labrum than the morphologically identifed as P. papatasi confrmed the European species with P. chinensis and P. longiductus species identifcation when compared with sequences having the closest values of the remaining Adlerius from GenBank using BLAST algorithm (99.46%–100% species. Length of appendices on the external geni- identity). Statistical parsimony analysis placed fve hap- talia (coxite, style, parameral sheath) varied but did lotypes into a single network. Two haplotypes (hap1 and not match any of the Adlerius species completely; the hap2) are dominant and present at most of surveyed number of coxite hairs was lower than for P. balcani- localities, while the other three (hap3, hap4 and hap5) cus and higher than for P. simici and P. chinensis. In 15 have a restricted occurrence, two of them unique at a analyzed females, the length of A3 exceeded all that of single locality, Malaesti and Crihana Veche, respectively. Adlerius species except for P. longiductus, which also Among these fve haplotypes, only fve polymorphic posi- has a similar morphology of the pharyngeal armature tions were found (Fig. 3). (Fig. 2). All six analyzed P. perfliewi specimens provided iden- tical sequences of ITS2, and a neighbor joining analysis

Table 1 Mean values obtained for the descriptive morphological characters for 3 males (A) and 15 females (B) of Phlebotomus sp. (Adlerius) in Moldova compared with other Adlerius species reviewed in Artemiev [3] and newly described P. creticus [13] (A) A3a A3/Labruma Style ­lengtha Coxite ­lengtha Parameral sheath Coxite / No. of coxite hairs ­lengtha parameral ­sheatha

Phlebotomus sp. 461.67 1.82 215. 95 436.75 183.67 2.38 60 (Adlerius) (439.44–493.20) (1.69–1.86) (207.10–223.00) (402.10–461.95) (176.47–191.60) (2.20–2.61) (57–64) P. balcanicus 372.00 1.39 200.00 424.00 188.00 2.28 106 (320.00–408.00) (1.17–1.52) (184.00–216.00) (388.00–464.00) (164.00–208.00) (2.08–2.52) (92–130) P. chinensis 419.00 1.59 202.00 364.00 175.00 2.06 24 (396.00–454.00) (1.54–1.64) (188.00–215.00) (344.00–380.00) (164.00–182.00) (1.95–2.09) (20–27) P. creticus 340.00 1.29 x 405.20 195.30 x 69 (285.00–406) (1.14–1.53) x (345.00–451.00) (178.00–219.00) x (54–85) P. longiductus 417.00 1.52 204.00 386.00 195.00 1.98 64 (340.00–500.00) (1.31–1.83) (180.00–236.00) (340.00–428.00) (172.00–240.00) (1.64–2.23) (51–81) P. simici 275.00 1.18 148.00 296.00 189.00 1.57 20 (240.00–290.00) (1.10- 1.20) (140.00–156.00) (270.00–320.00) (170.00–200.00) (1.55–1.59) (19- 22) (B) A3a A3/Labruma

Phlebotomus sp. (Adlerius) 397.80 1.26 (269.90–458) (0.98–1.52) P. balcanicus 304.00 0.90 (244.00–360.00) (0.69–0.99) P. chinensis 325.00 1.27 (280.00–368.00) (1.25–1.31) P. creticus 298.00 0.88 (268.00–326.00) (0.80–0.94) P. longiductus 388.00 1.06 (332.00–440.00) (0.93–1.30) P. simici 244.00 0.80 (200.00–270.00) (0.75–0.86)

Minimum and maximum values of each measurement and count are given in parentheses a Measurements in µm Șuleșco et al. Parasites Vectors (2021) 14:371 Page 7 of 17

Fig. 2 Morphology of Phlebotomus sp. (Adlerius) specimens from Moldova: a pharyngeal armature of a female (LCE6), b parameral sheaths of a male (LMG2), c male coxite with a feld of hairs (LMG2)

10 samples

1 sample

Andrusul_de_Jos Balabanesti papatasi_moldova_hap_4 Chetris Ciopleni Colibasi Crihana_Veche papatasi_moldova_hap_3 Lozova Magdacesti Onitcani

papatasi_moldova_hap_5 Rosu Slobozia_Dusca Ciobruci papatasi_moldova_hap_2 Costesi Ceadir_Lunga Dubasarii_Vechi Balatina Navimet Malaesti papatasi_moldova_hap_1 Corjova

Fig. 3 Haplotype network constructed for 36 P. papatasi specimens collected from diferent regions of Moldova. The relative frequency of haplotypes was refected by the size of the circle, and dashes represent the mutational steps

with sequences of P. perfliewi species complex available Te fnal alignment of the 12 cox1 sequences obtained from GenBank placed them in a lineage of P. perfliewi for the Phlebotomus sp. (Adlerius) specimens collected (s.s.) with populations from Greece and Ukraine. from Moldova was 547 bp long. All the Moldovan Phle- botomus sp. (Adlerius) specimens were revealed to Șuleșco et al. Parasites Vectors (2021) 14:371 Page 8 of 17

belong to the same species as they clustered together species in the reference database with a log score value in the NJ tree and to have diverged from the rest of the (LSV) > 2.0, which is accepted as the unambiguous assign- Adlerius species included in the analysis (mean K2P dis- ment. Similarly, four specimens identifed by morphol- tance: 6.5%–16.7%). Te clade comprised P. longiductus ogy as P. perfliewi (1 male and 1 female from Braniste, from China, Phlebotomus sp. (Adlerius) from Moldova 2 females from Balabanesti) produced spectra similar to and P. chinensis from China, which was divided into P. perfliewi in the reference database with LSV > 2.0 and three well-supported (100% bootstrap values) lineages thus were conclusively identifed as this species. Protein (Fig. 4). Concordantly, the mean K2P distances between spectra of one male and one female of Phlebotomus sp. Phlebotomus sp. (Adlerius) from Moldova and P. chinen- (Adlerius) originating from Magdacesti and Balaban- sis and P. longiductus from China were higher (13.4% and esti, respectively, showed diferences from spectra of all 6.5%, respectively) than those yielded between some spe- Adlerius species represented in the reference database (P. cies pairs within the subgenus Adlerius (Additional fle 4: arabicus, P. balcanicus, P. creticus, P. halepensis, P. simici), Table S4). Supporting these results, TCS identifed three indicating that Moldovan specimens represent another independent networks for these three taxa. Te frst net- species within the subgenus Adlerius. work included only the fve Phlebotomus sp. (Adlerius) Of material collected in 2017, most efort was there- haplotypes from Moldova, while the second and third fore focused on the further analysis of Phlebotomus sp. networks comprised P. longiductus (n = 2) and P. chin- (Adlerius). Seven of ten analyzed specimens (1 male from ensis (n = 5) haplotypes from China, respectively. With a Corten, 6 females from Ceadir-Lunga) produced identi- 95% connection limit, 32 mutational steps were required cal protein profles similar to those collected in 2016. to connect Moldovan Phlebotomus sp. (Adlerius) to P. In addition, protein profles of one male of P. papatasi longiductus and 64 mutational steps to connect Phleboto- (locality Ceadir-Lunga) and two females of P. perfl- mus sp. (Adlerius) from Moldova to P. chinensis (Fig. 5). iewi (locality Ceadir-Lunga and Slobozia Mare) were Te NJ tree clearly showed that the recently identifed obtained, all similar with protein spectra of respective P. creticus [13] is highly diverged from Phlebotomus sp. species obtained in the previous season. (Adlerius) from Moldova (mean K2P distance = 12.3%), Specimens of three recorded species that belong to clustering together with P. balcanicus sequences from diferent subgenera of the genus Phlebotomus produced Romania and Serbia. Tis group was placed as a sister reproducible, distinctive and substantially diferent pro- taxon to P. balcanicus from Turkey (Fig. 4). Te mean tein spectra (Fig. 6a), enabling rapid and conclusive spe- K2P distance between P. creticus and P. balcanicus from cies identifcation. No diferences in the protein spectra Romania and Serbia was lower (1.8%) than the one of specimens from diferent localities were observed. Te yielded between P. creticus and P. balcanicus from Turkey dendrogram constructed based on protein spectra of (4.5%) (Additional fle 4: Table S4). Tese results were in all 20 analyzed specimens shows clear clustering corre- concordance with the TCS results, which grouped P. cre- sponding to these three species (Fig. 6b). It also demon- ticus and P. balcanicus from Romania and Serbia in one strates that two specimens with spectra of compromised network and constructed an independent network for quality [LCE01, a P. perfliewi female from Ceadir-Lunga, P. balcanicus from Turkey (Additional fle 5: Figure S1). and LCU01, a Phlebotomus sp. (Adlerius) male from Cor- P. simici specimens originated from Austria and Greece ten, both collected in 2017] were successfully and cor- were clustered together, while Turkish and Israeli P. sim- rectly identifed, albeit positioned on long branches that ici sequences were placed in a distinct lineage in the NJ refect the lower quality of their spectra. Protein spectra tree, with high bootstrap support (> 95.0%). Rest of the of collected Phlebotomus sp. (Adlerius) specimens were taxa classifed in the subgenus Adlerius were each repre- compared with protein profles of three species of the sented by a single lineage (Fig. 4). subgenus Adlerius recently known to occur in Europe: Phlebotomus balcanicus and P. simici from an entomo- MALDI‑TOF mass spectrometric protein profling logical survey in Bosnia and Herzegovina, Montenegro Species identifcation of chosen specimens from col- and Northern Macedonia [14] and newly described P. lections performed in 2016 and 2017 was confrmed by creticus from Crete [13]. Figure 7 shows that specimens MALDI-TOF MS protein profling. Of 20 analyzed, all of all four species grouped in their own distinct clades, except two produced intense, species-specifc protein Phlebotomus sp. (Adlerius) forming a sister cluster with P. profles of high quality. simici from Northern Macedonia. Of material collected in 2016, protein profles of four specimens originating from two localities (Balabanesti Bloodmeal analysis of engorged females and Ciopleni) identifed by morphology as Phlebotomus A total of 100 engorged P. papatasi females were cap- papatasi indeed matched with protein profles of this tured in 10 villages in 2016. Analysis of bloodmeal Șuleșco et al. Parasites Vectors (2021) 14:371 Page 9 of 17

Fig. 4 Neighbor joining dendrogram based on cox1 sequences showing relations among Adlerius species Șuleșco et al. Parasites Vectors (2021) 14:371 Page 10 of 17

Fig. 5 Haplotype networks constructed for 12 Phlebotomus sp. (Adlerius) specimens from Moldova, six P. chinensis specimens from China (GenBank Accession numbers: KF137543, KF137546–KF137550) and three P. longiductus specimens from China (GenBank Accession numbers: KF137551– KF137553). The relative frequency of haplotypes is refected by the size of the circle; missing haplotypes are illustrated by the small circles, and dashes represent the mutational steps sources by RFLP assay on a part of vertebrate cyt b gene Mare, 2 humans from Corjova, 3 humans from Malaesti provided human restriction patterns in 98 specimens: Noi, 3 humans and 8 chickens from Ceadir-Lunga. In 91 females collected inside human dwellings by aspira- samples collected in 2016, 7 of 8 analyzed specimens tors, 4 females trapped by a CO­ 2-baited CDC trap out- successfully provided host peptide maps, confrming all side an animal shelter with rabbits and poultry and 3 identifcations of 5 human bloodmeals (2 from human engorged females trapped inside hen houses with poul- dwellings and 3 from animal shelters) and 2 chicken try. Two specimens collected inside a hen house provided bloodmeals (both from a hen house) as previously a restriction pattern of chickens (Gallus gallus). Sequenc- revealed by RFLP and cyt b gene sequencing. In samples ing analysis of cyt b amplicons from 2 specimens tested as collected in 2017, identifcations were obtained for 15 of feeding on chickens and from 20 randomly chosen speci- 17 analyzed specimens, showing 12 chicken bloodmeals mens originating from diferent localities confrmed all (all from hen houses) and 3 human bloodmeals (1 from host identifcations revealed by RFLP. human dwelling, 2 from a hen house). MALDI-TOF peptide mass mapping analysis of blood- fed females included specimens collected in 2016 (8 Discussion specimens previously analyzed by RFLP and cyt b gene Tis study provides frst data on sand fy species compo- sequencing as described above) and 2017 (17 speci- sition, spatial distribution and host preferences in Mol- mens), providing distinct host peptide maps for 22 of dova after > 50 years, substantially updating reports that 25 analyzed females. All successfully analyzed speci- indicated the presence of three species (P. papatasi, P. mens displayed 8–16 signals of host-specifc alpha- and perfliewi and P. chinensis) without providing sufcient beta-hemoglobin peptides, except one female with fewer information about their geographical distribution [41, host peptides, which nevertheless still enabled a conclu- 42, 45]. Our study confrmed low sand fy species diver- sive identifcation of the blood source (Additional fle 3: sity reported in Moldova in the past, reporting presence Table S3). Te sequencing of the peptide fragments by of three species. For their species identifcation, an inte- tandem mass spectrometry (MS/MS) and subsequent grative approach was deployed that enables morphologi- database searching provided amino acid sequences of cal and molecular identifcation in parallel, utilizing both these host peptides and assigned the following blood ori- DNA barcoding as a standard method of molecular typ- gin: 6 chickens (Gallus gallus) in samples from Slobozia ing and MALDI-TOF MS protein profling, a rapid and Șuleșco et al. Parasites Vectors (2021) 14:371 Page 11 of 17

a 6000 P. papatasi 5366 531 4 966 2 642 7 847 5 7979 917 4 928 3 973 5 8680 LBA_01 839 5 826 1 958 3 4000 5528 5085 768 9 9835 6279 680 5 8572 5248

2000 5477 6969 901 2 10851 12004 12384 0 5000 P. papatasi 6424

4000 839 4 5312 5364 8678 965 9 847 2 917 0 7977 928 2 LCE_33

3000 825 7 8568 6290 5083 9581 7688 9833 6805 2000 6734 9008 0 179 4 12000 1 12378 10842

15000 P. perfiliewi 526 9 685 3 923 7 952 5 8706 641 9 LBA_03 10000 5477 851 4 9692 9108 5315 5106 7676

5000 6275 943 2 674 8 710 2 10840 12390 0 P. perfiliewi 6000 641 7 5268 6852 870 3 9690 5475 952 2

9234 LSM_24 8548 910 4 9592

4000 5105 499 1 767 4 531 3 9429 710 0

2000 674 5 6273 657 9 12386 10838 0 8 8000 Phlebotomus sp. (Adlerius) 533 3 869 5051 559 7 926 1 847 1 827 3 6000 6247 LMG_02 9122 567 2 539 0 4000 8400 134 1 885 5 8570 1 9596 938 8 6799 7870 6981

2000 9854 642 0 12354 10845 757 5 12124 0

8000 Phlebotomus sp. (Adlerius) 5330 8695 8468 8270 9258 5594 6244 LCE_32 6000 9118 5669 5388 5048 8398

4000 133 7 885 2 1 959 4 938 5 6797 6980 9850

2000 12349 8567 786 7 1211 8 0 5000 7000 9000 11000 13000 m/z

b LBA_09 LBA_01 LBA_02 LCI_03 LCE_33 LCE_01 LBA_03 LBA_10 LBR_02 LBR_01 LSM_24 LCU_01 LCE_02 LCE_06 LCE_05 LCE_27 LCE_28 LCE_32 LBA_11 LMG_02

1000 900 800 700 600 500 400 300 200 100 0 Distance Level Fig. 6 MALDI-TOF MS protein profling of sand fy species in Moldova. a MALDI-TOF mass spectra of three typical Moldovan sand fy species. Zoomed mass range of 4–14 kDa is shown. b Cluster analysis of MALDI-TOF MS protein profles of 20 sand fy specimens collected in Moldova. Distances in the dendrogram are displayed in relative units Șuleșco et al. Parasites Vectors (2021) 14:371 Page 12 of 17

BC_189 FVA_04 BC_144 MO_11 MO_08 CrX_28 CrX_13 CrX_16 CrX_03 CrX_37 ) LCE_05 LCE_32 LCE_28 LBA_11 LMG_02 FSE_35 FSE_10 FSE_24 FSE_17 FSE_26

1000 900 800 700 600 500 400 300 200 100 0 Distance Level Fig. 7 Dendrogram comparing MALDI-TOF mass spectra of Phlebotomus sp. (Adlerius) from Moldova and three other European members of the subgenus Adlerius (Bosnia and Herzegovina: specimen codes BC, Montenegro: specimen codes MO, Northern Macedonia: specimen codes FVA and FSE, Greece: specimen codes CrX). Distances are displayed in relative units

cost-efective tool recently emerging as an alternative city in 1947 [45]. Te Ukrainian sand fy control eforts method for species identifcation of various organisms between 1951 and 1954 focused on treatment of build- including medically important arthropods. It provided ings in the villages afected by dichlorodiphenyltrichlo- 100% correct species determination with most protein roethane (DDT) and hexachlorocyclohexane, providing profles of a high quality and stable for specimens col- a rapid reduction of sand fy fever morbidity and sig- lected at diferent localities and during two consecutive nifcant decrease of sand fy densities [20]. Moreover, seasons, thus representing a sufciently robust identifca- between 1948–1956, the national malaria eradication tion tool. Moreover, correct species determination of few programs successfully used the same insecticides to com- specimens, which provided protein spectra of lower qual- bat the malaria vector Anopheles maculipennis (s.l.) in ity, was nevertheless also achieved. Tese results support Moldova. It can be assumed that such widespread use of the use of this combined taxonomical approach as suit- insecticides led to a decrease of the sand fy densities in able for processing sand fies collected through feld ento- Moldovan villages as well. Sand fy surveillance after the mological surveys. antimalaric campaign showed the presence of three sand Presence of two of three previously detected species fy species in four regions (Vulcanesti, Taraclia, Cahul in Moldova was confrmed by our sampling, P. papatasi and Ceadir-Lunga) in southern Moldova between 1957 being the most abundant and most widely distributed and 1967. In total, 453 sand fy specimens were collected species. Scarce historical data mentioned low numbers manually inside the buildings at 8 localities during that of P. papatasi (5 females and 2 males) in Giurgiulesti vil- decade with average number of specimens per building lage, Cahul region, southern Moldova, in 1946. In 1947, between 0.7 and 5.7 [45]. Our manual collections inside during the papataci fever outbreak in southern Ukraine the buildings in the same villages (Giurgiulesti, Slobozia (Reni region) and southern Moldova (Giurgiulesti), sand Mare, Colibasi) or villages located close to the historical fy surveillance revealed the presence of low numbers sampling sites show a higher average number of sand fies of P. papatasi in other villages located in Cahul region inside the buildings (Table 2). (Chislita, Colibasi, Vadul lui Isac, Manta, Crihana Veche Te presence of sand fies inside the human dwellings villages and Cahul town), but no exact numbers were and animal shelters was frst reported by residents from given. Only one P. papatasi male was caught in Chisinau southern Moldova in 2011 when the local population Șuleșco et al. Parasites Vectors (2021) 14:371 Page 13 of 17

Table 2 Comparison of historical sand fy collections in the Republic of Moldova (1957–1967) and recent collections (2015–2017) Locality Specimens collected Average no. of specimens per Specimens collected Average no. of specimens manually (1957–1967) building (1957–1967) manually (2015–2017) per building (2015–2017)

Etulia 43 2.4 Chismichioi 196 5.7 Alexandru Ioan Cuza (Suvo- 38 1.6 rovo, old name) Giurgiulesti 16 1.3 17a 5.6a Cislita-Prut 18 1.2 Valeni 8 0.7 Slobozia Mare 108 4.2 49 16.3 Colibasi Low ­numbersb Low ­numbersb 32 16 Cahul city Low ­numbersb Low ­numbersb 23c 7.7c Cairaclia 26 1.1 Not specifed Low ­numbersb Low numbers 243d 8.1d Total 453 292 a 2019 b 1947 c 2016 in Rosu (neighboring village to Cahul city) d 2015–2017 in Ceadir-Lunga city started sufering from sand fy bites during the summer until last collections in mid-September, suggesting that season. Our entomological surveys revealed the presence the actual indoor activity of P. papatasi may have been of P. papatasi in high numbers inside the human dwell- even longer. On the contrary, frst specimens from out- ings and hen houses in southern and central Moldova. door trapping were collected in early July, despite earlier Between 2013 and 2017, a total of 793 sand fies (34.2% trapping eforts. In 2015, no specimens were trapped males) were collected and identifed from 30 (51.7%) outdoors after mid-August, and in 2017, outdoor activ- out of 58 localities belonging to 12 regions of Moldova ity ceased by mid-September, despite later trapping (Fig. 1a) by CDC traps and manual aspirators (Addi- eforts (Additional fle 6: Figure S2). Low numbers of tional fle 1: Table S1). Te northernmost sites of P. papa- trapped specimens do not allow drawing robust conclu- tasi collections were recorded at latitudes 47° 35ʹ–47° sions regarding the seasonal dynamics, and few data for 47ʹ where the species was present in low numbers only the species recorded in Moldova are available from other in CDC trap collections; the northernmost record of P. countries of a similar latitude; no information on sea- papatasi sampling in the 1950s was in the Chisinau city sonality was provided by a recent study in Romania [5], at latitude 47°02’ [45]. and only scant remarks on trapping dates were given by a Comparison of historical records and our recent data sand fy survey in Hungary [16]. Recent study of seasonal shows a higher average number of sand fies inside the dynamics of P. mascittii, a species so far not recorded in buildings, and the biting burden reported by the resi- Moldova, at localities in Styria and Lower Austria of sim- dents living at sand fy-infested sites indicates an increase ilar or higher latitude showed sand fy activity from early of sand fy population in Moldova that probably suc- June until end of August, depending on the year and trap- cessfully recovered after ceasing wide-scale applications ping locality [27]. Further efort that will include more of insecticides. Moreover, while breeding of livestock in localities is needed to better understand the seasonal the households was common in the countryside before dynamics of sand fy populations in Moldova. the 1990s, deterioration of the economic situation led to We applied several methods to determine blood sources a reduction of livestock, which was replaced by a large of collected bloodfed females of P. papatasi. Bloodmeal number of backyard poultry in the villages. Perhaps both analysis of engorged P. papatasi females collected in a socioeconomic and environmental changes promoted the 2016 survey by RFLP assay demonstrated their strong increase of P. papatasi densities in the villages. anthropophily; 98% of successfully analyzed females were Continuous trapping in Ceadir-Lunga during the active feeding on humans. Tis fnding is not surprising as 91 of sand fy season in years 2015 and 2017 showed markedly 98 females determined to feed on humans were collected longer activity of P. papatasi indoors; specimens were by manual aspirators inside the houses. Interestingly, the collected from frst aspirations in the second half of June remaining 7 females with detected human blood were Șuleșco et al. Parasites Vectors (2021) 14:371 Page 14 of 17

sampled outside an animal shelter with rabbits and poul- geographically separated by prominent natural barriers try (4 specimens) or even inside hen houses with poultry such as the High Atlas Mountains in Morocco [21]. In (3 specimens), suggesting that P. papatasi females pre- our study, P. papatasi collected at various sites in Mol- ferred to feed on humans even when other hosts were dova showed a limited genetic variability in cytochrome available. Moreover, while domestic cats were present at oxidase I gene, providing fve haplotypes with only fve two indoor sampling sites, they were not determined to polymorphic sites, suggesting a genetically homogene- be a source of blood. Domestic chickens were detected as ous population. With respect to the geography of Mol- a second and less frequently utilized host, chicken blood dova and adjacent regions that lack natural barriers of being identifed in two females collected inside a hen dispersal, we may expect a rather unrestricted gene fow house. Tese fndings were confrmed by a sequencing with populations from neighboring countries. Te very analysis of chosen engorged specimens that included also low mean intraspecifc K2P distance (0.02%) obtained females with alternative blood sources to humans avail- for P. papatasi specimens collected from Moldova was able. Peptide mass mapping using MALDI-TOF mass comparable with those previously reported from Greece, spectrometry was recently introduced as a new and efec- Cyprus [10] and Turkey (Erisoz Kasap et al. [26]) and fur- tive tool for bloodmeal identifcation of hematophagous ther supports the conclusion that Moldovan P. papatasi arthropods [24]. Beside other advantages, this method comprise genetically close populations. in general provides very specifc identifcations in case Two other recorded species, P. perfliewi and Phleboto- of human bloodmeals within this study based on MS/MS mus sp. (Adlerius), were present sporadically in CDC trap sequencing of 3–4 unique peptides of human hemoglob- collections inside or outside the animal shelters. Phle- ins, thus excluding a misidentifcation induced by human botomus perfliewi is one of the main vectors of Leishma- skin debris contamination during the sample prepara- nia infantum in the Mediterranean basin and in Central tion, which may blur the results provided by DNA-based Asia [35]. It is regarded as a species complex of at least methods. With a vast majority of analyzed bloodmeals three species; however, recent taxonomic study failed to identifed as of human origin, we decided to apply this align their morphological and genetic characters, and MALDI-TOF mass spectrometry approach to re-analyze sequencing analyses of two chosen markers (ITS2 and cyt some samples collected in 2016 to exclude a possibility of B) provided incongruent phylogenies [9]. Unfortunately, contamination and analyze all engorged females collected cox1 was not used in that study; we therefore decided to in the following season. It proved to be well applicable, deploy ITS2. Sequences obtained from specimens col- providing 22 identifcations of 25 analyzed specimens, lected in Moldova suggest that they belong to P. perfliewi confrmed all identifcations of human and chicken blood (s.s.) and are closely related to populations from Greece previously revealed by RFLP and cyt b gene sequenc- and Crimea. ing among samples from 2016 and identifed 12 chicken Te subgenus Adlerius harbors over 20 described and bloodmeals and 3 human bloodmeals among sam- formally undescribed species, females of which are usu- ples from 2017. In conclusion, bloodmeal analyses of ally considered indistinguishable morphologically, and engorged females confrmed both humans and chickens their identifcation is often based on the identifcation as hosts of bloodfeeding sand fy females, emphasizing of associated males [1, 3]. Previous historical record of the apparent tight trophic connection of P. papatasi with P. chinensis in Moldova is questionable and shall be con- human dwellings. Our observations support the notion sidered with respect to later evolvement of taxonomy that this species is an opportunistic feeder with host pref- within the subgenus. Phlebotomus chinensis is a species erences varying according to the availability and abun- currently understood to occur only in China. In the past, dance of the blood sources but strongly anthropophilic however, this taxonomic designation was applied to sand when feeding close to or even inside human dwellings. fy populations from much broader geographical areas, A vast majority of females feeding on humans may be which were later described as new Adlerius species [3]. also partially explained by the absence of other domes- Morphological analysis of males from Moldova, albeit tic animals (sheep, goats, cattle) at catching sites in Mol- limited to only three trapped specimens, suggests that dova while these hosts were reported as additional blood they difer from P. chinensis as well as two of three Euro- sources to humans in previously studied foci of P. papa- pean Adlerius species, P. balcanicus and P. simici, in sev- tasi in other regions [25, 48]. P. papatasi is a species with eral characters including number of coxite hairs. Tis was markedly wide geographical distribution; however, stud- further confrmed by MALDI-TOF MS protein profling, ies based on several genetic markers showed that various which added evidence that also a third European Adle- geographical populations are relatively homogeneous and rius species, only recently described P. creticus, provides of limited genetic diferentiation [8, 15]. More genetic species-specifc protein profles distinctively diferent variation was observed mainly among populations from specimens from Moldova. In a dendrogram derived Șuleșco et al. Parasites Vectors (2021) 14:371 Page 15 of 17

from their protein spectra (Fig. 7), Phlebotomus sp. (Adle- in Ceadir-Lunga, a most comprehensively surveyed site rius) from Moldova clustered with P. simici while P. cre- located in the south of the country, suggest that sand fy ticus formed a sister clade with P. balcanicus. For the activity indoors may start earlier than outdoors and last cox1 sequences obtained for the Phlebotomus sp. (Adle- until mid-September. Following studies at diferent local- rius) (both male and female specimens) from Moldova, ities with sand fy presence will provide full understand- the BLAST search revealed these sequences to be 93.78– ing of seasonal sand fy dynamics in Moldova. 94.15% identical to P. longiductus from China (GenBank Moldova is so far regarded as a non-endemic country accession numbers: KF137551–KF137553). Te NJ analy- for leishmaniasis [37]. In the past, a single ofcially pub- sis placed the Moldovan Phlebotomus sp. (Adlerius) and lished imported human case was reported in a 1-year- P. longiductus in highly diverged lineages (Fig. 4), and the old child who traveled with his parents from Rustavi, mean K2P distance between these two lineages (6.5%) Georgia, to Straseni Region, Moldova, in the spring of was comparable to those previously reported for difer- 2013 [33]. In the 1980s, a total of 6000 samples of human ent sand fy species classifed in the subgenus Adlerius serum from 28 regions in Moldova were tested for the (Erisoz Kasap et al. [26]). Tese results are further cor- presence of antibodies to sand fy fever viruses. In the roborated by parsimony network analysis which identi- Vulcanesti region (southern Moldova), 7.0% and 10.5% fed two independent networks for these two lineages, of humans tested were positive for antibodies to sand- suggesting they may represent two diferent species. fy fever Sicilian virus (SFSV) and Neapolitan sandfy Supporting the sequencing data, both female and fever viruses, respectively [44]. Precise information on male Phlebotomus sp. (Adlerius) specimens were found the imported/autochthonous human and canine cases to be diferent from P. longiductus morphologically as of leishmaniasis in Moldova is not available. However, described above, although the statistical signifcance of autochthonous cases of canine leishmaniasis have been this diference was not assessed. P. longiductus is widely recently reported in several countries in proximity to distributed in China, Central and South Asia, and in the Moldova, which are also considered as non-endemic Middle East and has been previously reported from East- for leishmaniasis, namely Romania [11], Ukraine [18] ern Europe (reviewed in [35]). Terefore, examination of and Hungary [47]. Te Republic of Moldova continues representative specimens across its range is needed to to preserve the traditional rural lifestyle with domestic reveal if these morphological and molecular diferences pets, poultry and cattle in backyard animal shelters, but refect intraspecifc geographical variation of local pop- with limited veterinary care. Te presence of two proven ulations or the cryptic speciation within this taxon. Te vectors of leishmaniasis in the country and favorable close relationship between P. balcanicus sequences from environmental conditions may have an impact on the Eastern Europe (Romania and Serbia) and recently iden- emergence and local transmission of leishmaniasis in tifed P. creticus needs further evaluation as well as the Moldova. Intensive and continuous surveillance should divergence of two lineages within the widely distributed be carried out to regularly update the information on P. simici, which has been recently recorded in Austria, sand fy vectors and the presence of Leishmania parasites suggesting possible northward spread in Europe [28]. An in potential domestic and wild reservoirs and sand fies. integrative approach deploying several molecular meth- ods that study both DNA and proteins to complement Conclusions the traditional morphological “golden standard” was Tree sand fy species of the genus Phlebotomus were recently successfully applied when describing Phleboto- identifed in the Republic of Moldova based on inte- mus creticus, a novel species from the eastern Mediterra- grated morphological and molecular approaches. Phle- nean [13], as well as studying sand fy fauna in East Africa botomus papatasi was the most widely distributed [39]. Our fndings further advocate its use as a valuable and abundant species, feeding indoors and strongly tool that provides better understanding of the taxonomy anthropophilic as suggested on bloodmeal analyses. and biogeography of this morphologically challenging yet Haplotype network analysis showed low structuring medically important sand fy subgenus. of the P. papatasi population with only 5 haplotypes Notably, fve species of sand fies were recently of cox1 with minor diferences detected. Two other reported from Romania [5] and six species are known recorded species, P. perfliewi and Phlebotomus sp. from Ukraine (except Crimea) [3, 4, 42]. We may specu- (Adlerius), were present sporadically in CDC trap col- late that some of the species occurring in these countries lections. ITS2 sequences of collected P. perfliewi may be detected by further entomological surveys also in specimens identifed then as P. perfliewi (s.s.). Distinct Moldova, especially when wider surveillance is possible. morphological and molecular characters of Phleboto- While this study cannot provide robust data on the sea- mus sp. (Adlerius) specimens suggest the presence of sonality of three detected species, the results obtained Șuleșco et al. Parasites Vectors (2021) 14:371 Page 16 of 17

yet undescribed species that will be further studied to Declarations reveal their relationships with other species within the Ethics approval and consent to participate subgenus and their potential role in pathogen transmis- Not applicable. sion. Te presence of two proven vectors of leishmania- Consent for publication sis in the country emphasizes a need for intensive and Not applicable. continuous surveillance to update the information on sand fy populations. Competing interests All individual authors declare that they have no competing interests.

Supplementary Information Author details The online version contains supplementary material available at https://​doi.​ 1 Laboratory of Entomology, Institute of Zoology, Chisinau, Republic of Mol- org/​10.​1186/​s13071-​021-​04858-4. dova. 2 Department of Biology, Ecology Section, Faculty of Science, VERG Labo- ratories, Hacettepe University, Ankara, Turkey. 3 BioCeV, Institute of Microbiol- ogy of the Czech Academy of Sciences, Vestec, Czech Republic. 4 Department Additional fle 1: Table S1. Positive sand fy localities in the Republic of of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic. Moldova. Additional fle 2: Table S2. Information on Phlebotomus papatasi Received: 24 January 2021 Accepted: 21 June 2021 specimens used for haplotype network constructed by PopArt using TCS method. Additional fle 3: Table S3. Engorged P. papatasi females analyzed for identifcation of blood origin and indicates which methods were applied. 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RD: contributed to feld data homogeneity in diverse geographical populations of Phlebotomus collection and processing. TȘ, VD, OEK: wrote the manuscript. PV, PH, BA: papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA revised the manuscript. All authors read and approved the fnal manuscript. Epidemiological consequences. Infect Genet Evol. 2008;8:159–70. 9. Depaquit J, Bounamous A, Akhoundi M, Augot D, Sauvage F, Dvorak V, Funding Chaibullinova A, Pesson B, Volf P, Léger N. A taxonomic study of Phleboto- This study was supported by VectorNet, a European network for sharing data mus (Larroussius) perfliewi s. l. Infect Genet Evol. 2013;20:500–8. on the geographic distribution of arthropod vectors, transmitting human 10. 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