Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily, Southern Italy.

In this study, the presence of Leishmania DNA and blood feeding sources in phlebotomine sand fly species commonly present in Sicily were investigated. A total of 1,866 female sand flies including 176 blood fed specimens were sampled over two seasons in five selected sites in Sicily (southern Italy). Sergentomyia minuta (n = 1,264) and Phlebotomus perniciousus (n = 594) were the most abundant species at all the sites, while three other species from the genus Phlebotomus (i.e., P. sergenti n = 4, P. perfiliewi n = 3 and P. neglectus n = 1) were only sporadically captured. Twenty-eight out of the 1,866 (1.5%) sand flies tested positive for Leishmania spp. Leishmania tarentolae DNA was identified in 26 specimens of S. minuta, while the DNA of Leishmania donovani complex was detected in a single specimen each of S. minuta and P. perniciosus. Interestingly, seven S. minuta specimens (0.4%) tested positive for reptilian Trypanosoma sp. Blood sources were successfully identified in 108 out of 176 blood fed females. Twenty-seven out of 82 blood sources identified in fed females of P. perniciosus were represented by blood of wild rabbit, S. minuta mainly fed on humans (16/25), while the sole P. sergenti fed specimen took a blood meal on rat. Other vertebrate hosts including horse, goat, pig, dog, chicken, cow, cat and donkey were recognized as blood sources for P. perniciosus and S. minuta, and, surprisingly, no reptilian blood was identified in blood-fed S. minuta specimens. Results of this study agree with the well-known role of P. perniciosus as vector of L. infantum in the western Mediterranean; also, vector feeding preferences herein described support the hypothesis on the involvement of lagomorphs as sylvatic reservoirs of Leishmania. The detection of L. donovani complex in S. minuta, together with the anthropophilic feeding-behaviour herein observed, warrants further research to clarify the capacity of this species in the transmission of pathogens to humans and other animals.

Introduction

Phlebotomine sand flies (Diptera: Phlebotominae) are insects of great interest in human and veterinary medicine. They are vectors of viral and bacterial pathogens and recognized as the main hematophagous arthropods proven to transmit protozoa of the genus Leishmania such as Leishmania infantum, the causative agent of canine leishmaniosis (CanL) in dogs and visceral (VL) or cutaneous (CL) leishmaniosis in humans in the Mediterranean area [1].

Sand flies are distributed throughout many regions of the world and their biodiversity and phenology have been investigated particularly in Leishmania endemic areas. In western Europe, only sand flies of the genus Phlebotomus are competent vectors for Leishmania transmission and P. perniciosus is the most widespread species [2].

Surveillance on phlebotomine sand fly vectors is pivotal to assess the risk for transmission of endemic Leishmania species, but also crucial to monitor the risk for introduction of new Leishmania species in non-endemic territories [2-3].

Several studies have investigated the presence of Leishmania DNA in phlebotomine sand flies, and the reported infection rate in P. perniciosus varied from 0.13% to 50% depending on the epidemiological context [4-5]. Although L. infantum is the only species widespread in Europe that causes illness, human cases due to non-indigenous Leishmania species are increasingly reported [6].

In Italy, imported leishmaniosis cases are mainly associated with international travellers and/or refugees coming from endemic zones, and consist of chronic forms diagnosed many months after entering the country [7-8]. Noteworthy, anthroponotic species such as Leishmania tropica and Leishmania donovani are included among the causative agents of imported human cases, and the risk of introduction and/or wider establishment of these species into Europe is higher since suitable sand fly vectors are present [3,6,9].

Sicily, one of the two major Italian islands, is located in the centre of the Mediterranean Sea. The island is characterized by a typical temperate climate, with mild and wet winters and hot dry summers. It is a well-known hyper-endemic region for CanL with a prevalence of infection up to 40% in dogs which are regarded as the main domestic reservoir hosts and source for human infection [10-11]. According to available entomological surveys on L. infantum vectors in Sicily, P. perniciosus has been recognized as the most abundant species, with P. neglectus and P. perfiliewi much less frequently detected [12-14]. The presence of P. sergenti has also been sporadically reported along the east side of the Island [12-14] implying the risk for L. tropica transmission. Although no autochthonous cutaneous leishmaniosis cases (ACL) caused by L. tropica have been detected in south-western Europe so far, Sicily is an area potentially susceptible to the introduction of this protozoan species due to its proximity to northern Morocco, an emerging ACL area [15], and to the large migratory flow of people between the two territories. As matter of fact, the flow of immigrants from endemic regions of North Africa and the Middle East, together with the rising number of reports of L. tropica human infections in Italy [6,7,16-17], could constitute the first step in its potential spread in Sicily and subsequently across other areas of southern Europe where the competent vector has been described [7].

Female sand flies are hematophagous insects and take blood meals on many vertebrate hosts as reptiles, birds, a variety of domestic and wild mammals and humans, as well showing an opportunistic feeding behaviour [1,4,18-19]. Interestingly, lagomorphs have been found to be the most frequent blood source for P. perniciosus in a large outbreak of human leishmaniosis in Madrid [20-21], and, xenodiagnostic studies demonstrated the role of hares and wild rabbits as reservoir hosts of L. infantum. Together these findings suggest the possible involvement of lagomorph species in the epidemiology of Leishmania infection [22-25]. Therefore, the identification of blood meal sources in wild-caught sand flies provides information on host-feeding patterns under natural conditions, which, in turn, results in data on potential reservoir hosts and essential knowledge for the establishment of efficient control strategies.

Given all of the above considerations, the aim of the present study was to investigate Leishmania infection rates and blood sources of phlebotomine sand fly species caught in Sicily over two transmission seasons.

Materials and methods

Sand fly collection and identification

Sand flies in this study were sampled during two different entomologic surveys carried out in privately owned areas located in Sicily in 2017 and 2018 (Fig 1). In 2017, sand flies were captured in a suburban area (Site A. 38°13’59” N; 15°32’49” E; 263 m.a.s.l) nearby the didactic farm of the Department of Veterinary Science of the University of Messina using a classical light trap (CLT), equipped with a traditional incandescent lamp (12V, 8W) and five Laika 4.0 light traps with LED of different colours (Fig 1). Traps were placed from before sunset until sunrise for three consecutive nights each month from May to October [26]. In 2018, sand fly captures were performed in four different sites, i.e., in three shelters located in suburban areas of Syracuse (Site B. 37°04’54” N; 15°12’43” E; 69 m.a.s.l) (Site C. 37°04’30” N; 15°13’59” E; 416 m.a.s.l) and Catania (Site D. 37°36’45” N; 15°01’46” E; 948 m.a.s.l) and on a farm situated in a rural area of the municipality of Messina (Site E. 38°00’42” N; 15°25’18” E; 177 m.a.s.l). In each of the above sites, light traps (i.e., CLT) were placed from May to October and left working from before sunset until sunrise for two consecutive nights twice a month (Fig 1).

Fig 1

Geographical characteristics of sand fly sampling sites and presence of vertebrate hosts.

Access to the collection sites and sampling procedures were authorized by owners of the farms (Site A and Site E) and of the shelters (Sites B, C and D).

In the laboratory, sand flies were differentiated by sex with the aid of a stereomicroscope and thereafter processed for identification. Briefly, the head and posterior last tergites of females were dissected, cleared and slide-mounted for microscopic observation as described elsewhere [12], and identified to species level using morphological keys [27]. Females were further ranked as unfed (no visible blood in the abdomen), blood fed (presence of blood in the abdomen) and gravid (presence of eggs). Finally, the thorax and the abdomen of each specimen were individually transferred into 2 mL vials containing 70% ethanol and stored for DNA analysis.

DNA extraction and Leishmania spp. detection

Genomic DNA was extracted from portions (i.e., thorax and abdomen) of each sand fly specimen stored in 70% ethanol using the Citogene® Cell and Tissue kit (Citomed, Portugal) according to the manufacturer’s instructions. The DNA extracted was suspended in 30 μL of sterile water and stored at + 4°C until analysis.

Trypanosomatidae DNA amplification

The presence of Trypanosomatidae DNA in unfed, blood fed and gravid females was firstly screened using a one-step PCR protocol with a set of primers targeting sections of the ribosomal internal transcribed spacer (ITS-rDNA) [28]. For further molecular characterisation of ITS-rDNA positive samples, a two-step PCR protocol using a set of primers targeting conserved regions of the mitochondrial cytochrome b gene (cytB) was performed [29]. An additional one-step PCR with specific primers for small subunit ribosomal DNA gene (SSU-rDNA) partial amplification [30] was carried out, to characterise the samples where the presence of Trypanosoma spp. was suggested by BLAST analysis of cytB sequences (S1 Table).

Blood source identification

Identification of blood sources was conducted by the amplification of a 350 bp segment of the host mitochondrial cytB, using the modified vertebrate-universal specific primers (cytB1-F and cytB-2-R) on blood fed sand fly specimens [31]. The cytB PCR was carried out with 5 μL of extracted DNA in a final volume of 25 μL, using 12.5 μL of NZYTaq 2× Green Master Mix (Nyztech, Portugal) and 1.5 μL of each primer (10 pmol/μL). Amplification was performed as follows: one cycle at 94°C for 5 min, followed by 40 cycles consisting of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 1 min, followed by final elongation at 72°C for 7 min [4] (S1 Table). Electrophoresis of PCR products was carried out in 1.5% agarose gel stained with 2.5 μL Greensafe premium® (Nzytech, Portugal), using a 100 bp DNA ladder as a molecular weight marker and final amplicons were visualized under UV light.

Sequence analysis

PCR products were purified and sequenced by Sanger’s method (StabVida, Portugal), using the same primers used for the PCR reactions. Nucleotide sequences obtained were examined using 4Peaks v1.8 (Nucleobytes, Netherlands) and analysed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to find positional homologs. Obtained sequences were deposited at the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/).

Phylogenetic analysis

Multiple sequence alignments of nucleotide datasets were performed using the iterative G-INS-I method as implemented in MAFFT v7 [32]. The obtained alignments were optimized via Gblocks [33], followed by their manual correction considering the encoding reading frame. Phylogenetic trees were constructed using the Maximum Likelihood method under the best-fitting evolutionary model (GTR + G +I; GTR—general time reversal, G—gamma distribution, I—proportion of invariant sites), selected based on the corrected Akaike information criterion, as suggested by Mega v6 [34]. The stability of the obtained tree topologies was assessed by bootstrapping with 1000 resamplings of the original sequence data. The generated trees were edited for display using FigTree v1.4.3 (available at http://tree.bio.ed.ac.uk/software/figtree/).

Statistical analysis

Statistical analysis was performed only for data from sites where a large number of sand flies was captured. Variables were tested for normality of distribution using the Shapiro-Wilk test. As assumption of normality was not valid (P < 0.05), nonparametric analysis was carried out. Chi-square analysis was performed to evaluate the difference in host preference within and between sand fly species. A P value < 0.05 was considered as statistically significant. Data were analyzed using statistical software Prism v4.00 (GraphPad Software Ltd., USA).

Results

Sand fly identification and Leishmania/Trypanosoma infection rates

During the two entomological surveys, a total of 3,090 sand flies (n = 1,866 females and n = 1,224 males) were captured. Species and details on phlebotomine sand flies collected in each site are summarized in Table 1. Among the female sand flies, S. minuta was the most abundant species (n = 1,264; 67.7%); followed by four species from the genus Phlebotomus: P. perniciosus (n = 594; 31.8%); P. sergenti (n = 4; 0.2%); P. perfiliewi (n = 3; 0.2%) and P. neglectus (n = 1; 0.1%). One hundred seventy-six females were blood fed (9.4%) and 113 were gravid (6.1%) (Table 2).

Table 1

Total sand flies collected during the two entomological surveys.

Site (Year)	Sand fly species
	Sergentomyia minuta		Phlebotomus perniciosus		Phlebotomus sergenti		Phlebotomus perfiliewi		Phlebotomus neglectus
	Females	Males	Females	Males	Females	Males	Females	Males	Females	Males
Site A (2017)	62	48	157	141					1	2
Site B (2018)	154	99	54	81						2
Site C (2018)	153	172	1	22	1
Site D (2018)	248	162	9	78	3	5				2
Site E (2018)	647	144	373	239			3			27
Total	1,264	625	594	561	4	5	3		1	33

Table 2

Female sand flies sampled and molecularly analysed.

Site (Year)	Female sand fly species
	Sergentomyia minuta (n = 1,264)			Phlebotomus perniciosus (n = 594)			Phlebotomus sergenti (n = 4)			Phlebotomus perfiliewi (n = 3)			Phlebotomus neglectus (n = 1)
	Unfed	Blood fed	Gravid	Unfed	Blood fed	Gravid	Unfed	Blood fed	Gravid	Unfed	Blood fed	Gravid	Unfed	Blood fed	Gravid
Site A (2017)	60	1	1	153	4								1
Site B (2018)	121		33	43	6	5
Site C (2018)	137	11	5	1			1
Site D (2018)	239	4	5	7		2	2	1
Site E (2018)	587	21	39	223	128	22				2		1
Total	1,144	37	83	427	138	29	3	1		2		1	1

Leishmania DNA was detected in twenty-eight female sand flies out of 1,866 (1.5%). In detail, 26 out of 1,264 (2.1%) S. minuta specimens (n = 21 unfed; n = 4 blood-fed and n = 1 gravid) tested positive to Leishmania tarentolae DNA; whereas Leishmania donovani complex DNA was identified in a S. minuta unfed female (1/1264; 0.1%) and in a P. perniciosus blood-fed female (1/594; 0.2%). Seventeen out of the 26 L. tarentolae positive females (n = 13 unfed; n = 3 blood-fed and n = 1 gravid) were captured in the rural biotype (Site E), whereas 9 (n = 8 unfed and n = 1 blood-fed) were collected in periurban environments (Sites A-D). Both L. donovani complex positive females (n = 1 unfed and n = 1 blood-fed) were captured in the rural environment (Site E).

The 26 cytB sequences obtained from S. minuta revealed >99% sequence identity and 100% sequence coverage with reference sequences of L. tarentolae (accession number: LC092878). The cytB sequences obtained from S. minuta and P. perniciosus specimens, revealed > 99% identity and 100% sequence coverage with L. donovani complex sequences (accession numbers: CP022652; KX061917). All ctyB-rDNA obtained sequences were submitted to DDBJ (DDBJ Accession numbers: LC464942 to LC464968; LC471400). Based on phylogenetic analysis, 24 out of 26 obtained L. tarentolae sequences amplified from S. minuta, segregate together with L. tarentolae reference sequences in a monophyletic cluster, supported by a high bootstrap value (i.e., 99). Despite the heterogeneity of the sequences, phylogenetic analysis has not evidenced an unambiguous existence of different haplotypes. The two L. donovani complex sequences herein amplified from S. minuta and P. perniciosus extracts, segregate in the L. donovani complex cluster (Fig 2). Based on cytB phylogenetic tree, the sequences obtained from both specimens segregate independently. The existence of intra-groups was suggested within the L. donovani complex cluster, however none of them evidenced the monophyly of L. donovani/L. infanutm species.

Fig 2

Maximum likelihood phylogenetic tree based on unambiguous multiple-Leishmania cytB sequences alignment, using the GTR+G+I model of evolution.

In addition to Leishmania, Trypanosoma DNA was detected in seven specimens of S. minuta, captured in rural Site E (n = 1) and periurban biotopes (Site A, n = 3; Site B, n = 1; Site C, n = 2). The 7 cytB sequences obtained revealed > 95% sequence identity and a sequence coverage > 80% with a reference sequence of Trypanosoma lewisi of Chinese origin (accession number: KR072974) [35]. The sequences were submitted to DDBJ (DDBJ Accession numbers: LC471393 to LC471399). The SSU-rDNA nucleotide sequences showed both 100% identity and coverage with the sequence of Trypanosoma sp. isolated from Gecko Tarentola annularis (accession number: AJ620548) [36]. The SSU-rDNA obtained sequences were submitted to DDBJ (DDBJ Accession numbers: LC471401 to LC471407). The phylogenetic tree shows that the obtained sequences share the same common ancestry of Trypanosoma varani (Accession number: AJ005279) forming together with Trypanosoma sp. isolated from Gecko a monophyletic cluster supported by a high bootstrap value (i.e., 92); whereas two sequences are different. These two sequences also share the same common ancestry of T. varani but segregate independently (Fig 3).

Fig 3

Maximum likelihood phylogenetic tree based on unambiguous multiple-Trypanosoma SSU-rDNA sequences alignment, using the GTR+G+I model of evolution.

At specific branch nodes bootstrap values (from 1000 random replicates of the original dataset) ≥ 75% are shown. The size bar indicates the number of nucleotide substitutions per site. The tree was rooted using as an outgroup a reference sequence of Trypanosoma brucei brucei (M94286). The sequences obtained in this study are identified with “*” and their respective accession numbers underscored.

At specific branch nodes bootstrap values (from 1000 random replicates of the original dataset) ≥ 75% are shown. The size bar indicates the number of nucleotide substitutions per site. The tree was rooted using as an outgroup a reference sequence of Trypanosoma vivax (U22316). The sequences obtained in this study are identified with “*” and their respective accession numbers underscored.

Identification of sand fly blood meals

The majority of blood-fed females were captured in site E (149/176; n = 128 P. perniciosus and n = 21 S. minuta) and underwent statistical analysis, whereas the remainder of 27 blood-fed specimens caught at the other sites (A, n = 4 P. perniciosus, n = 1 S. minuta; B, n = 6 P. perniciosus; C, n = 11 S. minuta; D, n = 4 S. minuta, n = 1 P. sergenti) were not statistically analysed (Table 2). Host mitochondrial cytB was successfully amplified from 169 out of 176 engorged females (96%), and the vertebrate hosts of 108 blood meals (64%) were distinctly identified (S2 Table).

Wild rabbit (Oryctolagus cuniculus) (n = 28) and human (Homo sapiens) (n = 24) were the most frequent blood sources. Other vertebrate hosts also found were, i.e., goat (n = 16), horse (n = 13) pig (n = 9), dog (n = 9), chicken (n = 3), cow (n = 3), cat (n = 1), donkey (n = 1) and rat (n = 1). Blood sources for each sand fly species and site of capture are shown in Fig 4.

Fig 4

Vertebrate hosts identified per each sand fly species in selected sampling sites (A-E).

Identification of blood source was successfully achieved in two out of the four engorged S. minuta specimens positive for L. tarentolae DNA, as being human (Homo sapiens) and donkey (Equus asinus); whereas the host mitochondrial cytB partial gene could not be amplified from the blood-fed P. perniciosus positive to L. donovani complex DNA.

Wild rabbits (27/82; 32.9%) represented the most preferred mammal species for P. perniciosus, while S. minuta mainly fed on humans (16/25; 64%). Results of the statistical analysis on frequency of blood sources for P. perniciosus and S. minuta from site E are summarized in S3 Table, where a statistically significant higher frequency of blood meal on rabbit by P. perniciosus compared to S. minuta (χ = 183.1; P = 0.01), and on human by S. minuta compared to P. perniciosus (χ = 27.7; P < 0.001), was detected.

Discussion

Our study aimed to assess the presence of natural Leishmania infection in wild-caught sand fly species common in Sicily, Southern Italy; additionally, the source of blood meals of fed females was also determined to gain evidence on sand fly feeding habits and to allow the identification of potential/alternative reservoir hosts for Leishmania.

Four out of five sand fly species analysed in this study are proven vectors of human leishmaniosis. Among the genus Phlebotomus, P. perniciosus was the most abundant species, which is in agreement with other entomological surveys conducted in Southern Italy [13,29,37-38], and the presence of L. donovani complex DNA in P. perniciosus herein reported confirms the role of this species in the maintenance and spread of leishmaniosis in the Mediterranean area. Only a single P. perniciosus (0.2%; n = 594) tested positive for L. donovani complex. To the best of our knowledge, the sole data on the prevalence infection for L. infantum in P. perniciosus in Sicily came from a study conducted in the city of Catania where a higher infection rate (i.e., 11%; 8/72) has been detected [13]. This difference may be related to the high endemicity of leishmaniosis in the area of Catania [11,13] and suggest that the risk of transmission of Leishmania can be high even in urban areas.

Phlebotomus neglectus and P. perfiliewi specimens were occasionally collected in suburban and rural environments, and P. sergenti, the vector able to transmit L. tropica, was also sporadically collected, with its presence limited to the eastern side of the Island [12-14].

The detection of L. donovani complex DNA in a S. minuta unfed female herein recorded, spurs to better investigate the potential role of this species in the circulation and eventually transmission of the parasite [39]. The positivity of this sand fly species may be correlated to its feeding behaviour and the large availability of mammalian reservoir hosts positive to Leishmania in endemic foci [38]. Although Sergentomyia species mainly feed on cold-blooded vertebrates, its sporadic/opportunistic anthropophilic feeding behaviour has been already suggested [4] and, accordingly, a higher human blood preference has been herein statistically demonstrated. Scientific evidence on the competence of S. minuta in transmitting Leishmania spp. to warm-blooded mammals is not available so far; however, it has recently been demonstrated that L. donovani, L. infantum and L. major are not able to develop late-stage infections in Sergentomyia schwetzi sand fly species [40].Sergentomyia spp. are widespread Mediterranean sand fly species, and proven vectors of reptile Leishmania species (e.g., L. tarentolae) non-pathogenic to humans. The positivity of S. minuta for L. tarentolae herein recorded (2.1%; 26/1264) confirms that this species feeds on cold-blooded animals, as this protozoan parasite is widespread in Gekkonidae species [41].

In Italy, the presence of Trypanosoma spp. in sand flies has been little reported. In particular, Trypanosoma platydactyli was described in S. minuta [42], and the infection by a Trypanosoma belonging to Trypanosoma theileri group with very high homology to other trypanosomes detected in European cervids was recently reported in Phlebotomus perfiliewi [43]. In the Mediterranean area, Trypanosoma nabiasi, a rabbit trypanosome, and its co-infection with L. infantum was found in P. perniciosus female sand flies caught in the context of human leishmaniosis outbreak in Madrid [44], and natural infection of sand flies by trypanosomes of lizards, amphibians, birds and rodents has been already reported mainly from the American continent and Asia [45-48]. Despite Trypanosoma spp. being protozoan parasites transmitted by hematophagous insects [49-50] the potential of phlebotomine sand flies in transmitting trypanosomes is still unclear. The findings herein obtained, suggest that S. minuta might potentially be a vector of both Leishmania and Trypanosoma parasites in southern Italy. Nevertheless, the detection of the trypanosomatid DNA is not sufficient to incriminate the species as a competent vector since it may simply originate from a blood meal without undergoing further development and/or transmission. The detection of L. donovani complex DNA and, in addition, the detection of reptilian Trypanosoma DNA within S. minuta indicates that more attention is required when identifying parasitic organisms by PCR within sand fly vectors in areas where leishmaniosis is endemic. As a matter of fact, it is important to highlight that, when Trypanosoma and Leishmania species are present in the same geographical area, mixed infections could appear within the same host and/or vectors representing a challenge to their diagnosis [51-53].

In regard to the detection of L. donovani complex sequences in both P. perniciosus and S. minuta, a species-specific identification cannot be concluded based on cytB. In fact, the monophyly of L. infantum and L. donovani species has not been consistently shown [29].

Concerning the identification of blood meal sources in this study, several vertebrate hosts have been recognized as blood sources in the most abundant sand fly species (P. perniciosus, S. minuta), confirming the opportunistic feeding behaviour of these hematophagous insects [4,18-19].

Knowledge of the host preferences of sand flies under natural conditions is essential to understand how host choice and blood-feeding behaviour of sand flies influence their vector capacity in leishmaniosis foci. Even though Canis lupus familiaris is regarded as the main domestic reservoir host for zoonotic leishmaniosis, it seems not to be the preferred species on which they feed [4,18]. The results from the current study show that despite three out of the five selected sites being shelters with high an availability of dogs (i.e., from 300 to 500 dogs per site), canine DNA was not detected in engorged P. perniciosus specimens caught in any of them. This finding could however be biased by the broad use of insecticides and repellents on dogs as preventive measures against sand fly bites and Leishmania transmission. In this study, during the sand fly sampling period, about a third of all dogs hosted in the three shelters were treated with actives repellent against sand flies.

Of the total engorged females investigated in this study 85% were caught on a farm located in a rural area far away from human settlements. There a large variety of vertebrate hosts were available, with dogs and rabbits (either domestic or sylvatic) being the most abundant species. At this site, wild rabbits represented the most preferred mammalian species for P. perniciosus, which agrees with what has already been reported in Spain [20-22]. In fact, high levels of anti-P. perniciosus saliva antibodies in wild rabbits suggested their exposure and attractiveness to sand flies [54]. The large availability of lagomorphs could contribute to the maintenance of high density of P. perniciosus in areas where these mammal species are abundant [54] and, the role of lagomorphs in sustaining sylvatic Leishmania cycle independently from the domestic one has been strongly suggested [22,25].

The results obtained in the current study show a different blood feeding pattern of S. minuta compared to P. perniciosus, suggesting that the two sand fly species do not prefer the same vertebrate host. It is well-known that S. minuta feeds on cold-blooded reptiles; noteworthy, reptile DNA was not amplified in engorged females herein analysed, and only DNA of warm-blooded vertebrates was amplified. Interestingly, S. minuta caught in site E mainly fed on humans, though the presence of this vertebrate host inside the farm is limited to a few hours during the day, and no inhabitations were present nearby.

Conclusions

The current survey describes Leishmania infection rate in P. perniciosus and S. minuta and documents, for the first time in Sicily, Trypanosoma DNA presence in S. minuta unfed females.

The findings herein reported, highlight the well-known role of P. perniciosus as competent vector of L. infantum in western Mediterranean area; also, the identification of blood meal sources suggests the importance of wild rabbits in the maintenance of P. perniciosus and their potential role as sylvatic reservoirs of Leishmania.

The role of wild animals in the epidemiology of leishmaniosis as well as that of S. minuta as a vector of Leishmania spp. to humans is worthy of future investigations to achieve efficient control strategies.

PCR protocols performed for detection and characterization of Trypanosomatidae DNA and blood source identification.

aTrypanossomatidae kinetoplast DNA. bHost mitochondrial DNA. cPCR product was previously diluted 1:50 in nuclease-free water. Abbreviations: cytB, cytochrome b; ITS-rDNA, ribosomal internal transcribed spacer DNA; SSU-rRNA, small subunit ribosomal DNA; bp, base pars.

(DOCX)

Click here for additional data file.

CytB sequences analysis for each sample, percentages of BLAST identity and sequences accession numbers.

A: LC469805; LC469817; LC469821; LC469832; LC469834; LC469836; LC469839; LC469842-44; LC469846-48; LC469850-51; LC469853-56; LC469863; LC469865; LC469871; LC469875-76; LC469878; LC469882; LC469887. B: LC469829; LC469837; LC469840-41; LC469852; LC469857; LC469859-62; LC469867; LC469872; LC469874; LC469880-81; LC469891-93; LC469896; LC469898; LC469900-03. C: LC469807-10; LC469813-16; LC469818-20; LC469822-26. D: LC469806; LC469811; LC469827-28; LC469830; LC469833; LC469835; LC469869; LC469877; LC469884, LC469890; LC469897; LC469908. E: LC469838; LC469845; LC469849; LC469858; LC469868; LC469870; LC469873; LC469883; LC469899. F: LC469885-86; LC469888-89; LC469905; LC469907; LC469910-12. G: LC469812; LC469906; LC469909. H: LC469831; LC469894; LC469904. I: LC469866. L: LC469879. M: LC469895.

(DOCX)

Click here for additional data file.

Differences in host preference within the most abundant sand fly species caught in site E.

(DOCX)

Click here for additional data file.

3 Jan 2020

PONE-D-19-33599

Leishmania  detection and blood feeding preferences of phlebotomine sand fly species common in the Mediterranean area

PLOS ONE

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'Sand flies collection in 2018 season has been partially funded by Bayer Animal

Health. The founder did not play any role in the study design, data collection and analysis, decision to publish and preparation of the manuscript.

Molecular analysis on sand flies were partially founded by the grant Research and Mobility no.015063 awarded by the University of Messina'

We note that one or more of the authors have an affiliation to the commercial funders of this research study: Bayer Animal Health GmbH

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: General comments:

Clearly written and comprehensible article bringing interesting information.

Title: “…common in the Mediterranean area” – the title is exaggerated – it is definitely not an analysis of sand flies common in the Mediterranean – only in the western part and still not all common species, rather only selected ones, which were common on localities (in Sicily) where the authors made their catches (over only two seasons). PLS change “…common in Sicily”. Introduction should, therefore, also focus primarily on Sicily and, where appropriate, on southern Italy. Most parts describing the faunistic situation should be move from Intro to Discussion.

It is really interesting that it was not possible to detect/identify any reptilian blood - it would like to comment on it with at least one sentence.

It would be appropriate to include a paragraph where the authors comment on the existence of different haplotypes. There were always two haplotypes for both Leishmania species (L.t. and L.d.), as well as for unnamed trypanosome species - at least they would like to mention or comment this situation. This is particularly interesting in the case of L.donovani / L.infantum - is it really possible to assume that both species were captured/detected?

Specific comments:

26. etc. n = XXX

35. etc. (27/82) – not clear, need explanation

36. blood rat

45. why only warm-blooded ?

68. etc. X% or X %

136/137: for more info about the used traps add “see Fig. 1”

160. individually ? = 1866 DNA extractions?

246. You should add the information that obtained sequences (of L.t. and L.d.) were not 100% identical – both species form two “haplotypes”

255. … the same for two left sequences (a monophyletic cluster with another L.t. strain)

259. add information about occurrence in: unfed / blood fed / gravid

264. add Tarentola annularis from Senegal

269. two (2)

270. T. varani / T. sp.

Tab 2. n (instead no); n = XY; It would be beneficial to add info about Leishmania/Trypanosoma infection into this table.

316 (express it also in percentage … 96 % - congratulation; and 64 %)

320/Tab 3. rooster ? / hen-chicken-fowl

Fig. 4. Unify column color/pattern for presented animals (use the same pattern for the same animal); y axis – better “No. blood fed females”

Tabs 3. and 4. Move to Supplementary material ???

453-4. It is/was not clear that Trypanosoma DNA was detected only in blood fed S.m. females??? This info must be add 327/8 and PLS add also info which blood sources were detected.

Reviewer #2: The manuscript describes detection and identification of trypanosomatids and bloodmeals in Sicilian sand flies. Despite the results are interesting, their interpretation is not correct and some parts of the manuscript (mainly Abstract and Introduction) need to be rewritten.

General or major comments

1. Some sand fly species studied are NOT „commonly present in the Mediterranean area“ or “common in the Mediterranean area”. Please change the info on lines 22, 125-126, 345-346. Title is also misleading, more specific one would be useful (e.g. “Identification of trypanosomatids and bloodmeals of sand flies in Sicily”).

2. It is necessary to mention (on lines 32-33) that Trypanosoma DNA found in S. minuta clusters with reptile Trypanosomatids. Therefore, finding of Trypanosoma DNA in S. minuta should not be interpreted as a support for S. minuta role in transmission of human pathogens. There is no single evidence that Trypanosoma DNA found originate from species infecting mammals. The sentence on lines 42-43 needs to be changed.

3. Surprisingly, authors did not find a single reptile blood in S. minuta which contradicts frequent finding of reptile trypanosomes. Again, this should be mentioned in the Abstract.

4. Introduction is too long. Text on lines 104-108 and 116-119 could be deleted as most of information is repeated again in Discussion.

Table 3 should include also the numbers of females with nonidentified bloodmeals: P. perniciosus (56?) and S. minuta (12?).

Table 4 should be moved into Supplement.

Minor comments

Single finding of Leishmania DNA in P. perniciosus should not be interpreted as confirmation of the vector role of this sand fly species (line 39) or highlighting its role in Mediterranean area (lines 455-456). It may agree with well-known role of this vector with circulation of L. infantum in western Mediterranean. Please, change sentences accordingly.

The second sentence of Introduction should be changed. It is not correct in two aspects: 1. There is a single bacterial pathogen transmitted by sand flies (not “plethora” as written by the authors) and number of sand fly-borne viruses is much lower than in mosquitoes. 2. Leishmania of subgenus Mundinia are very likely transmitted by biting midges, not by sand flies.

Lines 60-61: Text should be changed to clarify that both information is valid only for western Europe.

Line 65: clarify what do you mean by “in free territories”

Lines 73-80: Part about imported cases should be reduced or changed. The reference by Fotakis et al (2019) does not concern the Italy. Moreover, this study (published in a good journal) suffers of serious mistakes: only sand fly heads were used for Leishmania DNA and despite of this nonsense extremely high percentage of sand flies were claimed as positive! Such results cannot be trusted and should not be cited. If authors decide to mention/discuss the potential spread L. tropica in Italy, then there are two recent papers about this topic published in Int. J. Parasitol, showing high susceptibility of P. perniciosus and P. tobbi to L. tropica.

Lines 363-364: P. sergenti is NOT “the sole vector able to transmit L. tropica”. The vectorial role of P. arabicus is well described in many papers since 2003. Moreover, there are two papers on other sand flies as potential L. tropica vectors published this year, see above.

Lines 383-385: the information about potential of L. tarentolae to infect humans is misleading and should be deleted (together with both references).

Finding of reptile Trypanosoma in S. minuta is not unexpected (as said on line 404).

**********

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Reviewer #2: No

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4 Feb 2020

Dear Editor and Reviewers,

Thank you very much for reviewing our manuscript PONE-D-19-33599 entitled “Leishmania detection and blood feeding preferences of phlebotomine sand fly species common in the Mediterranean area”

We have addressed all your concerns and a detailed revision note to reviewers’ comments is provided below.

We hope that the revised manuscript is now suitable for publication in PLOSE ONE journal.

On the behalf of all Authors

Yours sincerely

Emanuele Brianti

Editor’s comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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Authors’ response: Done.

2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing

Authors’ response: Done.

3. In your Methods section, please provide additional location information of the collection sites, including geographic coordinates for the data set if available.

Authors’ response: Done.

4. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the collection sites access and, if no permits were required, a brief statement explaining why.

Authors’ response: The study sites are not located in protected areas with constrains of flora and fauna protection, and no specific permission for sand fly collection is needed. Indeed, the suburban and/or rural areas from which sand fly specimens were collected were privately owned areas and, according to the Italian regulation, the owner’s consent to access and to sample in the area is enough.

In material and methods section, We specified that the two different entomologic surveys were carried out in privately owned areas located in Sicily and, the access to collection sites as well as the sampling procedures were authorized by the owners.

5. Thank you for stating the following in the Financial Disclosure section:

'Sand fly collection in 2018 season has been partially funded by Bayer Animal

Health. The founder did not play any role in the study design, data collection and analysis, decision to publish and preparation of the manuscript.

Molecular analysis on sand flies were partially founded by the grant Research and Mobility no.015063 awarded by the University of Messina'

We note that one or more of the authors have an affiliation to the commercial funders of this research study: Bayer Animal Health GmbH

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Authors’ response: Done.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Authors’ response: Done.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Authors’ response: Done.

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Authors’ response: Done.

6. We note that Figure 1 in your submission contains map/satellite images which may be copyrighted.

All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (a) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (b) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Authors’ response: We changed the map in the Fig. 1 by replacing it with a figure obtained from a resource of public domain suggested by Editor.

Reviewers’ comments and Authors’ responses

Reviewer #1: General comments:

Clearly written and comprehensible article bringing interesting information.

Title: “…common in the Mediterranean area” – the title is exaggerated – it is definitely not an analysis of sand flies common in the Mediterranean – only in the western part and still not all common species, rather only selected ones, which were common on localities (in Sicily) where the authors made their catches (over only two seasons). PLS change “…common in Sicily”. Introduction should, therefore, also focus primarily on Sicily and, where appropriate, on southern Italy. Most parts describing the faunistic situation should be move from Intro to Discussion.

It is really interesting that it was not possible to detect/identify any reptilian blood - it would like to comment on it with at least one sentence.

Authors’ response: We thank the Reviewer for her/his positive comments. The title and the introduction were changed according to the suggestions. Briefly, the introduction has been amended by focusing mainly on southern Italy and Sicily, while some parts describing the faunistic situation in the area of the study were moved to discussion.

It would be appropriate to include a paragraph where the authors comment on the existence of different haplotypes. There were always two haplotypes for both Leishmania species (L.t. and L.d.), as well as for unnamed trypanosome species - at least they would like to mention or comment this situation. This is particularly interesting in the case of L.donovani / L.infantum - is it really possible to assume that both species were captured/detected?

Authors’ response: Regarding L. tarentolae cluster, and according to boosttrap values (based on 1000 resamplings of the original sequence data; Fig. 2) the existence of different haplotypes cannot be assumed. In addition, and despite different haplotypes within L. donovani complex cluster are suggested, the phylogenetic tree does not evidence the monophyly of L. donovani and L. infantum. In fact, the investigations on the phylogenetic relationships and the genetic diversity of L. donovani/L. infantum strains based in four gene markers have concluded that the segregation of L. donovani/L. infantum species-specific clusters is not consistent (Pereira et al. 2020, ref. 29). Therefore, based on this conclusion it is not possible to assume that both species were detected.

Sentences:

In results:

Lines 259-260: Despite the heterogeneity of the sequences, phylogenetic analysis has not evidenced an unambiguous existence of different haplotypes.

Lines 262-265: Based on cytB phylogenetic tree, the sequences obtained from both specimens segregate independently. The existence of intra-groups was suggested within the L. donovani complex cluster, however none of them evidenced the monophyly of L. donovani/L.infanutm species.

In discussion:

Lines 425-428 “In regard to the detection of L. donovani complex sequences in both P. perniciosus and S. minuta, a species-specific identification cannot be concluded based on cytB. In fact, the monophyly of L. infantum and L. donovani species has not been shown to be consistent (Pereira et al. 2020, ref 29)

Specific comments:

26. etc. n = XXX

Authors’ response: We checked and standardized it according to journal guidelines.

35. etc. (27/82) – not clear, need explanation

Authors’ response: The sentence has been rewritten and now reads “Twenty-seven out of 82 blood sources identified in fed females of P. perniciosus were represented by blood of wild rabbit,…”

36. blood rat

Authors’ response: the sentence now reads “…the sole P. sergenti fed specimen took a blood meal on rat.”

45. why only warm-blooded ?

Authors’ response: We deleted “…warm-blooded…” in the sentence.

68. etc. X% or X %

Authors’ response: We checked and standardized it as “X%”.

136/137: for more info about the used traps add “see Fig. 1”

Authors’ response: We added “Fig 1” in lines 128, 132.

160. individually? = 1866 DNA extractions?

Authors’ response: Yes, DNA was individually extracted from each sand fly as specified in the Material and Methods section as following “Genomic DNA was extracted from portions (i.e., thorax and abdomen) of each sand fly specimen…”

246. You should add the information that obtained sequences (of L.t. and L.d.) were not 100% identical – both species form two “haplotypes”

Authors´ response: As previous answered, for L. tarentolae cluster, the boosttrap values of the phylogenetic tree does not support the existence of different haplotypes; similarly, and despite the two L. donovani complex sequences are different, the classification at species level cannot be assumed because the monophyly of these species was not consistently demonstrated.

255. … the same for two left sequences (a monophyletic cluster with another L.t. strain)

Authors´ response: Please see previous answer

259. add information about occurrence in: unfed / blood fed / gravid

Authors’ response: the required information has been added in the revised version.

264. add Tarentola annularis from Senegal

Authors’ response: Done.

269. two (2)

Authors’ response: Done

270. T. varani / T. sp.

Authors’ response: Sorry, but we are unable to catch this

Tab 2. n (instead no); n = XY; It would be beneficial to add info about Leishmania/Trypanosoma infection into this table.

Authors’ response: We replaced “no” with “n=”. As regards to add information about Leishmania/Trypanosoma infection in the table, we believe that this may overload the table and unclear for the reader. However, we leave the final decision to the Editor, and if it is necessary, we will find the way to add also these data in the table.

316 (express it also in percentage … 96 % - congratulation; and 64 %)

Authors’ response: Done.

320/Tab 3. rooster ? / hen-chicken-fowl

Authors’ response: changed to “chicken”

Fig. 4. Unify column color/pattern for presented animals (use the same pattern for the same animal); y axis – better “No. blood fed females”

Authors’ response: Fig. 4 has been amended as suggested by the Reviewer.

Tabs 3. and 4. Move to Supplementary material

Authors’ response: Agree, Tables 3 and 4 have been moved to Supplementary material.

453-4. It is/was not clear that Trypanosoma DNA was detected only in blood fed S.m. females??? This info must be add 327/8 and PLS add also info which blood sources were detected.

Authors’ response: Trypanosoma DNA was detected only in unfed Sergentomyia minuta females. We corrected the mistake in the text.

Reviewer #2:

The manuscript describes detection and identification of trypanosomatids and bloodmeals in Sicilian sand flies. Despite the results are interesting, their interpretation is not correct and some parts of the manuscript (mainly Abstract and Introduction) need to be rewritten.

General or major comments

1. Some sand fly species studied are NOT „commonly present in the Mediterranean area“ or “common in the Mediterranean area”. Please change the info on lines 22, 125-126, 345-346. Title is also misleading, more specific one would be useful (e.g. “Identification of trypanosomatids and bloodmeals of sand flies in Sicily”).

Authors’ response: We agree with the Reviewer and the manuscript has been changed in line with her/his suggestions. “Mediterranean area” has been changed with “Sicily” and/or “Southern Italy” in lines 23, 120, 344, and the title amended as well.

2. It is necessary to mention (on lines 32-33) that Trypanosoma DNA found in S. minuta clusters with reptile Trypanosomatids. Therefore, finding of Trypanosoma DNA in S. minuta should not be interpreted as a support for S. minuta role in transmission of human pathogens. There is no single evidence that Trypanosoma DNA found originate from species infecting mammals. The sentence on lines 42-43 needs to be changed.

Authors’ response: We thank the Reviewer for her/his suggestions with which we agree. The sentences (Lines 32-33 and 42-43) have been changed accordingly.

3. Surprisingly, authors did not find a single reptile blood in S. minuta which contradicts frequent finding of reptile trypanosomes. Again, this should be mentioned in the Abstract.

Authors’ response: The absence of reptile blood in blood-fed S. minuta specimens is now mentioned in the abstract.

4. Introduction is too long. Text on lines 104-108 and 116-119 could be deleted as most of information is repeated again in Discussion.

Authors’ response: We simplified the Introduction section according to Reviewer’s comments and suggestions.

Table 3 should include also the numbers of females with nonidentified bloodmeals: P. perniciosus (56?) and S. minuta (12?).

Authors’ response: We moved Table 3 to Supplementary material according to suggestion of Reviewer #1, and we added the numbers of females with non-identified blood meals (P. perniciosus n = 5; S. minuta n = 12) in the table.

Table 4 should be moved into Supplement.

Authors’ response: Tables 3 and 4 were moved to Supplementary materials.

Minor comments

Single finding of Leishmania DNA in P. perniciosus should not be interpreted as confirmation of the vector role of this sand fly species (line 39) or highlighting its role in Mediterranean area (lines 455-456). It may agree with well-known role of this vector with circulation of L. infantum in western Mediterranean. Please, change sentences accordingly

Authors’ response: We thank the Reviewer for her/his suggestion; the sentences were amended as suggested.

The second sentence of Introduction should be changed. It is not correct in two aspects: 1. There is a single bacterial pathogen transmitted by sand flies (not “plethora” as written by the authors) and number of sand fly-borne viruses is much lower than in mosquitoes. 2. Leishmania of subgenus Mundinia are very likely transmitted by biting midges, not by sand flies.

Authors’ response: We thank the Reviewer for spotting these inaccuracies, the sentence has been changed accordingly.

Lines 60-61: Text should be changed to clarify that both information is valid only for western Europe.

Authors’ response: Done as suggested by the Reviewer.

Line 65: clarify what do you mean by “in free territories”.

Authors’response: “Free territories” has been reworded in “non-endemic territories”.

Lines 73-80: Part about imported cases should be reduced or changed. The reference by Fotakis et al (2019) does not concern the Italy. Moreover, this study (published in a good journal) suffers of serious mistakes: only sand fly heads were used for Leishmania DNA and despite of this nonsense extremely high percentage of sand flies were claimed as positive! Such results cannot be trusted and should not be cited. If authors decide to mention/discuss the potential spread L. tropica in Italy, then there are two recent papers about this topic published in Int. J. Parasitol, showing high susceptibility of P. perniciosus and P. tobbi to L. tropica.

Authors’ response: We thank the Reviewer for her/his valuable comments; the reference Fotakis et al., 2019 has been deleted. Also, we read with interest and cited in the revised manuscript, the publication by Vaselek S and Volf P. 2019 showing high susceptibility of P. perniciosus and P. tobbi to L. tropica.

Lines 363-364: P. sergenti is NOT “the sole vector able to transmit L. tropica”. The vectorial role of P. arabicus is well described in many papers since 2003. Moreover, there are two papers on other sand flies as potential L. tropica vectors published this year, see above.

Authors’ response: We deleted “sole”.

Lines 383-385: the information about potential of L. tarentolae to infect humans is misleading and should be deleted (together with both references).

Authors’ response: Agree, done.

Finding of reptile Trypanosoma in S. minuta is not unexpected (as said on line 404).

Authors’ response: We thank the Reviewer for notifying us of this inconsistency; “…unexpected…” has been deleted.

Submitted filename: Response_to_Reviewers_PONE-D-19-33599R1.docx

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10 Feb 2020

Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily, Southern Italy

PONE-D-19-33599R1

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25 Feb 2020

PONE-D-19-33599R1

Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily, Southern Italy

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