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

Simple Summary Phlebotomine sand flies (Diptera: Psychodidae) are hematophagous insects, and many species serve as vectors of various human and animal pathogens, including Leishmania and Trypanosoma protozoa. In Thailand, the first case of autochthonous leishmaniasis was reported 62 years ago. At present, the number of human cases is increasing in different regions of the country, but most cases are reported from southern Thailand. Therefore, we studied the potential transmission of Leishmania and Trypanosoma by sand flies in three provinces of southern Thailand, and analyzed blood sources of engorged sand fly females. We detected Trypanosoma sp. DNA in Sergentomyia barraudi, S. indica, S. khawi and Idiophlebotomus asperulus but no Leishmania spp. DNA. Moreover, bloodmeal analysis revealed that Trypanopsoma-positive females of S. barraudi and Sergentomyia sp. fed on dogs and humans, respectively. The results of this study contribute to the knowledge of leishmaniasis and trypanosomiasis presence and sand fly feeding behavior in southern Thailand. Abstract An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.


Introduction
Phlebotomine sand flies (Diptera: Psychodidae) are hematophagous insects, and many species serve as vectors of Leishmania spp. [1,2] to humans and animals resulting in Leishmaniasis. The DNA of Trypanosoma spp. has also been detected in sand flies [2][3][4]; however, sand flies have not been incriminated as a Trypanosoma vector. Leishmaniasis is a vectorborne disease that continues to be a major health problem in many regions of the world [5]. In Thailand, four human-infecting Leishmania were reported [6]; Leishmania donovani and L. infantum belong to the long-time known subgenus Leishmania, L. martiniquensis and L. orientalis (originally described as L. siamensis) are classified within a newly established subgenus Mundinia [7]. Human diseases caused by these four species show various clinical symptoms, manifested as visceral leishmaniasis (VL), caused by L. donovani, L. infantum, L. martiniquensis and L. orientalis [8][9][10][11]; cutaneous leishmaniasis (CL), caused by L. donovani, L. infantum, L. martiniquensis and L. orientalis [7,[12][13][14]; and diffuse cutaneous leishmaniasis (DCL) caused by L. infantum, L. martiniquensis and L. orientalis [10,15,16]. Since the first imported case in 1960, autochthonous human leishmaniasis has increased to twenty cases [6]. Most of these local human cases were reported in southern Thailand in Phang-Nga, Satun, Krabi, Trang, Nakhon Si Thammarat, Songkhla, Phattalung, and Surat Thani provinces, highlighting the importance to investigate leishmaniasis prevalence in these regions. Two previous studies detected DNA of L. martiniquensis in S. barraudi, S. gammea and S. iyengari from endemic leishmania area in Trang province, southern Thailand [17,18]. Recently, the novel species L. orientalis was detected from a cutaneous leishmaniasis patient in northern Thailand [7], and L. orientalis has been detected in field-caught S. iyengari in southern Thailand. Although human trypanosomiases caused by species of the genus Trypanosoma have never been reported in Thailand, Trypanosoma evansi and Trypanosoma lewisi, two species that can be transmitted from animals to humans, and were incriminated in rare human infections [19][20][21][22], were detected in animals [23,24]. Recently, Trypanosoma sp. DNA was detected in S. khawi and P. stantoni in southern Thailand [2,25]. These findings suggest that sand flies in southern Thailand may be incriminated in transmission of both Leishmania and Trypanosoma protozoans. Although Trypanosoma DNA detection in sand fly species has been reported continuously, the complete information of Trypanosoma life cycle remains uncertain, while the life cycle of Leishmania in sand flies has been suggested as a natural vector of Leishmania [26,27]. However, little is known about the biology and feeding preferences of sand flies that may provide important insight into their transmission cycle and potential reservoir hosts.
Blood meal analyses provide important insight into the dynamics and transmission route of sand fly-borne diseases. The immunological techniques such as enzyme-linked immunosorbent assay (ELISA) were initially deployed to identify sand fly bloodmeals [28,29], later replaced by molecular tools with higher accuracy. The amplification of the cyt b gene using polymerase chain reaction (PCR) technique and subsequent sequencing is currently widely used for blood meal analysis. However, it remains less cost-and time-effective for analysis of field samples in larger field surveys [30]. The PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) of the cyt b gene decreases the cost of the analysis as it does not require the sequencing step and it has been successfully used to identify sand fly blood meals in many endemic regions, including Brazil [31,32] and Spain [33]. However, it has not yet been applied to sand flies in Thailand. The comparison of potential of PCR-RFLP of the cyt b gene and amplification of the cyt b gene using the PCR technique and subsequent sequencing has been studied [33], showing that the PCR-RFLP technique is an efficient and reliable alternative equal to PCR and subsequent sequencing, with lower cost and quicker results. The PCR-RFLP technique is based on amplifying the cyt b gene of the sandflies blood meal, then cleaving by restriction endonuclease enzymes on recognition sites and cleaving on DNA fragments. The cleaved DNA fragment pattern of the cyt b gene could be shown distinguishing between vertebrate species [33]. However, the PCR-RFLP technique necessitates the use of two or more restriction enzymes in order to obtain conclusive results [33]. Therefore, this study aimed to identify sources of blood in engorged Insects 2022, 13, 197 3 of 13 sand fly females to understand their host preferences. In addition, the specimens were also screened for the presence of Leishmania and Trypanosoma DNA.

Ethics Statement
The method for sand fly collecting and specimen preparation in this study was certified by the Institutional Animal Care and Use Committee, Prince of Songkla University, under reference number 2561-10-021.

Sand Fly Collection
Insects were collected at five localities in four provinces of southern Thailand: from four rural settlements in Phang-Nga (site PNA), Satun (site STN1), Songkhla (site SKA), Surat Thani (site SNI) provinces, and one Buddha cave in Satun province (site STN2) (Figure 1), using Center for Disease Control (CDC) light traps (John W. Hock Co., Gainesville, FL, USA) placed 5 to 10 cm above the ground. Ten traps were deployed at each site monthly in the early rainy season in June and August 2015, and in the late rainy season in January 2015, from sunset to sunrise (6.00 p.m.-6.00 a.m.). Insects were sacrificed with ethyl acetate in a plastic bag. Sand flies were collected from the total catch insects on Petri dishes under a stereomicroscope (Olympus, Tokyo, Japan) and preserved in a 70% ethanol solution until processing.
Insects 2022, 13, x FOR PEER REVIEW 3 of in engorged sand fly females to understand their host preferences. In addition, the spec mens were also screened for the presence of Leishmania and Trypanosoma DNA.

Ethics Statement
The method for sand fly collecting and specimen preparation in this study was cert fied by the Institutional Animal Care and Use Committee, Prince of Songkla Universit under reference number 2561-10-021.

Sand Fly Collection
Insects were collected at five localities in four provinces of southern Thailand: from four rural settlements in Phang-Nga (site PNA), Satun (site STN1), Songkhla (site SKA Surat Thani (site SNI) provinces, and one Buddha cave in Satun province (site STN2) (Fig  ure 1), using Center for Disease Control (CDC) light traps (John W. Hock Co., Gainesvill FL, USA) placed 5 to 10 cm above the ground. Ten traps were deployed at each si monthly in the early rainy season in June and August 2015, and in the late rainy season i January 2015, from sunset to sunrise (6.00 p.m.-6.00 a.m.). Insects were sacrificed wit ethyl acetate in a plastic bag. Sand flies were collected from the total catch insects on Pet dishes under a stereomicroscope (Olympus, Tokyo, Japan) and preserved in a 70% ethano solution until processing.

Sample Preparation
Blood-fed sand fly females were selected and placed individually in a 1.5 mL micro tube containing 70% ethanol. The head of each individual sand fly was dissected on

Sample Preparation
Blood-fed sand fly females were selected and placed individually in a 1.5 mL microtube containing 70% ethanol. The head of each individual sand fly was dissected on a sterilized glass slide and mounted into Hoyer's medium [34]. The individual thorax and abdomen were transferred to a sterile 1.5 mL microtube with 70% ethanol and stored at −20 • C for later DNA extraction.

Morphological Identification of Sand Flies
The species of sand flies was identified using taxonomic keys [35][36][37][38] based on their species-specific morphological characters of cibarium (shape and color of pigment patch, number of vertical and horizontal teeth) and pharynx under light microscope (Olympus, Tokyo, Japan).

Genomic DNA Extraction
The thorax and abdomen of each sand fly stored in 70% ethanol were transferred into a new 1.5 mL microtube, dried at room temperature to remove the remnants of ethanol and homogenized using a disposable polypropylene pestle. The genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, with a final elution by 200 µL of elution buffer, and then stored at −20 • C.

Polymerase Chain Reaction (PCR) Analysis and DNA Sequencing
The concentration of genomic DNA was determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cytochrome c oxidase subunit I (COI) gene was amplified using specific primers LepF (5 -ATTCAACCAATCATAAA GATATTGG-3 ) and LepR (5 -AAACTTCTGGATGTCCAAAAAATCA-3 ) to obtain the PCR products approximately 658 bp [39] to identify Trypanosoma DNA positive sand flies and confirm the species level. The PCR reaction was performed in a total volume of 25 µL, containing 10.35 µL nuclease-free water, 0.5 µL of each primer (10 µM), 7.65 µL of My Taq™ HS Red Mix (BioLine, Taunton, MA, USA) and 6 µL of DNA template. The PCR reactions were performed in Eppendorf Mastercycler ® Nexus thermal cycler (Eppendorf, Hamburg, Germany) following the published thermal profile as described previously [39]. The PCR products were stained with MaestroSafe TM Nucleic acid loading dye (Maestrogen, Hsinchu, Taiwan), loaded onto a 1.5% agarose gel and visualized under UV light.
PCR amplification of the internal transcribed spacer 1 (ITS1) region of Leishmania was done using primers LeF (5 -TCCGCCCGAAAGTTCACCGATA-3 ) and LeR (5 -CCAAGTCA TCCATCGCGACACG-3 ) [40]. However, this primer set is also complementary to other Trypanosomatidae sequences [40]. Therefore, the amplification of the ITS1 region of Trypanosoma was also performed using this primer set. The total volume of PCR reaction was 25 µL and consisted of 11.2 µL of nuclease-free water, 0.4 of each primer (10 µM), 7 µL of My Taq™ HS Red Mix (BioLine, USA) and 6 µL of DNA template. The PCR was performed following the profile as previously described [40].
The PCR products were purified using TIANquick Midi Purification Kit (TIANGEN BIOTEC, Beijing, China). The purified PCR products were sent to a sequencing service (Macrogen, Seoul, Korea) for direct sequencing using the same primers as those in the PCR reactions.

Identification of Blood Meal Source by PCR-RFLP
The cyt b gene of mitochondrial DNA was amplified from the genomic DNA. The specific primers of vertebrates were used: BM1 (5 -CCCCTCAGAATGATATTTGTCCTCA-3 ) and BM2 (5 -CCATCCAACATCTCAGCATGATGAAA-3 ) [31]. The total volume of PCR was 25 µL, containing 11 µL of nuclease-free water, 7 µL of My Taq™ HS Red Mix (BioLine, Taunton, MA, USA), 0.5 of each primer (10 µM) and 6 µL of DNA template. The reaction was performed in a thermal cycler (Eppendorf, Hamburg, Germany) following the PCR profile [41]. The PCR products sized 358 bp were obtained and used for blood meal source analysis by restriction fragment length polymorphism (RFLP) technique. The PCR products were cut at the specific site by restriction enzymes Hae III, Rsa I and Aci I (Thermo Fisher scientific, Inc., Waltham, MA, USA). PCR product digestion was performed in a total volume of 30 µL containing 17 µL of nuclease free water, 10 µL of PCR product, 2 µL of 10X FastDigest Green Buffer, 1 µL of FastDigest enzyme and 10 µL of PCR product, and then incubated at 37 • C for 5 min. The digested PCR product was loaded onto 1.5% agarose gel for fragment size evaluation alongside a molecular weight marker (BioLine, Taunton, MA, USA). The expected fragments of cyt b PCR products digested by endonuclease enzymes Hae III, Rsa I and Aci I [31] are shown in Table 1 and used for blood meal source identification. The patterns of digested DNA fragments of the three endonuclease enzymes in each blood meal source show distinct differences.

Phylogenetic Analysis of Sand Fly Species and Detected Parasites
The DNA sequences of sand flies and Trypanosoma were checked for ambiguous base calls and assembled by BioEdit version 7.2.5 [42]. The assembly sequences were compared to the GenBank database using BLASTn tool (https://blast.ncbi.nlm.nih.gov/ Blast.cgi accessed on 9 November 2021). Phylogenetic trees were constructed by the maximum likelihood (ML) method using Molecular Evolutionary Genetics Analysis version 6.0 (MEGA 6.0) software [43].

Detection of Leishmania and Trypanosoma spp. in Sand Flies
Screening of the genomic DNA of all 155 engorged females for Leishmania DNA by PCR amplification of ITS1 region provided eight positive samples (5.16%). Four of them were detected in samples from Satun province (ST43, ST134, ST143 and ST139), three from Songkhla province (SK97, SK109 and SK91) and one (PN71) from Pang-Nga province. ITS1 sequences obtained were compared with sequences from GenBank by nucleotide BLAST (blastn) search analysis, showing unexpectedly best match with Trypanosoma spp. rather than Leishmania spp. Phylogenetic analysis of ITS1 sequences by the maximum likelihood (ML) method showed that sequence LSK97 group together with Trypanosoma congolense, T. rangeli and T. lewisi, while the sequences LSK91 and LPN71 were closely related to Trypanosoma minasense (AB362411), and the sequence LST143 is closely related to Trypanosoma avium (AY929322) in Clade A. The samples LSK91, LSK97, LPN71 and LST143 showed low sequence identity (<50%) with their closest strains; therefore, the sequences LSK91, LSK97, LPN71 and LST143 were identified as Trypanosoma sp. based on ITS1 region ( Figure 2). When considering the sequences in Clade B, samples LSK109, LST43, LST134 and LST139 group together with Trypanosoma sp. (KJ467211) with 62% bootstrap (Figure 1). The sequence LST43 showed 86.8% sequence similarity to Trypanosoma sp. (KJ467211), while other samples in Clade B showed very low sequence similarity to Trypanosoma sp. (KJ467211) in the range of 48-55%. Therefore, the sequences LSK109, LST43, LST134 and LST139 were identified as Trypanosoma sp. The ITS1 sequences of Trypanosoma sp. in this study were deposited in the GenBank database under the accession number OL332783-OL332790.

Molecular Identification of Trypanosoma-Positive Sand Flies
Species identity of eight Trypanosoma-positive sand flies was confirmed using PCR amplification and sequencing analysis of COI gene. The phylogenetic tree was constructed based on COI gene sequences of sand flies and related sand fly sequences derived from the GenBank and BOLD databases using the ML method with 1000 replicates of bootstrapping. Based on the sequences obtained, four species with high sequence similarity (99.8-100%) were identified: S. barraudi (ST143), S. indica (SK91), S. khawi (SK97, SK109) and Id. asperulus (ST134, PN71). Two samples (ST43 and ST139) were identified as Sergentomyia sp. with no species designation due to lack of reference sequences ( Figure 3). These two sand flies were suspected to be novel species. The partial COI gene sequences of sand fly sequences were deposited in GenBank with the accession number OK576207-OK576214. Insects 2022, 13, x FOR PEER REVIEW 7 of 13

Molecular Identification of Trypanosoma-Positive Sand Flies
Species identity of eight Trypanosoma-positive sand flies was confirmed using PCR amplification and sequencing analysis of COI gene. The phylogenetic tree was constructed based on COI gene sequences of sand flies and related sand fly sequences derived from the GenBank and BOLD databases using the ML method with 1000 replicates of bootstrapping. Based on the sequences obtained, four species with high sequence similarity (99.8-100%) were identified: S. barraudi (ST143), S. indica (SK91), S. khawi (SK97, SK109) and Id. asperulus (ST134, PN71). Two samples (ST43 and ST139) were identified as Sergentomyia sp. with no species designation due to lack of reference sequences ( Figure 3). These two sand flies were suspected to be novel species. The partial COI gene sequences of sand fly sequences were deposited in GenBank with the accession number OK576207-OK576214.

Molecular Identification of Trypanosoma-Positive Sand Flies
Species identity of eight Trypanosoma-positive sand flies was confirmed using PCR amplification and sequencing analysis of COI gene. The phylogenetic tree was constructed based on COI gene sequences of sand flies and related sand fly sequences derived from the GenBank and BOLD databases using the ML method with 1000 replicates of bootstrapping. Based on the sequences obtained, four species with high sequence similarity (99.8-100%) were identified: S. barraudi (ST143), S. indica (SK91), S. khawi (SK97, SK109) and Id. asperulus (ST134, PN71). Two samples (ST43 and ST139) were identified as Sergentomyia sp. with no species designation due to lack of reference sequences ( Figure 3). These two sand flies were suspected to be novel species. The partial COI gene sequences of sand fly sequences were deposited in GenBank with the accession number OK576207-OK576214.

Identification of Blood Source by RFLP
The genomic DNA of all 155 blood-fed females was used as a template for the amplification of the cyt b gene followed by RFLP assay. The 358 bp PCR product was successfully amplified (Figure 4a) for 56 samples (36.13%): 50 of Sergentomyia spp., 3 of Id. asperulus and 3 of P. argentipes. RFLP patterns revealed that human was the main blood meal source, as it was detected in 33 samples, followed by rat and dog blood identified in 14 and 9 samples, respectively (Table 2). Briefly, the restriction pattern for cyt b PCR products of human blood samples by Hae III endonuclease produced bands of 233 and 124 bp, while in the case of PCR products of dog and rat blood samples, no cleavage site was found. The pattern of rat DNA digested by Rsa I generated fragments with sizes of 267, 59, 31 bp, whereas the restriction site on cyt b PCR product from human and dog samples was not found. The fragment pattern of human DNA digested by Aci I resulted in fragments of 189, 113, 55 bp. Dog and rat DNA samples were not digested by Aci I (Table 1). No mixed blood meals were identified. Human was the most often detected host for S. hamidi and S. silvatica. The fragment of cyt b gene could not be amplified from blood-fed P. stantoni, S. indica and S. iyengari. Of eight Trypanosoma-positive sand flies, blood source of only two samples (ST43 and ST143) was successfully determined (Figure 4b), while for six remaining samples (SK91, SK97, SK109, ST134, ST139 and PN71) amplification of the cyt b gene fragment failed. Based on the RFLP patterns of samples ST43 and ST143, the source of blood was determined as human and dog, respectively.
The genomic DNA of all 155 blood-fed females was used as a template for the amplification of the cyt b gene followed by RFLP assay. The 358 bp PCR product was successfully amplified (Figure 4a) for 56 samples (36.13%): 50 of Sergentomyia spp., 3 of Id. asperulus and 3 of P. argentipes. RFLP patterns revealed that human was the main blood meal source, as it was detected in 33 samples, followed by rat and dog blood identified in 14 and 9 samples, respectively (Table 2). Briefly, the restriction pattern for cyt b PCR products of human blood samples by Hae III endonuclease produced bands of 233 and 124 bp, while in the case of PCR products of dog and rat blood samples, no cleavage site was found. The pattern of rat DNA digested by Rsa I generated fragments with sizes of 267, 59, 31 bp, whereas the restriction site on cyt b PCR product from human and dog samples was not found. The fragment pattern of human DNA digested by Aci I resulted in fragments of 189, 113, 55 bp. Dog and rat DNA samples were not digested by Aci I (Table 1). No mixed blood meals were identified. Human was the most often detected host for S. hamidi and S. silvatica. The fragment of cyt b gene could not be amplified from blood-fed P. stantoni, S. indica and S. iyengari. Of eight Trypanosoma-positive sand flies, blood source of only two samples (ST43 and ST143) was successfully determined (Figure 4b), while for six remaining samples (SK91, SK97, SK109, ST134, ST139 and PN71) amplification of the cyt b gene fragment failed. Based on the RFLP patterns of samples ST43 and ST143, the source of blood was determined as human and dog, respectively.

Discussion
In this study, twelve sand fly species, namely Idiophlebotomus asperulus, P. stantoni, P. argentipes, S. anodontis, S. barraudi, S. hamidi, S. hodgsoni, S. hodgsoni hodgsoni, S. indica, S. iyengari, S. khawi, S. silvatica and Sergentomyia sp. were collected from Phang-Nga, Satun, Songkhla and Surat Thani provinces, which are regions so far endemic for human leishmaniasis. All eleven sand fly species except S. hodgsoni hodgsoni were found in rural settlements. In contrast, S. hodgsoni hodgsoni was the most abundant species specifically found in a Buddha cave in Satun province, while S. khawi was not found in any cave. This finding agrees with earlier work suggesting that S. hodgsoni hodgsoni is the most common species collected from caves in Kanchanaburi province, Thailand [44], so we confirm that it Insects 2022, 13, 197 9 of 13 is indeed a cavernicolous species. The rest of the identified sand fly species were collected from both rural settlements and caves. The presence of 12 species at the studied localities comprises nearly half of all sand fly species reported in Thailand so far. In general, sand fly fauna in Thailand is still not fully investigated and rather poorly documented. The last published checklist includes 26 species of four genera [44], but later records and taxonomical reassessments added several more species such as S. hivernus [25] or S. raynali [45]. We may expect that future research that combines complementary morphological and molecular approaches will provide further insight into the true local diversity, and potentially may lead to recognition and taxonomical description of new species of sand flies in Thailand. As some species seem to be cave-dwellers, special attention will be given to vast cavernicolous habitats which remain largely unexplored and may reveal a score of so far undescribed species.
Sequencing analysis of vertebrate mitochondrial cytochrome b gene has been widely used to identify blood sources of various hematophagous insects, including sand flies [46]. In our study, we deployed this approach to successfully identify blood meals in approximately one-third of engorged females collected. This success rate reflects the fact that amplification of the host gene is difficult in later stages of blood meal digestion due to the negative effect of progressing digestive enzymes. This shortcoming could be overcome in future studies by the deployment of protein-based MALDI-TOF peptide mapping that has recently been demonstrated to also allow successful blood identification in blood meals with more advanced blood digestion [47]. In our study, humans were the major blood meal source of sand flies, followed by rats and dogs. No mixed blood meals were detected. Several species demonstrated opportunistic feeding behavior: blood of two or even three hosts were detected in different females of P. argentipes, S. anodontis, S. barraudi, S. silvatica and S. hodgsoni hodgsoni. This S. hodgsoni hodgsoni which, despite its exclusive occurrence in a cavernicolous habitat, was demonstrated to feed on dogs, humans and rats alike. As sand flies are generally regarded as rather poor fliers with limited range of host seeking, we may assume that they readily bite occasional human visitors of the caves. Moreover, humans were the most preferred hosts of S. silvatica and S. hamidi, although the low number of analyzed females does not allow credible assumptions about the anthropophily of these species. Blood meal analysis of females identified as S. khawi, S. indica and Id. asperulus that were shown to harbor DNA of Trypanosoma sp. unfortunately failed to provide identification of their blood meals, thus not providing any insight regarding the potential involvement of these three species in the transmission of these protozoans.
Hematophagous sand fly females serve as vectors of different pathogens, most importantly protozoans of the genus Leishmania that cause disease with varying clinical outcomes. Albeit Thailand is traditionally not regarded as a country heavily burdened by human leishmaniases, numbers of autochthonous cases increased in the last six decades, and four Leishmania species were recorded as their causative agents. So far, none of the sand fly species naturally occurring in the country were conclusively proven as a vector; however, various sand flies of the genera Sergentomyia spp. and Phlebotomus spp. are suspected vectors, since Leishmania DNA was detected by PCR assays within the field-collected specimens of these species: DNA of L. martiniquensis has been detected in S. khawi [2], and DNA of L. siamensis (later synonymized with L. martiniquensis) in S. gemmea, S. barraudi and S. iyengari [17,18,48]. In this study, we did not detect Leishmania DNA in any of 155 analyzed specimens. This correlates with a previous study from Satun province, which did not detect any Leishmania DNA in field-collected specimens of P. stantoni, P. argentipes, S. gemmea, S. indica, S. barraudi, S. iyengari, S. bailyi, S. perturbans and S. silvatica [49].
Besides Leishmania, which is the most important sand fly-borne parasitic protozoan, sporadic infections of Trypanosoma spp. were previously reported in Phlebotomus spp. [50] and Sergentomyia spp. [51] from various geographical regions. In Thailand, Trypanosoma sp. was recently detected in a single specimen of P. stantoni [25], while another study detected T. noyesi in S. anodontis and P. asperulus and another yet undescribed Trypanosoma sp. in these two sand fly species, along with P. betisi collected in endemic and nonendemic leishmaniasis areas in southern Thailand [2]. The results from our study correlate to those detected Trypanosoma sp. infected Id. asperulus, and may indicate the potential transmitter of Trypanosoma spp. in Thailand, as recently reported in terms of an infected Trypanosoma sp. found in S. barraudi, S. indica and S. khawi. Trypanosoma sp. LST43 detected in Sergentomyia sp. from Satun province in the current study belongs to Trypanosoma sp. (KJ467211) sequence from GenBank database with high sequence identity. Trypanosoma sp. (KJ467211) DNA was detected in P. stantoni from Songkhla province, southern Thailand [25]. From this result, we conclude that two sequences of Trypanosoma sp. LST43 may be the same species as Trypanosoma sp. (KJ467211). Although to date no human cases of trypanosomiasis were reported in Thailand so far, trypanosomiasis caused by Trypanosoma evansi and Trypanosoma lewisi were reported in animals [52,53]. Thus, precise species identification of sand fly-borne Trypanosoma spp. and further assessment of the potential role of different sand fly species as their vectors remain important factors to consider for adequate prevention planning. The specific primer for species-level identification of Trypanosoma spp. will be designed in our future work.

Conclusions
This study reports for the first time a detection of Trypanosoma sp. DNA in Sergentomyia barraudi, S. indica and S. khawi in Thailand, suggesting that several Trypanosoma spp. may circulate in natural and rural habitats with domestic canines serving as reservoirs. The analysis of blood meals in engorged females provides new data about blood sources for nine sand fly species, showing opportunistic feeding habits of some, and the utilization of human blood by several, including the cavernicolous species S. hodgsoni hodgsoni.