Molecular Detection and Analysis of Trypanosoma (Megatrypanum) spp. Diversity in Tabanidae (Diptera) Collected in Lithuania

Simple Summary Horse flies are known as vectors of various species of trypanosomatids. The aim of this study was to investigate Trypanosoma in the Tabanidae family collected in different localities in Lithuania. Our objectives were to examine the genetic diversity of Trypanosoma sp. detected in tabanids, assessing the relationships between parasite lineages and tabanid species. The present study reports on the molecular detection and genetic diversity of trypanosomes in 10 species of tabanids, with two of them being reported for the first time. The findings suggest that different strains of the T. theileri complex are not related to different tabanid species. Abstract Trypanosomatids from the Tabanidae family have not been studied in Lithuania in any detail. In this study, a nested PCR amplifying the DNA fragment coding the SSU rRNA was used to determine the Trypanosoma spp. prevalence and diversity in the Tabanidae family collected in Lithuania in 2018–2019. In total, 101 Tabanidae individuals were investigated from six areas in Lithuania, and 14 different species were identified. The overall positivity of Trypanosoma spp. DNA in tabanids was 50.5% (51/101). Tabanus maculicornis was the most abundant species and yielded the highest prevalence of trypanosomatids (84.62%, 22/26), while Hybomitra nitidifrons showed a high prevalence as well, reaching 77.8% (14/18). In flies of some species (Hybomitra lapponica and Hybomitra lurida), Trypanosoma was detected for the first time. Nine different haplotypes were detected as being distributed in different tabanid species. Analysis showed that most sequences obtained during our study were identical or extremely close to two major T. theileri subclades: TthI and TthII. Our data analysis suggests the presence of different Trypanosoma genotypes in the same tabanid species, meaning that different lineages of Trypanosoma could be more related to the vertebrate host and not the fly species. This is the first study of trypanosomatid parasites in tabanids from Lithuania, and our results are valuable in providing data on the diversity of these parasites in different Tabanidae species.


Introduction
The family Tabanidae (Diptera) consists of haematophagous insects which, due to their lifestyle and feeding behavior, play a role as vectors in the mechanical and biological transmission of a large variety of disease agents.However, distinct species of Tabanidae can act as vectors of different pathogens.
Thirty-nine Tabanidae (horsefly and deer fly) species have been detected thus far in Lithuania [1], although there are no data regarding the abundance of each species in the country.Tabanids are known as biological and mechanical vectors of viruses, such as equine infectious anemia and hog cholera, bacteria such as Bacillus anthracis, Francisella tularensis and Anaplasma marginale, protozoans such as Trypanosoma and Besnoitia and filarial nematodes [2].The aim of this study was to determine the prevalence of Trypanosoma spp.and the diversity in the Tabanidae members collected in Lithuania.
Insects 2024, 15, 581 2 of 10 Tabanids are known to mechanically transmit different trypanosomatids, such as Trypanosoma (Trypanozoon) evansi (Steel, 1885) Balbiani, 1888, the causal agent surra of camels, horses and other mammals.Tabanids are also known to mechanically transmit T. (Duttonella) vivax Ziemann, 1905 (South America) and T. (Trypanozoon) brucei Plimmer and Bradford, 1899 (parts of Africa) [2].Tabanids are considered vectors of the Trypanosoma (Megatrypanum) theileri Laveran, 1902 species complex [3,4] with the species T. theileri.The pathogen can be transmitted by laying infected feces on the skin of the mammalian host or through ingestion of infected insects [5].The T. theileri species complex consists of several species (T.theileri, T. melophagium, T. cervi, T. cf.cervi and T. trinaperronei) and various trypanosome genotypes and lineages reported from cervids, bovids and insects, but the exact taxonomy of these is not yet possible to determine [6][7][8].The status of trypanosomatid species remains unclear, as it does for all organisms with predominantly clonal reproduction [9], and the taxonomy of the T. theileri species complex is still controversial.All trypanosomes of this group have unique T. theileri-like morphology and are the largest mammalian blood trypanosomes with small kinetoplasts.Based on their morphology, they were merged into the subgenus Megatrypanum and classified according to the hosts they infect as well.The most common hosts are domestic and wild ruminants [10].The phylogeny based on the SSU rRNA of the T. theileri group showed a strongly supported and monophyletic clade [11] which, despite high similarity, was separated from other T. (Megatrypanum) trypanosomes (T.conorhini, T. legeri, T. minasense, T. pestanai and T. binneyi).Rodrigues et al. [11] indicated that the name T. (Megatrypanum) should only apply to members of the T. theileri clade and not to other species.
Megatrypanum, found in bovids and cervids, are transmitted by tabanid flies, whereas those infecting sheep and goats are transmitted by host-specific louse flies (Hippoboscidae).Interrupted feeding could be the most important factor determining the role of tabanids as effective mechanical vectors of various pathogens [12].Trypanosoma (Megatrypanum) are commonly found in the hindgut of tabanids but are absent from the salivary glands, meaning that the transmission occurs primarily through feces entering the bite wound or perhaps the ingestion of infected tabanids by the animal [5].The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and the lack of a genotype shared by different host species, suggest that the evolutionary history of trypanosomes of this subgenus is strongly constrained by their ruminant hosts [11].Recent phylogenetic studies segregated T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers [13,14].
Species of the T. theileri complex are distributed in many regions, but only few investigations confirmed their occurrence in tabanids.Trypanosomatid infections (T.theileri-like) in four species of female horseflies (Hyb.tarandina, Hyb.muehlfeldi, Hyb.bimaculata and Chrysops divaricatus) were detected in northwest Russia [15].Detection of Trypanosoma (Megatrypanum) in Haematopota pluvialis, T. bromius, T. maculicornis and T. distinguendus was reported in Poland [16], and Hyb.ciureai and T. bromius have been found to be positive for T. theileri in the Czech Republic [6].In one study, fifty-five percent of host-seeking Chrysops in North America were infected with trypanosomatid parasites [17].The infective stages of T. theileri were identified using transmission experiments [5], and the development stages of two species of the T. theileri complex in tabanids were described [18].
Tabanids in Lithuania were not studied earlier for the presence of trypanosomatids.We collected tabanids from 2018 to 2019 at several sampling localities in Lithuania in order to find which species of trypanosomatids are present in the area and to determine their prevalence in tabanids of different species.
The objectives of this study were (1) to collect tabanids in different localities in Lithuania and to investigate them for the presence of Trypanosoma and (2) to examine the genetic diversity of the detected Trypanosoma sp. and to assess the relationships between the parasite lineages and tabanid host species.To address these questions, we evaluated the genetic diversity of Trypanosoma isolates from 14 different species of Tabanidae.The knowledge of pathogens, which can be detected in insects of particular species, would give new insights Insects 2024, 15, 581 3 of 10 into insect feeding preferences as well as potential vector species and would allow better understanding of the transmission regularities of pathogens in the wild.
Species identification was carried out using a taxonomic key [20] before processing.Morphological identification of flies with uncertain identification was confirmed using PCR.Fragments of the cytochrome oxidase I gene were amplified using universal primers LCO1490 and HCO2198.They were then sequenced, and the identities of the obtained sequences were checked using the "Basic Local Alignment Search Tool" (National Centre of Biotechnology Information website: http//www.ncbi.nlm.nih.gov/BLAST(accessed on 1 May 2024).The obtained sequences (accession numbers PP946791-P946795) matched the sequences from GenBank more than 99% of the time.Voucher specimens were deposited at Vilnius University's Department of Zoology.

The Detection of Trypanosoma in Tabanid Flies Using PCR
The total DNA was extracted from each individual insect using a DNeasy Blood&Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.A nested PCR protocol was applied to amplify a 748 bp-long DNA fragment encoding SSU rRNA using the outer primers Tryp763 and Tryp 1016 and the inner primers Tryp99 and Tryp957 [21].All PCRs were performed in a total volume of 25 µL: 2 µL total genomic DNA template, 12.5 µL DreamTaq Master Mix (Thermo Fisher Scientific, Lithuania, Vilnius), 8.5 µL nucleasefree water and 1 µL of each primer.The temperature profiles in all PCRs were the same, as provided by Sehgal et al. [21].All amplifications were evaluated using electrophoresis.One positive control (Trypanosoma sp.infected insect, confirmed by sequence analysis) and one negative control (ultrapure water) were used during each amplification.

Sequence Analysis
The PCR-positive samples were purified and sequenced using a Big Dye Terminator V3.1 Cycle Sequencing kit and ABI PRISM TM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA), and the Trypanosoma sequences were identified using the "Basic Local Alignment Search Tool" available from the NCBI.The sequences were submitted to GenBank (GenBank accession no.PP945809-PP945817).Other sequences for the Megatrypanum trypanosomes included in the phylogenetic analyses were obtained from GenBank (Table 1).The newly generated sequences were aligned with other Megatrypanum trypanosomes which belonged to the TthI and TthII clades of Trypanosoma theileri.
To clarify the intra-species relationships among the T. theileri species group, a phylogenetic median-joining (MJ) network [22] was created using Popart-1.7 software (http://popart.otago.ac.nz, accessed on 17 February 2023 [23]).This method, described in detail in [22], includes nodes to represent inferred sequences by iteratively adding "median" sequence vectors, and it combines the features of Kruskal's algorithm.The MJ method begins with the minimum spanning trees, all combined within a single network while sequentially adding a few consensus sequences of three mutually close sequences at a time.

Prevalence of Trypanosomatids in Tabanidae
In total, 101 Tabanidae members were investigated from six areas in Lithuania, and 14 different species were identified.Identification of some species (Hyb.muehlfeldi, Hyb.distinquenda, Hyb.bimaculata, Hyb.nitidifrons and T. bromius) was double-checked using both morphology and barcode sequencing.The most abundant species were T. maculicornis and Hyb.nitidifrons (Table 2).
In all, 30 newly determined partial SSU rRNA sequences (748 bp) of Trypanosoma spp.were obtained.The overall positivity of Trypanosoma spp.DNA in the tabanids was 50.5% (51/101) (Table 2), and the DNA of Trypanosoma spp. was detected in horseflies of 10 species out of the 14 investigated.Trypanosoma DNA was detected in 22 individual T. maculicornis specimens out of 26 (84.62%), and lower positivity was observed in Hyb.nitidifrons (14/18, 77.8%) and T. bromius (6/13, 46.2%).Although flies of other species, such as Haem.pluvialis and Haem.subcylindrica, were abundant, trypanosomatid DNA was detected only in a few of them (Table 2).Four horsefly species were represented by a single individual each (Hyb.distinquenda, Hyb.lapponica, Hyb.lurida and Hyb.muehlfeldi) and were positive for T. theileri.No trypanosomes were detected in flies belonging to the genus Chrysops (C.italicus, C. relictus and C. caecutiens) or Haem.crassicornis during this study, supposedly because of the low number of investigated flies for these species.To the best of our knowledge, the trypanosomatid detections in Hyb.lurida and Hyb.lapponica are the first ones ever recorded.Five samples from Hyb. nitidifrons and one from T. maculicornis, positive for Trypanosoma spp., carried a mix of either two or more different trypanosomatid haplotypes showing double peaks in the chromatograms of their sequences.
Insects 2024, 15, x FOR PEER REVIEW 6 of 10 8), another was 99.9% similar to sequences belonging to TthII (Figure 1, circles 1 and 2), and one sequence from T. maculicornis (Figure 1, circle 9) was more closely related to T. melophagium (GenBank accession no.HQ664912) [26], a parasite of the sheep Melophagus ovinus, with a measured similarity of 99.73%.It was confirmed that T. melophagium is more related to the lineage of TthII, and these SSU rRNA sequences are highly conserved among Megatrypanum trypanosomes.The differences are only a few substitutions, despite the fact that T. melophagium is a different species and has different hosts.Several isolates of Trypanosoma from the hosts Hyb.lurida and Hyb.nitidifrons (Figure 1, circle 4) exhibited 100% similarity to the T. theileri isolate G24 from Glossina fuscipes fuscipes (Central African Republic, GenBank accession no.KR024688) [25] and from the horseflies Hyb.muehlfeldi (MK156792), Chrysops divaricatus (MK156793) in the Republic of Karelia [15].Both isolates from these horseflies belong to the complex TthI.Several sequences of Trypanosoma isolated from T. maculicornis were identical to those referred to as T. cf.cervi (GenBank accession no.JX178196) from Odocoileus virginianus (USA) (Figure 1, circle 3).These isolates occurred between two T. theileri complexes in the network (Figure 1) as well as an isolate from the same white-tailed deer, named as a separate species (T.(Megatrypanum) trinaperronei).Glossina fuscipes fuscipes), T. trinaperronei found in white-tailed deer and T. melophagium inferred using SSU rRNA (748 bp) sequences and median-joining network method for intraspecific phylogenies.Network demonstrates evidence of TthI (circle 4) and TthII (circles 1 and 2) phylogenetic lineages, where most sequences belong.Numbers in circles correspond to those in Table 1.

Discussion
Species of the T. (Megatrypanum) theileri group putatively are non-pathogenic mammalian parasites and are transferred by an infected vector.Böse et al. [5] experimentally proved that the transmission of T. theileri in the field usually takes place through contamination of the oral mucosa with metacyclic stages from the gut contents or feces of tabanids.Kostygov et al. [18] studied the development of two different species of the T. theileri complex in the intestine of the vector and concluded that the observed differences were associated with the species of the vector and not with the phylogenetic position of the parasites.Ganyukova et al. [15] studied four species of infected horseflies and detected four morphotypes of T. theileri-like trypanosomes in the hindgut.PCR-based methods are used for detection of these parasites in bloodsucking insects, and Werszko et al. [16] Figure 1.Median-joining network of Trypanosoma spp.found in insects (tabanids, culicids and Glossina fuscipes fuscipes), T. trinaperronei found in white-tailed deer and T. melophagium inferred using SSU rRNA (748 bp) sequences and median-joining network method for intraspecific phylogenies.Network demonstrates evidence of TthI (circle 4) and TthII (circles 1 and 2) phylogenetic lineages, where most sequences belong.Numbers in circles correspond to those in Table 1.

Discussion
Species of the T. (Megatrypanum) theileri group putatively are non-pathogenic mammalian parasites and are transferred by an infected vector.Böse et al. [5] experimentally proved that the transmission of T. theileri in the field usually takes place through contamination of the oral mucosa with metacyclic stages from the gut contents or feces of tabanids.Kostygov et al. [18] studied the development of two different species of the T. theileri complex in the intestine of the vector and concluded that the observed differences were associated with the species of the vector and not with the phylogenetic position of the parasites.Ganyukova et al. [15] studied four species of infected horseflies and detected four morphotypes of T. theileri-like trypanosomes in the hindgut.PCR-based methods are used for detection of these parasites in bloodsucking insects, and Werszko et al. [16] detected Megatrypanum trypanosomes in Haem.pluvialis, T. bromius, T. maculicornis and T. distinguendus in Poland by molecular means, with the overall positivity of Trypanosoma spp.being 33.68%.Trypanosoma theileri was detected in the tabanids of two species (Hyb.ciureai and T. bromius) as well as in other bloodsucking Diptera (mosquitoes and sandflies) in the Czech Republic [6], and the prevalence of T. theileri in tabanids was reported to be 44%.In Lithuania, only Trypanosoma in raptor birds and biting midges have been investigated thus far [27,28], and the subgenus Megatrypanum in mammals or tabanids has not been studied yet.
Our study focused on the molecular detection of trypanosomatids in Tabanidae, and we examined a wider range of species and more individuals than in previous studies [6,15,16].In flies of some species (Hyb.lapponica and Hyb.lurida), Trypanosoma was detected for the first time.T. maculicornis was the most abundant species, and this tabanid also yielded the highest prevalence of trypanosomatids (84.62%).Similarly, high prevalence for Trypanosoma spp. was detected for this species in Poland (60%) [16].Hyb.nitidifrons showed 77.8% prevalence of T. theileri and confirmed the results of a previous study on the development of T. theileri in this species of tabanids [18].Our findings suggest that T. maculicornis and Hyb.nitidifrons could be effective vectors of T. theileri.Some species of tabanids, such as T. bromius, have been previously confirmed as vectors of T. theileri [5], where 14% of tabanids were positive for trypanosomatids.A high prevalence in T. bromius (46.2%) was detected during our study as well as in a study by Brotánková et al. (50%) [6], but this species was not as prevalent (17.95%) for trypanosomatids in Poland [16].Some abundant tabanid species from our study (Haem.pluvialis and Haem.subcylindrica) demonstrated low prevalence of T. theileri, which is quite surprising because Haem.pluvialis showed the highest infection rate (57.14%) in a study carried out in Poland [16].
Five SSU rRNA sequences of Trypanosoma spp.obtained from Hyb. nitidifrons and one from T. maculicornis demonstrated double peaks in their chromatograms, suggesting mixed trypanosomatid infections in the same insect.No infections of Trypanosoma spp.were detected in the Chrysops flies during our investigation, while the presence of T. theilerilike trypanosomatids was reported in these flies in earlier works [5,15].This observation suggests that larger tabanids such as Tabanus or Hybomitra transfer more secretion to another host during feeding, possibly influencing the amount of infection of different strains or species of Trypanosoma spp., while smaller tabanids, like Chrysops, transfer fewer secretions to another host during feeding [12], making these tabanids less important in pathogen transmission.
There is no information concerning the occurrence of T. theileri parasites in wild animals in Lithuania, but data available on Polish cervids [29] show the presence of trypanosomes in red deer (Cervus elaphus), suggesting that trypanosomes can be found in cervids in Lithuania as well.
Trypanosoma sequences from tabanids obtained in the present study showed low genetic polymorphism, and similar to previous studies [11], we detected homogeneity in the T. theileri trypanosomes as well.Despite the fact that Schoener et al. [8] reported T. theileri as a complex of species that cannot be resolved using the SSU gene, nine different haplotypes distributed in different tabanid species were detected by us (Tables 1 and 2).It is known that the T. theileri group consists of several species (T.theileri, T. melophagium, T. cervi and T. trinaperronei) [6].
Analysis showed that most sequences obtained during our study were identical or quite close to two major T. theileri clades: TthI and TthII (Figure 1, circles 1, 2 and 4).The majority of the sequences were included in the TthII clade (Figure 1 and Table 1) along with a sequence from cervids [13], despite the fact that some authors [7,24] think that complex TthII is genetically closer to bovine tryposomatids and TthI is closer to cervids.The sequence of T. melophagium from Croatia (Figure 1, circle 10) was more related to the lineage TthII, as was confirmed by Martinković et al. [26].The clade TthI contains sequences obtained only from tabanids (Figure 1, network number 4), with only one sequence from Glossina fuscipes fuscipes [25].Some sequences from T. maculicornis, T. bromius, Hyb.bimaculata and Hyb.lapponica differed from the sequences available in GenBank and appeared to be closer to the TthI lineage, showing that this group can vary more genetically.Two different clusters in the constructed network (Figure 1) can be observed.It is worth pointing out that most of the obtained sequences were clustered with other genotypes of two T. theileri clades.One sequence from T. maculicornis and two isolates from white-tailed deer, described as T. trinaperronei n.sp.[7] and T. cf.cervi, clustered between complexes TthI and TthII, indicating similarity to both clades (Figure 1, circles 3 and 11).Similar results, where the lineage from the deer was recognized as a separate lineage, were demonstrated by Rodrigues et al. [11].Our network showed close relationships for the trypanosomes of the subgenus Megatrypanum and small polymorphism between the lineages.
Confirming previous studies [30], our data analysis suggests the presence of different Trypanosoma genotypes in the same tabanid species, meaning that different lineages of Trypanosoma could be more related to the vertebrate hosts but not the tabanid species.Trypanosoma spp.spread among hosts through tabanids, which feed on blood from a range of species, and thus the existence of several defined genotypes in the same species is probable.The sequences from T. maculicornis had six different haplotypes (Table 2), and any degree of host specificity was not found.This tabanid species is abundant and is infected by trypanosomatids more often (84.6%)than other tabanid species.
To the best of our knowledge, this is the first study on Trypanosoma genetic diversity in Tabanidae from Lithuania.Although widespread, the T. theileri group is largely neglected due to its low economic importance and causing no pathology.However, pathologies might have resulted from coinfections or stress when fever, anorexia, and anemia were reported as symptoms in several bovid infections [31,32].

Conclusions
The present study reported on the molecular detection and genetic diversity of trypanosomes in 10 species of tabanids, with two of them being reported for the first time.The findings suggest that different strains of the T. theileri complex are not related to different tabanid species but rather to different host species.Further research is needed to shed light on the genetic diversity and vector-parasite specificity of the T. theileri species complex in different tabanid species based on partial cathepsin L-like protein (CATL) gene, ITS and Tth625 fragment sequences.

Table 1 .
List of species where trypanosomes were detected and sequence numbers from the GenBank database used in this study.Sequences in bold (GenBank accession nos.PP945809-PP945817) were determined in the present study.Numbers in network as shown in Figure1.

Table 2 .
Prevalence of Trypanosoma spp. in Tabanidae of different species.* There are 9 haplotypes in total, as different tabanid species have the same haplotypes. )