Molecular Characterization of Lipoptena fortisetosa from Environmental Samples Collected in North-Eastern Poland

Simple Summary Lipoptenafortisetosa is an invasive, hematophagous insect, which lives on cervids and continues to spread across Europe. The species originated from the Far East and eastern Siberia. Besides wild animals, these ectoparasites can attack humans, companion animals, and livestock. These insects may also play a role in transmitting infectious diseases. The objective of this study was to confirm the presence of L. fortisetosa in north-eastern Poland and to characterize the examined population with the use of molecular methods. Deer keds were collected from six natural forests in the region of Warmia and Mazury. DNA of L. fortisetosa was extracted and subjected to molecular studies. Two species of deer keds (Lipoptena cervi and L. fortisetosa) were obtained in each location during field research. There were no differences in the sex distribution of these two ectoparasite species. During the research, more L. cervi than L. fortisetosa specimens were obtained. The studied insects were very closely related to specimens from Lithuania, the Czech Republic, and Japan. Our study indicates various ectoparasite lineages, and such research contributes to the improvement of basic knowledge on the distribution, genetic structure, and variability of the invasive ked fly L. fortisetosa. Abstract Recent years have witnessed an increase in the population of Lipoptena fortisetosa in Central Europe. The genetic profile of this ectoparasite has not been studied in Poland to date. The aim of the present study was to confirm the presence of L. fortisetosa in north-eastern Poland and to characterize the examined population with the use of molecular methods. Deer keds were collected between June and July 2019 in six natural, mixed forests. A fragment of the rRNA 16S gene was used as a marker to identify L. fortisetosa by polymerase chain reaction (PCR). DNA samples were sequenced in the last step. Six new locations of L. fortisetosa were confirmed. No significant differences were observed in the sex ratios of L. cervi and L. fortisetosa (L. cervi p-value = 0.74; L. fortisetosa p-value = 0.65). Significant differences were noted between the total size of L. cervi and L. fortisetosa populations (p-value < 0.001). The similarity to GenBank sequences ranged from 95.56% to 100%. The obtained nucleotide sequences were very closely related to L. fortisetosa sequences from Lithuania, the Czech Republic, and Japan. Molecular analyses revealed considerable genetic diversity, which could indicate that various ectoparasite lineages have spread throughout Europe.


Introduction
Deer keds are hematophagous ectoparasites of birds and mammals. The genus Lipoptena consists of 32 species, including L. capreoli, L. cervi, L. depressa, L. mazamae, and L. fortisetosa, which are of great importance in veterinary medicine. Lipoptena fortisetosa has been insufficiently investigated in cervids, despite the Japanese sika deer (Cervus nippon) that is its original host. The parasite also colonizes non-specific hosts, including cattle and humans [1,2]. Deer keds shed wings when they find a definitive host. Hippoboscidae reproduce by adenotrophic viviparity, and their life cycle is influenced mainly by climatic conditions [3,4]. Lipoptena spp. bites are painful and may lead to skin inflammation and alopecia [5,6].
There is evidence to suggest that L. fortisetosa continues to spread across Europe. The species originated in the Eastern Palearctic, in particular, the Far East and eastern Siberia. Lipoptena fortisetosa was first identified in 1965 in Japan by Maa [7]. In Central Europe, this deer ked is considered a cryptogenic species, but it is increasingly classified as an invasive species [8]. Lipoptena fortisetosa was first recorded in Europe in the 1960s [9]. Currently, this insect was spotted in several European countries including the Czech Republic [10], Estonia [11], Italy [12,13], Moldova [2], and Slovakia [14]. This ectoparasite was not detected in Poland during a program monitoring the ectoparasite burden of wild animals in 1973-1980 [15]. In Poland, L. fortisetosa was first observed in the region of Lower Silesia in the late 1980s [16]. Later, in 2007-2012, L. fortisetosa was identified in cervids in northern Poland and in the Tatra Mountains [8,[17][18][19][20]. In 2017, this ectoparasite was found even in companion animals in central Poland [21].
Lipoptena fortisetosa is a potential vector of infectious diseases. Deer keds can act as carriers of Bartonella spp., and the vertical transmission of this pathogen poses a particularly serious problem [22][23][24]. In recent research, Bartonella spp. of Japanese origin were isolated from L. cervi in the region of Warmia and Mazury in Poland [22]. According to Szewczyk et al. [22], the isolated Bartonella spp. strain had originated from Japanese sika deer that were introduced to Europe, and it was transmitted to the local environment by various vectors. Molecular studies confirmed that the insects harbored genetic material of Anaplasma phagocytophilum, A. ovis, Borrelia spp., Coxiella-like bacteria, Theileria luwenshuni, T. ovis, and Trypanosoma spp. [25][26][27][28][29][30][31]. Despite the above, little is known about the ectoparasite's ability to transmit pathogens other than Bartonella spp. to new hosts.
The observed migration patterns of the analyzed ectoparasites suggest that their geographic range should be monitored because of the potential vectorial capacity of this ectoparasite [32]. Salvetti et al. [12] noted that the geographic distribution of deer keds in Europe should be analyzed for social and environmental implications because Lipoptena spp. may cause problems for wildlife management. However, a detailed evaluation of ancestral lineages and migration routes requires larger and more frequent sampling. There is a considerable gap in the genetic distribution of L. fortisetosa between Western and Eastern Europe. Andreani et al. [33] did not detect any obvious and stable morphological and molecular differences in specimens from the geographical areas of Asia and Europe. In Italy, they identified the monophyletic clade of L. fortisetosa, along with two Central European and two Korean haplotypes [33]. These ectoparasites have been largely neglected in Polish research, and potentially important genetic data, including genetic diversity, are scarce. Most Polish studies of L. fortisetosa were based on macroscopic analyses, which is why the genetic and phylogenetic characteristics of the species, including DNA rRNA 16S gene remain unknown. The locations colonized by L. fortisetosa in north-eastern Poland have not been evaluated to date [4]. This Polish region abounds in forests, and it is characterized by increasingly frequent deer ked flights and mass attacks on animals and humans. Differences in sex ratios of host-seeking insects were not investigated in this location. This may be important because of the fact that females are more likely to harbor the genetic material of pathogens [32]. The spread of L. fortisetosa outside of Asia could have been accelerated by the introduction of sika deer to the European continent, whose small but stable populations can be found in the central part of the region of Silesia and in the eastern part of the region of Warmia and Mazury. The aim of this study was to confirm the occurrence of L. fortisetosa in north-eastern Poland and to characterize the specimens collected during host-seeking flights based on the results of molecular analyses.

Sample Collection
The occurrence of Lipoptena spp. was monitored in natural mixed forests in the region of Warmia and Mazury in north-eastern Poland. Ectoparasites were monitored in the following municipalities: Ełk (54 • Figure 1.

Sample Collection
The occurrence of Lipoptena spp. was monitored in natural mixed forests in the region of Warmia and Mazury in north-eastern Poland. Ectoparasites were monitored in the following municipalities: Ełk (54°19′10.1″ N 22°22′38.7″ E), Gołdap (54°19′10.1″ N 22°22′38.7″ E), Kolno (53°57′34.3″ N 21°04′46.0″ E), Jedwabno (53°28′24.8″ N 20°34′04.9″ E), Pieniężno (54°15′52.1″ N 20°11′14.2″ E), and Ruciane-Nida (53°35′47.4″ N 21°33′13.3″ E). A detailed map of the surveyed region is presented in Figure 1. The selected sampling sites are characterized by extensive forests and large cervid populations, including red deer (Cervus elaphus), European roe deer (Capreolus capreolus), and moose (Alces alces). The study was conducted between June and July 2019 between 7 and 10 a.m. This study period was chosen because of a noticeable increase in hippoboscidae activity. The number of Lipoptena spp. individuals was expressed by the sum number of winged deer keds that were captured on the investigator's clothing per day. Five daily measurements were conducted in each sampling site. A total of 30 measurements were conducted. The investigator walked through the forest wearing brown cotton clothing covering the entire body. Lipoptena spp. were captured immediately after landing on the clothing. During the walk, the investigator checked protective clothing every 20 m and collected insects from creases and folds on the fabric. Keds that escaped were not counted. Similar methods have been used by other researchers to acquire environmental samples of ticks and Lipoptena spp. [34,35]. The collected ectoparasites were placed in individual test tubes containing 70% ethanol. The selected sampling sites are characterized by extensive forests and large cervid populations, including red deer (Cervus elaphus), European roe deer (Capreolus capreolus), and moose (Alces alces). The study was conducted between June and July 2019 between 7 and 10 a.m. This study period was chosen because of a noticeable increase in hippoboscidae activity. The number of Lipoptena spp. individuals was expressed by the sum number of winged deer keds that were captured on the investigator's clothing per day. Five daily measurements were conducted in each sampling site. A total of 30 measurements were conducted. The investigator walked through the forest wearing brown cotton clothing covering the entire body. Lipoptena spp. were captured immediately after landing on the clothing. During the walk, the investigator checked protective clothing every 20 m and collected insects from creases and folds on the fabric. Keds that escaped were not counted. Similar methods have been used by other researchers to acquire environmental samples of ticks and Lipoptena spp. [34,35]. The collected ectoparasites were placed in individual test tubes containing 70% ethanol.

Species Identification
The collected samples were transported to the Biological Hazard Laboratory at the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn. Species and sex were identified based on morphological features, including body dimensions, wing venation, the length and structure of the palpi, and the number of erect hairs on the mesonotum, under the Leica M165C stereoscopic microscope (Leica, Wetzlar, Germany). Measurements were performed in Leica Application Suite 4.4 (Leica, Wetzlar, Germany) based on the taxonomic keys for Lipoptena spp. developed by Borowiec and Maa, and the morphological descriptions provided by Andreani et al. [13,36,37].

Statistical Analysis
The sex ratio of Lipoptena spp. and the number of L. cervi and L. fortisetosa specimens from each measurement were analyzed with Student's t-test for independent samples. The following statistical parameters were calculated for the analyzed populations of L. cervi and L. fortisetosa using overall data from daily observations (n = 30): mean (M), median (ME), standard deviation (SD), standard error (SE), variance (V), minimum (Min), and maximum (Max). Differences were regarded as statistically significant at p-value < 0.05. Data were processed statistically in Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA).

DNA Extraction
Five samples of L. fortisetosa (n = 30) from each sampling site were randomly selected for molecular analyses. The representative number of samples was estimated with the use of formulas for computing the minimum sample size to guarantee the reliability of the results (sample size: N P = number of collected specimens, α = 95%, f = 0.5, e = 15%). Keds were air-dried at room temperature for 15 min, and they were crushed with a sterile rod in sterile Eppendorf tubes. Genomic DNA was extracted from each sample with the Sherlock AX kit (A&A Biotechnology, Poland) according to the manufacturer's instructions. DNA was eluted in 40 µL of Tris-EDTA (TE) buffer, and the concentration of the extract was checked in the Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was stored at −20 • C until analysis.

Polymerase Chain Reaction
The DNA sequence of the mitochondrial DNA rRNA 16S gene of L. fortisetosa with an estimated length of 412-bp was amplified by PCR. The amplification primers were L700F (5 -AAAGTTTAACCTGCCCACTGAT-3 ) and L1213R (5 -CTGAACTCAGATCACGTAAGAAT-3 ) [22]. The following cycling conditions were applied: initial denaturation at 92 • C for 3 min; 35 cycles of denaturation at 95 • C for 10 s; annealing at 60 • C for 10 s; extension at 68 • C for 30 s; final extension at 68 • C for 5 min [22]. Each reaction was performed in a final volume of 25 µL containing 2.5 µL of 10× Standard Taq Reaction Buffer (Biolabs, NC, USA), 0.5 µL of 10 mM deoxyribonucleotide triphosphate (dNTPs) (Biolabs, USA), 0.5 µL of 10 µM of each primer, 1 µL of Lipoptena spp. DNA template, 0.125 µL of Taq DNA Polymerase (Biolabs, NC, USA), and 19.875 µL of double-distilled water. Double-distilled water was the negative control. Lipoptena cervi DNA extracted during a previous study was the positive control. PCR products were subjected to electrophoresis on 2% agarose gel. They were stained with ethidium bromide and visualized in a UV transilluminator.

Sequencing
After PCR, DNA samples were purified by the ethanol precipitation method described by Weinberger [38]. DNA was sequenced with the use of the described primers (L700F/L1213R) and the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), and the results were analyzed in the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The acquired nucleotide sequences were edited in the BioEdit program [39] and compared with GenBank sequences in the BLAST-NCBI program (attached in result section). Sequences were aligned with the ClustalW algorithm. A phylogenetic analysis of rRNA 16S sequences and the corresponding GenBank sequences was performed by the maximum likelihood method and the Tamura-Nei model in MEGA 10.1.17 [40]. Sequences of L. cervi served as the outgroup. Model selection was based on Akaike's information criterion (AIC). Bootstrap confidence values for assessing the reliability of branches were calculated in 10,000 replicates.

Results
The  Table 1. The phylogenetic tree is presented in Figure 2. The length of the alignment used to build the phylogeny was 445-bp. Twenty-eight sequences (MT182645-MT182658, MT182660-MT182661, MT182663) were characterized by 98.32% to 100% similarity with L. fortisetosa sequences from the Czech Republic and Lithuania (MF495939, MN889549). The similarity between sequences MT182659 and the L. fortisetosa sequence from Japan (AB632589) reached 95.56%. Detailed data, including the ID of the closest match, country of origin, and percent sequence identity, are presented in Table 2. The distribution of sequences in each sampling site is presented in Table 3.     Table 3. Molecular characteristics of Lipoptena fortisetosa in sampling sites.

Discussion
Autochthonous L. cervi accounted for 79.88% of the collected ked samples, which suggests that it is the dominant species in the region of Warmia and Mazury. However, the proportions of Lipoptena species should be regularly monitored in the studied region because of the possible increase in the population of L. fortisetosa. In Europe, L. fortisetosa is considered a multivoltine species, whereas L. cervi is regarded as a univoltine species [8]. The phenomena described above could influence the rate at which these ectoparasite populations develop in the future, and it could increase the number of host-seeking L. fortisetosa. Because of phenological differences between the species, the number of collected individuals can also be influenced by the month of sampling. Both ectoparasites species should be sampled in a period when they are most active. This study demonstrated that adult L. fortisetosa and L. cervi are active in the same period of time, and similar observations were made by Oboňa et al. [14]. Neither species was characterized by significant differences in the sex ratio. In around 30% of ectoparasitic species, the sex ratio does not significantly depart from unity [44]. Adult keds find hosts immediately after emergence. However, the sex ratio of Lipoptena-spp.-inhabiting hosts could differ from that noted in this study because of a potentially shorter life span of adult males [44].
The migration patterns of Lipoptena spp. in Europe have not been fully elucidated, but the geographic range of these ectoparasites was probably expanded by natural migration from the east. It cannot be ruled out that this insect may have been brought to Europe along with imported animals [33]. Lipoptena fortisetosa had been previously identified in only several European countries, including the Czech Republic [10], Moldova [2], and Poland [4,8]. Recent reports from Estonia [11] and Italy [12,13,33] indicate that the geographic range of Lipoptena spp. has expanded both northward and southward since 2017. Andreani et al. [33] suggested that the relatively recent migration of sika or hybrid individuals to Italy from neighboring countries may have contributed to the ectoparasite transmission. There is no conclusive evidence to indicate whether this geographic shift has resulted from an increase in the cervid population or climate change. The impact of global climate change on the geographic expansion of Lipoptena spp. was postulated by Kurina et al. [11]. Keds are poor fliers that cover a distance of only up to 50 m when searching for hosts [45], which is why hosts play an important role in the transmission of these ectoparasites. The presence of L. fortisetosa in highly diverse regions also suggests that these insects are eurytopic species that tolerate a wide range of environmental conditions [4,46].
The importance of Lipoptena spp. in veterinary medicine has not been fully established, which is why these ectoparasites should be studied in greater detail. The existing knowledge about the impact of L. fortisetosa on wild animals is superficial, and these ectoparasites could affect game management in the future. Polish cervids and other wild animals were found to be heavily infested with hippoboscids. Deer keds were detected in 64% of European roe deer [47], 76% of fallow deer, and 78% of European red deer [48]. One animal was infested with 9.9 insects on average [48]. Vikøren et al. [49] found that one moose can carry more than 16,000 keds. Andreani et al. [33] detected infestation of European deer species by L. fortisetosa. The stability of the L. fortisetosa population can be attributed to adaptive genetic variation and the benefits associated with colonizing different cervid species as hosts. These observations point to high plasticity in host selection, which is influenced by the availability and population density of a given host species. For this reason, there are no obvious barriers to the future expansion of the discussed ectoparasites [11,33].
The available GenBank data show that the sequences originating from the Czech Republic or Lithuania (cited in the manuscript) were closely similar to ours. However, the GenBank database contains only several 16S rRNA sequences of European L. fortisetosa. Sequence MT182659, obtained in the present study, was most similar to the sequence originating from Japan (AB632589) [43]. The insect harboring the above sequence was captured in the municipality of Pieniężno where a stable population of sika deer has existed since the beginning of the 20th century. Lipoptena fortisetosa could have been accidentally introduced to that region by sika deer, and a distinct population of these ectoparasites could have evolved over time. Similar conclusions were formulated by Andreani et al. [33] and Mihalca et al. [50]. Interestingly, two Korean haplotypes of this insect were detected in Italy [33]. This confirmed our assumptions. Polish deer keds could belong to an isolated and genetically diverse population. The genetic polymorphism of L. fortisetosa is probably associated with the segregation of haplotypes that are relatively popular in this species, and the stability of the L. fortisetosa population points to the presence of dynamic evolutionary mechanisms.
Similar levels of genetic diversity were reported in a study carried out on L. mazamae [51]. Trout et al. [51] identified six unique haplotypes using 30 DNA sequences of a 259-bp region of the mitochondrial DNA rRNA 16S gene amplified by PCR. Such molecular differences were also reported in other ectoparasites, including Cimex lectularius, tsetse flies (Glossina spp.), and ticks [52][53][54][55]. Szalanski et al. [52] observed high mitochondrial DNA diversity with moderate to high levels of gene flow in C. lectularius. Krafsur et al. [53] identified 26 haplotypes in tsetse flies with the use of 12S rRNA and 16S rRNA markers, where most haplotypes occurred only once or twice. Only six haplotypes were identified in the same locations [53]. 16S rRNA and Cytochrome c oxidase I (COI) gene sequences are also useful markers for analyzing genetic polymorphism in ticks and for identifying and differentiating the populations of Dermacentor nuttalli, Ixodes persulcatus, and I. pavlovskyi [54,55]. These findings indicate that the mitochondrial DNA rRNA 16S gene can be reliably used to study the population genetics of ectoparasites. Previous research has demonstrated that Lipoptena spp. could be paraphyletic; therefore, further research is needed to explore the genetic variation and taxonomy among this genus [56]. Kurina et al. [11] and Andreani [33] found that the COI sequences of L. fortisetosa deposited in GenBank are genetically diverse and have different geographic lineages in Europe. Similar conclusions can be formulated based on the results of this study.
The current study revealed considerable genetic diversity of L. fortisetosa in six sampling sites in the region of Warmia and Mazury, which indicates that the genetic structure of its population is far more complex than previously thought. Several genetically distinct original populations probably migrated or were introduced to Europe. The present findings suggest that a decrease in deer ked populations in forests could contribute to their diversity. The presence of progressive evolutionary mechanisms indicates that L. fortisetosa could further adapt to the environmental conditions of Central Europe [4]. Lipoptena fortisetosa is an invasive species that strongly competes for hosts with the native population of L. cervi, which could influence the size of both insect populations in the future. Further genetic analyses of louse flies, combined with pathogen detection, are needed to identify areas with a potentially high risk of infectious disease transmission by L. fortisetosa [51]. The structure of deer ked populations should be analyzed in greater detail to adequately manage and protect wild animals and to monitor the transmission of pathogens between distant regions. The identification of Bartonella spp. of Japanese origin by Szewczyk et al. [22] and L. fortisetosa of Japanese origin in this study demonstrates how the dispersion of deer keds in the environment can contribute to the spread of infectious diseases transmitted by these vectors. The results of this study provide additional evidence that L. fortisetosa populations in Central Europe have different genetic lineages. Detailed genetic characterization of deer keds in Central Europe will enable researchers to track and predict the spread of L. fortisetosa. Migrations to new areas should encourage new research into the dynamics and genetics of L. fortisetosa populations because, according to Kurina et al. [11], a thorough understanding of the distribution and bionomics of this invasive migratory species is crucial for its effective control in Europe.

Conclusions
The results of the present study and previous research suggest that L. fortisetosa has formed stable and genetically diverse populations throughout Poland. The population genetics of L. fortisetosa should be further explored to track the migration of these ectoparasites to Northern and Western Europe. The occurrence of L. fortisetosa should be regularly monitored to detect an increase in the number of individuals in the total deer ked population. Further research is needed to detect new locations and analyze the genetic lineages of L. fortisetosa. The sequences identified in this study indicate that the population of L. fortisetosa continues to spread despite geographic limitations. Funding: This project was financially co-supported by the Ministry of Science and Higher Education under the program entitled Regional Initiative of Excellence for the years 2019-2022 (project no. 010/RID/2018/19; amount of funding PLN 12,000,000). The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are presented in this paper.