Morphological and Molecular Identiﬁcation of Physaloptera alata (Nematoda: Spirurida) in a Booted Eagle ( Aquila pennata ) from Portugal

Simple Summary: Birds of prey are important predators and scavengers at the top of the food chain, but their parasite diversity has not been well studied. The aim of this study was to identify several nematode specimens found in the gizzard of a booted eagle ( Aquila pennata ) during necropsy. Following morphological and molecular analysis, they were identiﬁed as Physaloptera alata , a circumstance which represents the ﬁrst report of this species in a booted eagle from Portugal. A new genetic sequence for this parasite is now available in GenBank for future research on birds of prey. This information is crucial for understanding the parasitological fauna of these birds in Portugal, as well as for wildlife rehabilitation centers, disease ecologists


Introduction
Nematode parasites belonging to the genus Physaloptera can infect a broad range of hosts. Physaloptera spp. are distributed worldwide and can be found in the gastrointestinal tract of many carnivores, including birds of prey [1][2][3][4]. In felines, Physaloptera spp. can cause inflammatory and degenerative pathological changes [5,6]. They can have a complex life cycle, with arthropods such as cockroaches and beetles acting as intermediate hosts [7]. Small vertebrates could play a role in the life cycle as paratenic hosts by ingestion of an infected arthropod, while predators are usually definitive hosts [6,[8][9][10][11].
Birds of prey play an important role as top predators in their biotope ecology [12]. Because of this, changes in the fitness of the raptor population can have important consequences for their ecosystem [1]. However, studying the pathogens that affect these predators involves a large variety of challenges. Many species of birds of prey have large territories of difficult access, some of them are migratory, and many of them are under a protected status in their countries of origin [4,13]. For these reasons, parasite diversity in raptorial birds has not been studied in depth [4,13,14]. A few reports aimed at elucidating the parasitic species infecting raptors have been conducted [1][2][3][4][13][14][15]. This makes the confirmation of morphological identifications via phylogenetic studies difficult.
We report the presence of adult Physaloptera alata in a booted eagle (Aquila pennata) for the first time in Portugal. Additionally, we confirm the morphological identification of the parasite using phylogenetic analysis.

Case Report
In August 2018, a young booted eagle (A. pennata) was admitted to CERAS (Centro de Estudos e Recuperação de Animais Selvagens, Quercus, Associação Nacional de Conservação da Natureza (Center for Studies and Recovery of Wild Animals, Quercus National Association for Nature Conservation)), a wildlife rehabilitation center located in Castelo Blanco, eastern-central Portugal. The animal was originally found in the municipality of Abrantes, also in eastern-central Portugal. The eagle weighed 453 g and was in very poor condition (1 on a scale from 0 to 5). Additionally, the specimen presented traumatic injuries due to collision with an unknown object. These injuries consisted of dislocation of the left shoulder, in addition to fractures of the elbow joint and ankylosis. The eagle was euthanized due to poor condition, sustained trauma, and permanent loss of flight ability. During necropsy, 31 adult nematode-like worms were found in the gastrointestinal tract (gizzard). The worms were collected in water and preserved in 90% alcohol for further analysis. Histological observations were not part of this study.

Morphological Identification of Parasite Species
Nematodes were mounted in glycerol and observed under a light microscope, Olympus BX 50 (Olympus, Tokyo, Japan). Parasite genus identification was performed using several morphological keys [16][17][18].

Parasite DNA Extraction and Amplification of the 18S Small Subunit rRNA Gene and the Cytochrome C Oxidase Subunit I Gene
Genomic DNA (gDNA) was extracted and purified from the nematodes using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. For molecular identification of the parasite species, two genes were targeted: the 18S small subunit tRNA gene (SSU 18S rRNA), and the cytochrome c oxidase subunit I (cox1) gene. For the amplification of SSU 18S rRNA, the primers Nem_18SF (5 -CGCGAATRGCTCATTACAACAGC-3 ) and Nem_18SR (5 -GGGCGGTATCTGATCGCC-3 ) were used to amplify a ≈ 900-base-pair (bp) fragment according to Floyd et al. [19]. PCR reactions consisted of 2.5 µL of DreamTaq™ Green Buffer (10x; Thermo Fisher Scientific, Dreieich, Germany), 0.2 µM of each deoxynucleoside triphosphate (dnp), 0.5 µM of each primer, and 2 U of DreamTaq Polymerase (Thermo Fisher). Additionally, 3 µL of worm gDNA was used as the DNA template. Finally, nuclease-free, DNA-free water was added up to a final volume of 25 µL. PCR conditions were as follows: an initial denaturation cycle of 5 min at 94 • C followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 54 • C for 30 s, and extension at 72 for 1 min. A final extension cycle of 72 • C for 7 min was added.
Amplification of the cox1 gene (≈650 bp) was performed using a primer cocktail as described by Prosser et al. [20]. PCR reactions and thermal cycling conditions were conducted exactly as described before [20]. gDNA from a previously identified Spirurida nematode (Diplotriaena obtuse) [21] was used as positive control for both PCR assays. Finally, all PCR reactions were run in a Biometra Tadvanced ® Thermal cycler (Analytik Jena, Jena, Germany) and visualized in a 1.5% agarose gel.

Cloning, Sequencing, and Phylogenetic Analysis
Fresh SSU 18S tRNA PCR products were cloned into a pCR™2.1 vector using the TA Cloning™ Kit (Invitrogen, Dreieich, Germany) following the kit's instructions. Afterwards, plasmid DNA was prepared using the GeneJet Plasmid Miniprep Kit (Invitrogen, Dreieich, Germany) according to the instructions in the kit. Plasmid minipreps were commercially sequenced (Microsynth Seqlab, Göttingen, Germany) in both directions using the SSU 18S tRNA primers mentioned above.
Produced sequences were analyzed using the MEGA X software (version 10.1.5) [23] and blasted in the GenBank database. Annealing was conducted using the MUSCLE Software (https://drive5.com/muscle/ accessed on 12 May 2023) and evolutionary relationships between sequences were constructed using the maximum likelihood method and Kimura 2-parameter model [24]. Finally, uncorrected pairwise (p) distances between gene sequences were also calculated using the MEGA X software.

Morphological Identification of Physaloptera sp.
The specimens had a thick cuticle and were striated transversely and detached from the body, with well-marked annulations ( Figure 1A). At the anterior part, a cuticle dilatation was reflected over the base of the lips, forming a cephalic collar with small teeth ( Figure 1B,C). The mouth had two large lateral lips ( Figure 1B,C) with teeth on their internal surface and with papillae externally. The buccal cavity was short, and the esophagus was divided into two parts: a shorter muscular clear portion, and a longer glandular opaque portion ( Figure 1A). The nerve ring encircled the esophagus in the beginning of its third posterior portion ( Figure 1D). These anatomical characteristics allowed us to identify the specimens as belonging to the genus Physaloptera.

Molecular Identification of Physaloptera spp. and Phylogenetic Analysis
The presence of bands of specific lengths during agarose gel visualization confirmed the successful amplification of the SSU 18S rRNA and cox1 genes. Sequences of the SSU 18S rRNA amplicon clones showed the highest identity percentage (99.31%) and a query cover of 100% when aligned to a P. alata 18S rRNA sequence (GenBank accession number: AY702703.1) with a query cover of 100%. This significant alignment was followed by partial sequences of the SSU 18S rRNA gene from Physaloptera apivori (98.85% identity, 100% query cover, accession number EU004817.1), and Physaloptera sp. (98.27% identity, 100% query cover, accession number: MG808040.1). The sequence produced in the present study was deposited in the GenBank database under accession number MN855524.  Figure 2D). Posterior end with five pairs of pedunculated papillae and four pairs of sessile papillae ( Figure 2E).

Molecular Identification of Physaloptera spp. and Phylogenetic Analysis
The presence of bands of specific lengths during agarose gel visualization confirmed the successful amplification of the SSU 18S rRNA and cox1 genes. Sequences of the SSU 18S rRNA amplicon clones showed the highest identity percentage (99.31%) and a query cover of 100% when aligned to a P. alata 18S rRNA sequence (GenBank accession number: AY702703.1) with a query cover of 100%. This significant alignment was followed by partial sequences of the SSU 18S rRNA gene from Physaloptera apivori (98.85% identity, 100% query cover, accession number EU004817.1), and Physaloptera sp. (98.27% identity, 100% query cover, accession number: MG808040.1). The sequence produced in the present study was deposited in the GenBank database under accession number MN855524.
A dataset of 11 sequences of the SSU 18S rRNA gene, including the ones generated in this study, was used to build phylogenetic trees ( Figure 3). The selected sequences belonged to other species of suborder Spirurina. Additionally, a Cyrnea mansioni sequence also from birds of prey was used to root the tree. Phylogenetic analysis clustered the sequence of this project within the genus Physaloptera (order Spirurida) and closely related to a P. alata (accession number: AY702703) sequence obtained from birds of prey in Germany [3] (Figure 3).
Animals 2023, 13, x 6 of 10 A dataset of 11 sequences of the SSU 18S rRNA gene, including the ones generated in this study, was used to build phylogenetic trees ( Figure 3). The selected sequences belonged to other species of suborder Spirurina. Additionally, a Cyrnea mansioni sequence also from birds of prey was used to root the tree. Phylogenetic analysis clustered the sequence of this project within the genus Physaloptera (order Spirurida) and closely related to a P. alata (accession number: AY702703) sequence obtained from birds of prey in Germany [3] (Figure 3).
Separately, the highest cox1 gene identity percentage for our sequence was 85.07% with a sample from Physaloptera sp. (accession number MW5146) collected from a stray cat [27], followed by Abbreviata caucasica (syn. Physaloptera mordens) (accession number: MT23195) from a chimpanzee with 81.71% similarity [28]. After the estimation of evolutionary relationships, the cox1 gene sequence from this study was found to be clustered in the Physaloptera spp. branch (Figure 4).  Separately, the highest cox1 gene identity percentage for our sequence was 85.07% with a sample from Physaloptera sp. (accession number MW5146) collected from a stray cat [27], followed by Abbreviata caucasica (syn. Physaloptera mordens) (accession number: MT23195) from a chimpanzee with 81.71% similarity [28]. After the estimation of evolutionary relationships, the cox1 gene sequence from this study was found to be clustered in the Physaloptera spp. branch (Figure 4).

Discussion
We report P. alata in a booted eagle from Portugal. Booted eagles live in South Africa and migrate to the Iberian Peninsula and other Mediterranean regions to breed. Nevertheless, they are scarcely found in Europe [29]. A study found P. alata in one booted eagle from Spain in 1993 [1]. To our knowledge, this is the only European record that precedes this present report.
Besides booted eagle, P. alata has been recorded in peregrine falcons [3], Asiatic sparrowhawks (Accipiler nisus) [26], and sharp-skinned hawks (Accipiter velox) [26]. In Portugal, P. alata has been reported in birds of the orders Falconiformes and Strigiformes [30]. The present study is the first report in a Portuguese booted eagle.
Despite the available information, most of these previous studies were restricted to sole reports of parasite presence in a certain species, with small sample sizes [1][2][3][4][13][14][15]18,31,32]. In these studies, parasite species differentiation was based on morphological features only. Morphological traits are a pivotal and important tool for taxonomic classification; however, they can be difficult to recognize for non-experienced parasitologists.
The molecular identification of a determined species can help to confirm previous studies based on morphological features. In addition, they could also bring more

Discussion
We report P. alata in a booted eagle from Portugal. Booted eagles live in South Africa and migrate to the Iberian Peninsula and other Mediterranean regions to breed. Nevertheless, they are scarcely found in Europe [29]. A study found P. alata in one booted eagle from Spain in 1993 [1]. To our knowledge, this is the only European record that precedes this present report.
Besides booted eagle, P. alata has been recorded in peregrine falcons [3], Asiatic sparrowhawks (Accipiler nisus) [26], and sharp-skinned hawks (Accipiter velox) [26]. In Portugal, P. alata has been reported in birds of the orders Falconiformes and Strigiformes [30]. The present study is the first report in a Portuguese booted eagle.
Despite the available information, most of these previous studies were restricted to sole reports of parasite presence in a certain species, with small sample sizes [1][2][3][4][13][14][15]18,31,32]. In these studies, parasite species differentiation was based on morphological features only.
Morphological traits are a pivotal and important tool for taxonomic classification; however, they can be difficult to recognize for non-experienced parasitologists.
The molecular identification of a determined species can help to confirm previous studies based on morphological features. In addition, they could also bring more information into the evolutionary relationships of different organisms. To our knowledge, only one study in nematodes from birds of prey has used genetic data to identify nematode species, including one Physaloptera sp. [3]. Currently, phylogenetic classifications of Physaloptera spp. in birds have been studied using the SSU 18S rRNA gene [3] and the cox1 gene [20,30].
Honisch and Krone [3] used the SSU 18S rRNA gene to further study the phylogenetic relationships of Spirurina found in raptors; the authors produced SSU 18S rRNA partial sequences of one Physaloptera nematode: P. apivori (accession number: EU004817). Additionally, they used an unpublished sequence identified as P. alata (accession number: AY702703, found in a peregrine falcon) in their study.
During the phylogenetic analysis, the sequence generated in our investigation was found to be closely related to the P. alata sequence (AY702703). However, these data come from unpublished results and no morphological identification of the specimen was available. Additionally, not all of the Physaloptera spp. reported in birds of prey have been sequenced. This is why our combination of morphologic evaluation and phylogenetic analysis of a female and a male specimen was pivotal for the identification and confirmation of P. alata as the species found in this eagle.
To our knowledge, no cox1 sequence from the Physaloptera worms of raptors was available for comparison. This only allowed the identification of the specimen to the genus level. A similar challenge was found by Kalyanasundaram et al. [33], who solely used the cox1 gene to determine the species of a spirurid nematode detected on bobwhites. However, since there are limited resources in the gene database of Spirudia cox1, the authors could only identify the bobwhite parasite as Physaloptera sp. With this study, we provided the first P. alata partial sequence from raptors for the cox1 gene, with the hope that future samples join ours and a larger database can be built.
Raptor research faces a considerable number of challenges, which makes conservation efforts, including health monitoring, difficult [34]. Given their pivotal role as top predators in the ecosystem, it is necessary to increase measures in order to elucidate raptor health. In the particular case of the booted eagle, although they migrate to southern Europe to breed each year, they are poorly studied and rarely found [29]. The lack of reports of parasites infecting these birds is most likely due to the scarcity of samples and specimens to study. Animals that are legally found and sent to rehabilitation centers, such as in this case, represent a unique opportunity to collect data about raptor health status and their parasitic fauna.

Conclusions
The present study is a small but necessary contribution to current efforts aimed at elucidating raptor health. Additionally, these results add information to the current database of Physaloptera spp. in these birds of prey. Finally, we encourage wildlife professionals, veterinarians, parasitologists, and ecologists to report and communicate similar cases.