New Data on Pterygodermatites (Pterygodermatites) plagiostoma Wedl, 1861 (Nematoda, Rictulariidae) Parasite of the Algerian Hedgehog Atelerix algirus Linnaeus, 1758 (Eulipotyphla: Erinaceidae) from the Canary Islands

Simple Summary A redescription of Pterygodermatites (Pterygodermatites) plagiostoma (Nematoda, Rictulariidae) is made by means of light and scanning electron microscopy, including the first data on male specimens. The morphologic study was based on specimens recovered from two Algerian hedgehogs from Tenerife and Gran Canaria islands (Canary Archipelago, Spain). The main differential characteristics of P. (P.) plagiostoma males are the number of cuticular projection pairs, the number of precloacal fans, and the size of spicules. The cloacal papillae are arranged according to the Ascaridida type, with two precloacal pairs, an unpaired precloacal papilla, one pair lateral to the cloaca, six postcloacal pairs, and a pair of phasmids. Females are mainly characterized and differentiated by the number of prevulvar pairs of cuticular projections, by the total number of cuticular projection pairs, by the level of differentiation from combs to spines and by the position of the vulva in relation to the esophagus–intestine junction. The comparison with species of the subgenus P. (Pterygodermatites) shows P. (P.) plagiostoma as a species clearly differentiated from the remaining species of this subgenus. Abstract A redescription of the rictulariid nematode Pterygodermatites (Pterygodermatites) plagiostoma Wedl, 1861, is made by means of light and scanning electron microscopy, including the first data on male specimens. The morphologic study was based on specimens recovered from two Algerian hedgehogs (Atelerix algirus) from Tenerife and Gran Canaria islands (Canary Archipelago, Spain). The main characteristics of P. (P.) plagiostoma males are the presence of 49–53 pairs of cuticular projections, the presence of one or two midventral precloacal fans (generally one), and the size of two unequal spicules, measuring 98–123 µm (right spicule) and 185–236 µm (left spicule). The cloacal papillae are arranged according to the Ascaridida type. They include two precloacal pairs, an unpaired precloacal papilla, one pair lateral to the cloaca, six postcloacal pairs, and a pair of phasmids near the tail tip. Females are mainly characterized by the presence of 71–77 pairs of cuticular projections, with 43–46 pairs of prevulvar combs, by the differentiation from combs to spines at the level of or slightly posterior to the vulva and by the position of the vulva, located posteriorly to the esophagus–intestine junction. Clear differences were found between P. (P.) plagiostoma and related species of the subgenus P. (Pterygodermatites).

The aim of the present study is to describe for the first time the male of P. (P.) plagiostoma and contribute with new data on females from a hedgehog A. algirus from the Canary Islands. In addition, the sequence of the mitochondrial cytochrome c oxidase subunit I gene (MT-CO1) of P. (P.) plagiostoma is provided.

Specimens
Two Atelerix algirus Linnaeus, 1758 (Eulipotyphla: Erinaceidae), were found dead on the road in El Rosario (Tenerife Island) on 3 June 2021 and in Jinámar (Gran Canaria Island) (Canary Archipelago, Spain) on 9 October 2019 and then they were scanned for intestinal helminths. A total of 149 rictulariid nematodes (38 males and 111 females) were found in the gastrointestinal tract of the two hedgehogs. They were identified as P. (P.) plagiostoma following the available literature [7,13].

Scanning Electron Microscopy Study
Six P. (P.) plagiostoma males and eight females were preserved for scanning electron microscopy (SEM) examination, fixed in 70% ethanol and posteriorly dehydrated in an ethanol series and critical point dried with carbon dioxide in an Emitech K850X (Quorum Technologies Ltd., Laughton, East Sussex, UK). Finally, specimens were mounted on stubs with conductive adhesive tape and colloidal silver, coated with carbon in an Emitech K950X (Quorum Technologies Ltd., Laughton, East Sussex, UK) evaporator, and examined using a Field Emission SEM JSM-7001F (Jeol) (Jeol Ltd., Tokyo, Japan) at 10 kV in the "Centres Científics i Tecnològics" of the University of Barcelona (CCiTUB).
DNA amplification by PCR was conducted using the primer cocktail as described by Prosser et al. [21], for the barcode region of the mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). The PCR amplification contained 1X Buffer (Bioline, London, UK), 0.2 mM of each dNTP (Bioline), 0.5 µL of each primer cocktail (10 µM of a three-forwardprimers mix, and 10 µM of a three-reverse-primers mix), 1U of Taq DNA polymerase (Bioline), 1.5 mM MgCl 2 (Bioline), and 20-30 ng of total genomic DNA in a total volume of 50 µL. Amplification was conducted with XP Cycler (Bioer Technology) using the following parameters: 94 • C for 1 min; five cycles at 94 • C for 40 s, 45 • C for 40 s, 72 • C for 1 min; followed by 35 cycles at 94 • C for 40 s, 51 • C for 40 s, 72 • C for 1 min; and a final extension at 72 • C for 5 min [21]. The resulting amplifications were visualized on 1.2% agarose gel at 100 V for 45 min.
The product of PCR was sequenced in Macrogen (Madrid, Spain) with primers NemF1_t1 and NemR1_t1 [21]. The analysis of the sequences was carried out with software MEGA X [22], using the multiple alignment program ClustalW included in MEGA X, and minor corrections were made by hand.
A phylogenetic analysis based on the MT-CO1 gene sequences of P. (P.) plagiostoma and other Pterygodermatites species available in GenBank was performed using Neighbor-Joining distance method (NJ) with the p-distance model [23] and Maximum-Likelihood (ML) method with Tamura-Nei model [24], both with at least 1000 bootstrap replications in MEGA X [22]. The sequence Plectus aquatilis KX017524 was used as the outgroup. Mitochondrial cytochrome c oxidase subunit I gene (MT-CO1) sequence: a fragment of 700 bp was obtained for the MT-CO1 of P. (P.) plagiostoma isolated from A. algirus in El Rosario, Tenerife. A 551 bp fragment was successfully sequenced and submitted to the GenBank database under the accession number ON502379.

Phylogenetic Tree
The phylogenetic trees created using NJ and ML ( Figure 1

Phylogenetic Tree
The phylogenetic trees created using NJ and ML ( Figure 1) methods based on the MT-CO1 gene showed similar results. P. (P.) plagiostoma is included in a clade together with P. (Paucipectines) zygodontomis and P. (Pa.) jägerskiöldi with a high bootstrap value (100%), and clearly separated from these two species. In the other clade P. (Mesopectines) whartoni and P. (Me.) nycticebi are included. Plectus aquatilis was used as the outgroup.

Discussion
Within the subgenus P. (Pterygodermatites) there are only six described species (see Table 1). Considering male specimens, there is data for only three species, namely P. (P.) aethechini, P. (P.) mexicana, and P. (P.) shaldybini [13,16,17]. In the original description of P. (P.) plagiostoma [8], there was no additional data on males other than a caudal extremity showing the presence of two unequal spicules. In the present study, for the first time, we extensively describe the male of P. (P.) plagiostoma.   [7] in Erinaceus europaeus from mainland Spain; 3 according to Jägerskiöld [3] in Erinaceus auritus lybicus (=Hemiechinus auritus) from Egypt.
In the Rictulariidae, the number of cloacal papillae of males is constant (19 papillae arranged in nine pairs of papillae and an unpaired papilla [13]). Thus, there are two precloacal pairs, an unpaired precloacal papilla, either another precloacal pair or a pair located laterally to the cloaca and six postcloacal pairs. Additionally, near the tail tip there is a pair of phasmids. Quentin [13] analyzed the disposition of male cloacal papillae in numerous rictulariids and described three types of arrangement of papillae: the type Ascaridida presenting some pairs of papillae not aligned and slightly dorsolateral, the type Spirurida with all pairs of papillae aligned, and a third type with pedunculated and grouped papillae. In males of P. (P.) plagiostoma, the cloacal papillae arrangement corresponds to the type Ascaridida [13,27] having the pairs 1, 4, and 8 in a dorsolateral position, particularly the pair 8. According to Quentin [13], the type Ascaridida, which corresponds to the most primitive arrangement of cloacal papillae, is present in rictulariids included in both genera Pterygodermatites and Rictularia, e.g., P.  [28][29][30][31]. With respect to the subgenus P. (Pterygodermatites), Le Roux [17] reported only three precloacal and five postcloacal pairs of cloacal papillae in P. (P.) aethechini. However, the original description of Le Roux was illustrated with a lateral view of the male caudal extremity showing probably the unpaired precloacal papilla. In a posterior study concerning P. (P.) mexicana, Caspeta-Mandujano et al. [16] described in five sublateral pairs of cloacal papillae (pairs 1, 2, 4, 8, and 9) and four subventral pairs (pairs 3, 5, 6, and 7) but no unpaired precloacal papilla are mentioned.
Within the P. (Pterygodermatites) subgenus, males present three characters that are useful to discriminate between species: the number of cuticular projection pairs (combs and spines), the number of midventral fans, and the size of spicules [13,19,32]. Considering the species of the subgenus P. (Pterygodermatites) for which information on males is available, there are differences in the male of P. (P.) plagiostoma in the characters spicules size, number of fans, and number of cuticular projection pairs (see Table 1). In fact, in comparison to P. (P.) aethechini [17], the spicule sizes, particularly the right spicule is 98-123 µm in P. (P.) plagiostoma vs. 118-132 µm in P. (P.) aethechini. Additionally, in comparison to P. Concerning females, the most discriminant characters between species are the prevulvar and total number of cuticular projection pairs, the body level where the transition from combs to spines occurs, and the position of the vulva in relation to the esophagus-intestine junction [13,19,32]. In fact, the differentiation of cuticular projections (from combs to spines) occurs at the level of the vulva in P. (P.) plagiostoma whereas it occurs at a posterior level in P. (P.) aethechini [17]. On the other hand, the vulva position is posterior to the esophagusintestine junction in P. (P.) plagiostoma whereas it is anterior in P. (P.) atlanticaensis [18]. There is also a higher number of both prevulvar and total pairs of cuticular projections (combs and spines) (43-46/71-77) in P. (P.) plagiostoma in comparison to the number of these characters (40/66) in P. (P.) mexicana [16] although the number of cuticular projection pairs in P. (P.) plagiostoma (71-77) is surpassed by the 84 pairs described in P. (P.) shaldybini [13]. However, none of these discriminant characters of females differ between P. (P.) plagiostoma and P. (P.) spinosa [13].

Conclusions
The present work on the rictulariid nematode P. (P.) plagiostoma contributes with the first morphoanatomical study of male specimens since its original description and provides further information on females. Moreover, the current finding in Tenerife and Gran Canaria islands (Canary Archipelago, Spain) enlarges the geographical distribution of this rictulariid.
The most useful characteristics to differentiate P. (P.) plagiostoma from the remaining species of the subgenus P. (Pterygodermatites) are the total number of pairs of cuticular projections, the size of spicules and the number of midventral fans for males, and the number of prevulvar and total cuticular projection pairs and the position of the vulva in relation to the esophagus-intestine junction for females. The parasitized hosts and the geographical distribution are also useful criteria. The arrangement of cloacal papillae in males of P. (P.) plagiostoma follows the type Ascaridida and this type seems to be a characteristic of the subgenera P. (Pterygodermatites), P. (Paucipectines), and P. (Neopaucipectines).
The analysis of available data on the species of P. (Pterygodermatites) emphasizes the need for more research, particularly in respect to male specimens, for which data are scarce or unknown for several species.