Microbothrium cirrhigalei, Ota, 2025

Microbothrium cirrhigalei n. sp.

( Fig. 3 View Fig ) [New Japanese name: Hige-tsuno-zame-kagi-nashi-hadamushi]

Holotype. MPM Coll.-No. 25320.

Paratypes. Four specimens ( MPM Coll.-No. 25321), 6 specimens ( KMNH IvR 600084–600089), and 4 specimens (KAUM-AT-3318) .

Description. Body lanceolate, 2041–2717 (2361; n = 15) long including haptor, 931–1292 (1144; n = 15) wide at anterior margin of testis. Haptor muscular sucker to accommodate shape of host dermal denticle, without sclerotized armature, attached to tip of dermal denticle of host, 240–359 (301; n = 15) long, 275–419 (352; n = 15) wide. Eyes absent. Mouth opening at terminal of anterior body; two protrusions on anterior side of mouth; prepharynx sometimes associated with small buccal cavity, surrounded by anterior glands on each side; pharynx pyriform, 147–283 (227; n = 15) long, 190–347 (281; n = 15) wide; esophagus short or absent; bifurcate intestinal cecum with numerous diverticula leading laterally, not confluent posteriorly.

Testis subspherical, intercecal, midline at top of lower body, 357–641 (537; n = 15) long, 402–653 (519; n = 15) wide. Vas deferens exiting from anterior margin of testis, immediately swelling to form seminal vesicle at anterior of testis, extending anteriorly on dorsal side, curving along penis, and entering ejaculatory bulb. Ejaculatory bulb weakly muscular, spherical to oval. Sclerotized tube extending from left side of ejaculatory bulb, turning three times (n = 14), entering base of penis, and extending to penis tip, 838–1149 (980; n = 14) long. Penis weakly muscular, tapered, 150–218 (185; n = 15) long along the curve.

Germarium spherical to ovoid, intercecal, slightly right side at mid-body level, 107–230 (157; n = 15) long, 118– 189 (149; n = 15) wide. Oviduct arising from anterior part of germarium and receiving ducts from vitelline reservoir and seminal receptacle. Ovovitelline duct extending anteriorly, connecting with oötype base. Oötype with thick wall, rounded, opening ventrally at unarmed common genital pore located in upper quarter of body. Vaginal pore muscular, unarmed, on ventral surface between left intestinal cecum and middle of oötype at upper third of oötype. Vaginal tube not muscular, extending downward, turning right, anterior to seminal vesicle, running along anterior margin of seminal vesicle, and connecting to seminal receptacle. Seminal receptacle midbody, left of germarium. Egg ovate, with short filament, 107–138 (123; n = 4) long, 72–107 (85; n = 4) wide without filaments, in oötype.

Type host. Mandarin dogfish, Cirrhigaleus barbifer Tanaka, 1912 ( Squaliformes: Squalidae ).

Type locality. Oita Marine Palace Aquarium (Umitamago). The other compatible fishes with C. barbifer in the tank had no infected any microbothriid during regular health check-ups; thus, the ancestors of type specimens were likely derived from the East China Sea off the Danjo Islands at 140 m depth, Higashi-Hama-machi, Goto City, Nagasaki Prefecture.

Sites of infection. Body surface (dermal denticles).

Etymology. The specific name is derived from the generic name of the type host, Cirrhigaleus barbifer . The new Japanese name, “hige-tsuno-zame” refers to the host in Japanese, and “kagi-nashi-hada-mushi” means microbothriid.

Representative DNA sequences. INSDC accession numbers: LC805887 (28S rDNA, 1297 bp), LC805888 (18S rDNA, 1860 bp), LC805889 ( ITS1 - 5.8 S-ITS2, 914 bp), and LC805890 (cox1, 426 bp) . Each sequence obtained from two paratypes ( MPM Coll.-No. 25321) were identical .

Remarks. Microbothrium cirrhigalei n. sp. was assigned to the genus because of the presence of defined genus characteristics (see Nitta and Nagasawa 2017; Remarks of M. myzolepis n. sp.). The new species is distinguished from M. lepidorhini by the vaginal pore positioned on the left side of the ventral body (vs. right in M. lepidorhini : Guiart 1938; Brinkmann 1940, 1952a). Microbothrium cirrhigalei n. sp. readily separated from M. tolloi by the vagina is devoid of sclerotization (vs. sclerotized in M. tolloi : Brinkmann 1952b). The sclerotized tube associated with the penis of the new species completes three turns, but those of its congeners turn zero to two times ( Table 3 View Table 3 ). Furthermore, the vagina of M. cirrhigalei n. sp. is not muscular (only the vaginal opening is muscular), while those of M. apiculatum and M. myzolepis n. sp. are muscular ( Saint-Remy 1891; MacCallum 1926a, b; Price 1938; Table 3 View Table 3 ).

Molecular data comparison

The genetic distance between the 18S rDNA sequences of M. myzolepis n. sp. and M. cirrhigalei n. sp. was 0.7%, M. myzolepis n. sp. and Leptocotyle minor (Monticelli, 1888) was 5.8%, M. myzolepis n. sp. and H. japonica was 8.7%, M. cirrhigalei n. sp. and L. minor was 5.8%, and M. cirrhigalei n. sp. and H. japonica was 8.5% (1852 bp). The genetic distance between the 28S rDNA sequences of M. myzolepis n. sp. and M. cirrhigalei n. sp. was 2.1% (1261 bp). The genetic distances of ITS1-5.8S-ITS2 and cox1 sequences between M. myzolepis n. sp. and M. cirrhigalei n. sp. were 4.8% and 20.1%, respectively; those between M. myzolepis n. sp. and H. japonica were 49.3% and 28.6%, respectively; and those between M. cirrhigalei n. sp. and H. japonica were 50.7% and 30.7% (819 bp and 387 bp), respectively. The pairwise sequence divergences among the microbothriid species for the 18S rDNA, 28S rDNA, ITS1-5.8S-ITS2, and cox1 sequences are summarized in Tables 4–6 View Table 4 View Table 5 View Table 6 .

Phylogenetic analysis

The phylogenetic trees based on partial 28S rDNA sequences are shown in Fig. 4 View Fig . In accordance with the findings of Perkins et al. (2009) and Nitta and Nagasawa (2017), the tree utilized Calicotyle japonica Kitamura, Ogawa, Shimizu, Kurashima, Mano, Taniuchi, and Hirose, 2010 ( Monocotylidae ) as an outgroup. It is noteworthy that the topologies of the trees constructed through both ML and BI analyses exhibited congruence.

Phylogenetic trees illustrated a distinct separation within the microbothriid lineage, resulting in the formation of two major clades. The first major clade encompassed species from Dermopristis Kearn, Whittington, and Evans-Gowing, 2010 , Dermophthirius MacCallum, 1926 , and Haplocotyle Nitta and Nagasawa, 2017 , with a notable affinity between the species of the former two genera. The second major clade includes Pseudoleptobothrium Young, 1967 , Asthenocotyle Robinson, 1961 , Leptocotyle Monticelli, 1905 , and Microbothrium . Microbothrium myzolepis n. sp. and M. cirrhigalei n. sp. were placed in the same clade.