Proteocephalid tapeworms (Cestoda: Onchoproteocephalidea) of loaches (Cobitoidea): Evidence for monophyly and high endemism of parasites in the Far East Author Tomáš Scholz Author Alain de Chambrier Author Takeshi Shimazu Author Alexey Ermolenko Author Andrea Waeschenbach text Parasitology International 2017 66 871 883 journal article http://dx.doi.org/10.1016/j.parint.2016.09.0161383-5769 ea0932c3-01e6-4c74-90a2-4d1999acbdaf 886409 3.2.3. Proteocephalus demshini n. sp. Figs. 4 , 6 Description (based on 5 whole-mounted specimens and 2 scoleces studied using SEM; for morphometrical data — see Table 2 ): Proteocephalidae, Proteocephalinae. Testes , vitelline follicles, ovary and uterus medullary. Medium-sized tapeworm, body length 18– 33 mm (n = 5), maximum width up to 1.1 mm (n = 5). Strobila acraspedote, anapolytic, consisting of about 80–100 proglottids: 55–60 immature, 1 mature, 16–24 pregravid and 6–13 gravid proglottids. Immature, mature and pregravid proglottids wider than long, gravid longer than wide. Table 2 Comparative measurements of species of Proteocephalus (Cestoda: Onchoproteocephalidea) from loaches (Cobitoidea) in the Palaearctic region.Measurements in micrometres unless oth- erwise stated.

Species	P. sagittus a	P. demshini n. sp.	P. midoriensis b	P. misgurni n. sp.
Authors	Grimm, 1872	This paper	Shimazu, 1990	This paper
Hosts	Barbatula barbatula	Barbatula toni (Dybowski,	Lefua echigonia Jordan &	Misgurnus anguilli –caudatus
(Linnaeus, 1758)	1869)	Richardson, 1907	(Cantor, 1842)
Total length	Up to 43 mm	18–33 mm (n = 5)	9–17 mm	> 33 mm
Width of proglottids (WP)	1.37 mm	1.09 mm (n = 5)	370	530
Width of scolex	324–675	420–445 (n = 2)	285–310	425–535 (n = 4)
Diameter of suckers	122–244	170–190 (n = 4)	115–140	1 95–285 (n = 16)
Ratio of width of suckers to width	32–50%	40–44% (n = 8)	45–47%	42–49% (n = 16)
of scolex
Width of neck	240–370	360	215–300	300–470
Number of testes	41–94	62–89 (x = 78, n = 9)	32–52	32–44 (x = 38, n = 8)
Length of testes	45–60 × 35–40	45–60 × 45–50 (n = 15)	30–60	35–55
Size of cirrus-sac (CS)	100–172 × 53–107	110–145 × 50–60	140–220 × 60–70	80–105 × 60–85
Ratio of length of CS/WP	16–35%	15–25% (x = 19, n = 14)	20–27%	19–32% (x = 25%, n = 12)
Ratio of CS length/width	36–41%	37–50% (x = 44%, n = 9)	57–85% (x = 70%)	75–93% (x = 83%, n = 10)
Length of cirrus/CS length Relative position of genital pore c Relative width of ovary d Relative length of ovary e Ovary superficie f	65–83% 33–54% (x = 43%) 72–83% 23–30% (x = 26%, n = 12) 13.2%	Up to 65% 27–45% (x = 33%, n = 10) 76–83% (x = 79%, n = 13) 26–41% (x = 34%, n = 26) 16.6%	< 50% 41–50% 75–82% 27–33% (x = 30%, n = 20) 16.5%	<35% 28–41% (x = 35%, n = 13) 74–88% (x = 79%, n = 13) 24–41% (x = 32%, n = 20) 14.6%
Part of ovary overlapped by	17–21%	0–9%	39–52%	16–20%
vitelline follicles (VF) Relative size of Mehlis gland g	13–13% (x = 14%, n = 8)	14–23% (x = 17%, n = 12)	19–29% (x = 23%)	17–25% (x = 21%)
Position of vagina to CS	Anterodorsal or anterior	Anterior (n = 84)	Anterior	Anterior (n = 57)
Ratio of width of band of VF to	8–14% (x = 10%, n = 17)	15–19% (x = 17%, n = 8)	19–22%	10–12% (n = 2)
width of proglottid
Ratio of length of poral band of VF	72–81% (x = 74%, n = 9)	71–83% (x = 77%, n = 13)	69–77% (x = 74%, n = 6)	65–71% (x = 68%, n = 11)
to length of proglottid
Ratio of length of aporal band of	69–81% (x = 74%, n = 8)	68–86% (x = 81%, n = 13)	68–83% (x = 74%, n = 6)	67–74% (x = 72%, n = 11)
VF to length of proglottid
Uterine branches	9–14 on each side	3–8 on each side	9–12 on each side	6–11 on each side
Diameter of embryophore	16–27	23–25	26–29	25–29
Diametrer of oncosphere	12–20	15–16	21–22	15–17
Length of embryonic hooks	N/A	5–7	8	8–10

N/A — not available. Measurements that differentiate sister species from each other, i.e. P.demshini n.sp. from P.sagittus , and P.misgurni from P.midoriensis , respectively, are highlighted by bold. a Data from Scholz et al. [13] supplemented by new measurements. b Data from Shimazu [19] supplemented by new measurements. c Ratio of distance of the genital pore from the anterior margin of the proglottid to its length. d Ratio of width of the ovary to the width of the proglottid. e Ratio of the length of the ovary to the length of the proglottid. f Ratio of the surface of the ovary to the surface of the proglottid (see de Chambrier et al., 2012 for method of calculation). g Ratio of the length of Mehlis'gland to the length of the proglottid. Scolex wider than neck. Suckers spherical, with deep cavity ( Fig.4 A ). Apical organ absent; apex of scolex and region posteromedian to suckers with gland cells ( Fig. 4 A ). Neck (proliferation zone) 360 wide. Scolex covered with acicular filitriches and gladiate spinitriches (on suckers — Fig. 6 F ) and acicular filitriches (between suckers and on scolex apex); neck covered with gladiate spinitriches.Inner longitudinal internal musculature developed, anastomosed. Ventral osmoregulatory canals thin-walled, 15–25 wide, ventral to lateral bands of vitelline follicles ( Fig. 4 C ). Dorsal osmoregulatory canals thick-walled, narrow, about 5–8 wide. Testes 62–89 in number, spherical, in two irregular layers, form one compact field between lateral bands of vitelline follicles and ovary ( Fig. 4 B ), present also in gravid proglottids. Vas deferens strongly coiled, with loops forming elongated field crossing median line of proglottid ( Fig. 4 B ). Cirrus-sac pyriform to elongate ( Fig. 4 C), thick-walled. Sperm duct (internal vas deferens) coiled. Cirrus muscular. Common genital atrium narrow, deep ( Fig. 4 C). Genital pores alternating irregularly, pre-equatorial ( Fig. 4 B ). Ovary medullary, bilobed, with long and narrow lateral wings. Relative size of ovary, i.e. its surface in relation to surface of pregravid and gravid proglottids (see [33]), about 17%. Mehlis' gland 85–160 in diameter. Vaginal canal almost straight in distal part, with terminal part ( pars copulatrix vaginae ) surrounded by chromophilic cells; vaginal sphincter absent ( Fig. 4 C ). Vitelline follicles medullary, forming two wide lateral bands interrupted ventrally on poral side at level of cirrus-sac and vagina; bands widened posteriorly and reaching to anterior margin of ovary only, rarely with few follicles slightly (less than by 10%) overlapping ovarian wings on ventral side ( Fig. 4 B ). Uterus medullary, with development of type 2 [9]. Lumen of uterine stem occupies up to 49% of proglottid width in last immature proglottids. In mature proglottids, lumen occupies up to 55% of proglottid width; apex lined with numerous chromophilic cells ( Fig. 4 C ). In gravid proglottids, uterine diverticula occupy up to 90% of proglottid width. Eggs spherical, with hyaline outer envelope and bilayered embryophore; oncosphere containing embryonic hooks ( Fig. 4 D). Taxonomic summary Type and known only host : Barbatula toni (Dybowski, 1869 ) ( Cypriniformes : Nemacheilidae ). Site of infection : intestine. Type locality : River Komissarovka near Kamen-Rybolov, Primorsky Region, Russia (44̊57′22″ N , 131̊42′0 0″ E ). Prevalence and intensity of infection ( mean with range and number of fi sh examined in parentheses ): 19% (n = 32); 1.7 (1–4). Deposition of specimens : Holotype (one whole-mounted specimen from host field No. RUS 190 collected on 28 June 2011 ; Coll. No. MHNG-PLAT 79145); three paratypes (whole-mounted specimens from hosts field Nos. RUS 191, 194a and 195 collected on 28 June 2011 ; IPCAS C-727; MHNG-PLAT 79150); one voucher (wholemounted specimen from host field Nos. RUS 190 collected on 28 June 2011 ; Coll. No. IPCAS C-727); 2 scoleces from host field No. RUS 192 (MHNG-PLAT 79149) used for SEM observations. Fig. 3. Proteocephalus midoriensis Shimazu, 1990 from Lefua echigonia , Japan (IPCAS C-362/1) (A, C–E) and from Lefua costata , Russia (“ Proteocephalus sp. n. ” of N.I. Demshin; not accessioned) (B). A, B. Scoleces. C. Mature proglottid, dorsal view, note vitelline follicles overpassing the ovary ventrally; uterus not illustrated. D. Detail of the posterior part of the proglottid, ventral view. E. Detail of terminal genitalia, ventral view. Etymology : The species is named in honour of the late N . I . Demshin, Vladivostok, Russia , for his extraordinary contribution to helminthology in the Russian Far East, especially life-cycles of cestodes. Differential diagnosis. The new species differs from P . midoriensis and P . misgurni from M . anguillicaudatus (see below) by the shape of the proglottids and the cirrus-sac, width of the lateral bands of the vitelline follicles and their posterior extent (see Remarks on P . midoriensis and compare Figs. 3–5 ). Proteocephalus demshini is strikingly similar to P . sagittus from the congeneric host and a few other loaches in its morphology including the shape of proglottids, which are wider than long except for the last gravid proglottids, wide bands of the vitelline follicles widened posteriorly, and an elongate to pyriform cirrus-sac. These two species can be distinguished from each other by (i) the number of uterine diverticles on each side, 3–8 in P . demshini versus 9–14 in P . sagittus ; (ii) the ratio of the width of the band of the vitelline follicles at the level of the ovary in relation to the width of the proglottid, which is 15–19% (x = 17%) in the new species versus 8–14% in P . sagittus (x = 10%); (iii) shape of the anterior end of the body, with the neck region widened anteriorly in P . demshini versus of the same width in P .sagittus ; and (iv) the posterior extent of the vitelline follicles, which overlap the ovary only slightly in P . demshini (up to 1/10 of the length of the ovary) versus up to 1/ 5 in P . sagittus ( Table 2 ). Fig. 4. Proteocephalus demshini n. sp. from Barbatula toni , Russia. A.Scolex (host field No. RUS 195; IPCAS C-727). B. Mature proglottid, ventral view (host field No.RUS 191; IPCAS C-727); uterus not illustrated. C. Detail of terminal genitalia, ventral view (host field No. RUS 192; MHNG-PLAT 79149). D. Eggs drawn in distilled water (host field No. RUS 190; IPCAS C-727). Discrete species status of these apparent sibling species is supported by molecular data (see Fig. 1 and Suppl. Figs. 2 and 4 ); uncorrected pdistances are 0.5% and 9.9% for lsrDNA and cox1 , respectively (Suppl. Table 1 ). The type host of this new species, B . toni , occurs in Asia and lives in all rivers flowing to the Arctic and Pacific Oceans between the rivers Ob and Huang He [1]. Its helminth fauna is relatively depauperate [3, 34–36], but includes also three species of cestodes, yet none of them being specific to, or typical of, this fish. Scholz and Ergens ( 1990 ) [37] reported juveniles of a species of Proteocephalus that possessed a vestigial muscular apical sucker, thus apparently not belonging to P . demshini , which is devoid of an apical sucker. T .Shimazu (unpubl. data) examined 100 specimens of B . toni from Hokkaido in 1984 and 2011 but no proteocephalidean cestodes were found, which indicates that P . demshini is endemic to the Primorsky Region of Russia .