Staphylocystis clydesengeri, Makarikov, Arseny A. & Kinsella, John M., 2013

Makarikov, Arseny A. & Kinsella, John M., 2013, Morphological and molecular differentiation of Staphylocystis clydesengeri n. sp. (Cestoda, Hymenolepididae) from the vagrant shrew, Sorex vagrans (Soricomorpha, Soricidae), in North America, Zootaxa 3691 (3), pp. 389-400 : 391-396

Staphylocystis clydesengeri n. sp.

(Figures 1–3)

Site in the host: small intestine.

Type host: Sorex vagrans (Baird, 1857) ( Soricomorpha : Soricidae ).

Type locality: Pattee Canyon, Missoula County, Montana, USA (46°, 49” N, 113°, 56” W).

Other localities: vicinity of Spokane, Washington (exact coordinates not known).

Material deposited: Holotype: USNPC 106801 (labelled Sorex vagrans, Pattee Canyon, Missoula County, MT , 14 July 2011, coll. J.M. Kinsella), paratypes: USNPC 106802-106808 (labelled Sorex vagrans, Pattee Canyon, Missoula County, MT , coll. J.M. Kinsella, different dates between May of 1998 to August 2009). Voucher specimens: USNPC 106809 (labelled Sorex vagrans, Pattee Canyon, Missoula County, MT , coll. J.M. Kinsella, 13 August 2011), USNPC 106810 (labelled: vicinity of Spokane, WA, 27 July 2009, coll. V. Tkach).

Etymology: This species is named in honour of Clyde Senger who was the first to note the morphological differences between Staphylocystis schilleri and the form described here as a new species.

Description: Based on 11 stained mounted specimens and 7 scoleces cleared in Berlese’s medium. Strobila 17.5 mm (14.4–24; 19.5; 6) mm long, with maximum width 1320 (1320–1600; 1497; 4) mm at level of gravid proglottides. Strobila consisting of 190 (138–190; 169; 6) craspedote proglottides. Scolex wider than long, 166 × 360 (166–190 × 352–430; 175 × 382; 6), clearly distinct from neck. Scolex with protracted rostellum conical, scolex with retracted rostellum slightly flattened antero-posteriorly. Suckers very muscular, thick-walled, unarmed, rounded or slightly oval, 100 × 85 (100–122 × 85–106; 109 × 97; 7). Rostellar pouch 140 × 209 (130–160 × 209– 242; 143 × 232; 5), with muscular walls, its bottom reaches approximately middle of suckers. Rostellum very muscular, apex not invaginable 92 × 99 (88–96 × 99–118; 93 × 111; 7). Rostellum armed by a single crown of 37 – 42 (average 40; 7) hooks with strongly developed dorso-ventrally flattened guard. Hook number in holotype could not be counted precisely due to overlap of several hooks in lateral regions of crown. Hook length 42 (38–44; 41.5; 13), hook blade length 21(19–22; 21.4; 12), hook handle length 21 (19.5–23; 21.2; 12) and hook guard length 17 (16–18; 17.3; 12).

FIGURE. 1. Staphylocystis clydesengeri n. sp. A. Holotype, dorso-ventral view of scolex. B. Paratype, rostellar hooks in profile and view from posterior surface showing enlarged hook guard. C. Holotype, male mature proglottis. D. Holotype, hermaphroditic mature proglottis. E. Hermaphroditic mature proglottis of Staphylocystis schilleri from Sorex palustris . (scale bars: A = 100 µm; B = 20 µm; C–E = 250 µm).

FIGURE. 2. Staphylocystis clydesengeri n. sp. A. Paratype, genital ducts. B. Holotype, pregravid proglottis, showing uterus development. C. Paratype, gravid proglottis. D. Paratype, egg. E. Paratype, embryonic hooks (from left to right: median, antero-lateral, postero-lateral) (scale bars: A = 100; B, C = 250 µm; D = 20 µm; E = 10 µm).

FIGURE. 3. Rostellar hooks of S. schilleri and S. clydesengeri n. sp. A. Staphylocystis schilleri from Sorex palustris collected near Missoula, Montana. B, C. Paratype of S. clydesengeri n. sp. Note that all photographs are taken at the same magnification. (scale bars: A–C = 20 µm).

Osmoregulatory canals penetrate through rostellar pouch wall. Neck narrower than scolex. Ventral osmoregulatory canals 30 (28–40; 32; 7) at level of hermaphroditic proglottides, transverse anastomoses not observed. Dorsal osmoregulatory canals thin, 12 (9–12; 11.5; 7) at level of hermaphroditic proglottides, usually situated directly above ventral canals. Genital pores unilateral, dextral, genital ducts pass dorsally to both ventral and dorsal longitudinal osmoregulatory canals. Development of proglottides gradual, male and female gonads developing at approximately same rate. All segment in the strobila transversely elongated, but the length:width ratio increases from young to gravid proglottides.

Mature proglottides 110 × 782 (87–130 × 782–911; 110 × 846; 5), trapezoid (Fig. 3D). Testes relatively small, three, spherical or somewhat elongated, situated in a relatively tight triangle, one poral and two antiporal. Poral testis 51 × 57 (50–58 × 57–102; 54 × 90; 5), middle testis 51 × 67 (50–58 × 67–103; 54 × 92; 5), aporal testis 48 × 57 (48–66 × 57 – 110; 56 × 89; 5). Cirrus pouch 148 × 38 (135–168 × 37–47; 151 × 41; 9) in hermaphroditic proglottides, normally only reaching or somewhat overlapping poral osmoregulatory canals, but not crossing them. Genital atrium simple, infundibular, deep, opens laterally approximately at level of border between first and second thirds of lateral proglottis margin. Evaginated cirrus not observed. Internal seminal vesicle elongate, 103 × 33 (91– 103 × 33–42; 96 × 36; 8), occupying more than half of cirrus pouch length. External seminal vesicle ovoid, 90 × 38 (90–130 × 38–66; 106 × 50).

Ovary 90 – 296 (72–95 × 272–360; 83 × 318; 5) wide, median, usually consisting of three large lobes, sometimes with secondary smaller lobes, ventral to male genital organs, occupying half to two-thirds of median field, usually overlapping testes. Vitellarium irregularly shaped, sometimes lobed 47 × 82 (40–61 × 82–132; 52 × 107; 7), postovarian, median. Copulatory part of vagina not clearly distinct from seminal receptacle; ventral to cirrus pouch. Seminal receptacle elongated, 250 long (178–260 × 54–80 × 63; 5), was not filled with sperm in mature hermaphroditic proglottides of holotype, but was filled in gravid proglottides. In majority of other specimens seminal receptacle was filled in mature hermaphroditic proglottides.

Uterus first appears as transversely elongated sac, not extending beyond osmoregulatory canals, situated dorsally to other organs. With proglottis development, uterus grows and forms numerous lateral pockets and dorsoventral diverticula. Testes and vitellarium persist in postmature proglottides; cirrus pouch and vagina persist in gravid proglottides. Gravid proglottides 340 × 1286 (340–420 × 1286–1490; 382 × 1395). Fully developed uterus labyrinthine, extending into both lateral fields, saccate; walls of gravid uterus usually deeply folded or invaginated sometimes creating impression of being perforated. Uterus contains numerous small eggs. Eggs 40 – 44 × 32 – 39 (42 × 35; 8), spherical or subspherical; embryophore thin, without polar filaments, close to surface of oncosphere; oncosphere 22 – 28 × 18 – 24 (25 × 22; 8). Median embryonic hooks 16, antero-lateral and postero-lateral hooks 14.5–15. Antero-lateral embryonic hooks much more robust than slender postero-lateral and median hooks.

Remarks: Morphological differentiation. The only other Staphylocystis species previously reported in North America is S. schilleri originally described from Sorex cinereus in Wisconsin (Rausch & Kuns 1950). The two species share general morphological characteristics such as the hook shape, overall scolex and strobila anatomy including triangular arrangement of testes and gravid uterus expanding into lateral fields of proglottides. But S. clydesengeri n. sp. differs from S. schilleri in several significant morphological features. The two species can be most easily distinguished based on the size and number of their rostellar hooks. Staphylocystis clydesengeri has 37 – 42 (average 40) hooks 39 – 44 µm (average 40.7 µm) long while S. schilleri has 22 hooks 27 – 30 µm long according to the original description. In our material of S. schilleri from Montana the hook number was higher (28 – 30, average 29). We could not count hooks in the holotype of S. schilleri very precisely due to their dense arrangement in the crown, but it looked like the number 22 is correct or at least very close to the real situation. In our experience, counting rostellar hooks in the laterally positioned crowns of Staphylocystis on total mounts usually results in underestimations of their numbers comparing with the crowns of the same species from the same sample cleared and apically oriented (or squished) in Berlese’s medium. The cirrus pouch in mature (hermaphroditic) proglottides of S. schilleri distinctly crosses the poral ventral osmoregulatory canal. In S. clydesengeri the cirrus pouch only barely reaches or, rarely, slightly crosses the poral ventral osmoregulatory canal. Gonads in S. clydesengeri occupy the central part of the middle field of the proglottis and do not reach the canals on either side. In fully mature proglottides of S. schilleri gonads normally fill the middle field of the proglottis entirely and reach or even overlap osmoregulatory canals on either side of the proglottis. Mature proglottides in heat-killed relaxed specimens of the two species distinctly differ in shape. In the new species proglottides are relatively shorter than in S. schilleri (Fig. 1 D, E). Finally, the two species differ in the early development of the uterus and its extent in gravid proglottides. In the new species the early uterus has numerous pockets and chambers from the very beginning while in S. schilleri the uterus at is initially formed as a two-winged saccular structure. The fully developed uterus in the new species extends well into the lateral fields while in S. schilleri it is confined to the median field of the proglottides and may only somewhat overlap the osmoregulatory canals on either side.

Staphylocystis clydesengeri clearly differs from all Palearctic species parasitic in Sorex . While there are multiple characters separating these species, the rostellar hook number and size are discriminative enough, therefore we are not providing other differentiating features. The new species has 37 – 42 hooks 39 – 44 µm long while S. furcata has 23 – 30 hooks 23 – 27 µm long (Vaucher 1971, Genov 1984), S. sibirica has 28 – 30 hooks 32 – 35 µm long (Morozov 1957, Gulyaev & Shakhmatova 1990) and S. amurensis has 34 hooks 19 – 22 µm long (Karpenko 1984, 2004).

Vampirolepis novosibirskiensis Sawada & Kobayashi, 1994 described from Sorex araneus , Sorex caecutiens and Sorex minutus in the Novosibirsk region and Altai Krai, Russian Federation (Sawada & Kobayashi 1994), clearly belongs to Staphylocystis . By the combination of morphological characteristics such as rostellar hook shape, size and number, organization of mature segments and other features, it is morphologically indistinguishable from Staphylocystis furcata . Therefore, we consider V. novosibirskiensis a junior synonym of S. furcata .

We do not provide here differentiation of the new species from Staphylocystis spp. parasitic in crocidurine shrews because of the very high level of specificity among shrew hymenolepidids to the host genus (see the detailed discussion below). These two groups of shrews do not share their cestodes even when they occur in the same habitat, let alone on different continents. In the absence of any crocidurine shrews in North America we are certain that our specimens described herein as a new species cannot belong to one of the numerous Staphylocystis species known from Crocidura and related genera. In addition, Staphylocystis from the two groups of shrews deminstrate a high level of genetic divergence (see the Molecular differentiation below).

Molecular differentiation. The aligned trimmed sequenced rRNA fragments from 5 specimens of S. clydesengeri (sequence length 2856 base pairs), one S. schilleri (2866 base pairs) and one S. furcata (2862 base pairs) comprised a short portion of the 18S gene, complete ITS1 spacer, complete 5.8S gene, complete ITS2 spacer, and partial 28S gene. Due to introduced gaps the length of pairwise alignments between the three species varied from 2862 to 2872 base pairs. No intraspecific variability was detected among 4 replicates of S. clydesengeri . The two North American species S. clydesengeri and S. schilleri differed in only 3 bases while both these species differed from the Palaearctic S. furcata in 7 bases.

Sequences of mitochondrial genes nad1 and 16S provided further convincing evidence of the status of S. clydesengeri as a new species. The nad1 alignment of 4 sequences of S. clydesengeri (2 from Montana and 2 from Washington), one S. schilleri and one S. furcata was 663 bases long and had no gaps. Among 4 sequences of the new species only one specimen from Montana had a single base substitution. Results of pairwise comparisons are presented in Table 1 . Staphylocystis clydesengeri and S. schilleri differed in 33 bases while the two North American species differed from S. furcata in 45 – 49 positions ( Table 1 ).

The 16S alignment of 3 sequences of S. clydesengeri (2 from Montana and one from Washington), one S. schilleri and one S. furcata was 452 bases long and had no gaps. No intraspecific variability was detected in 16S sequences of 4 sequenced specimens of S. clydesengeri . Results of pairwise comparisons are presented in Table 1 . Staphylocystis clydesengeri and S. schilleri differed in 11 bases while the two North American species differed from S. furcata in 11 – 16 positions ( Table 1 ). Thus, molecular data strongly support the status of S. clydesengeri as a new species.

We do not provide a detailed molecular differentiation between S. clydesengeri and members of the genus known from crocidurine shrews because the new species shows much greater levels of interspecific sequence divergence from Staphylocystis parasitic in crocidurine shrews than from Staphylocystis parasitic in Sorex . For instance, the approximately 1400 base pair long sequence of the nuclear ribosomal 28S gene of S. clydesengeri obtained in the present study was identical to that of S. furcata collected in the Ukraine. In the same DNA region, S. clydesengeri had 41 nucleotide differences from Staphylocystis brusatae (Vaucher, 1971) collected from lesser white-toothed shrews Crocidura suaveolens Pallas in the Ukraine (GenBank JQ 260805 ) and in 29 nucleotides from Staphylocystis sp. collected from Asian house shrews Suncus murinus Linnaeus in Thailand (V. Tkach, unpublished data). This provides additional evidence of the significant differences between Staphylocystis from soricine and crocidurine shrews.

Tables

TABLE 1. Number of variable sites with (%) based on pairwise comparison of 663 base-pair long fragment of mitochondrial nad 1 gene (above diagonal), and number of variable sites with (%) based on pairwise comparison of 542 base-pair long fragment of mitochondrial 16 S gene (below diagonal).

Cestode species	S. clydesengeri n. sp.	S. schilleri	S. furcata
S. clydesengeri n. sp.	--------	33 (5.0%)	49 (7.4%)
S. schilleri	11 (2.4%)	--------	45 (6.8%)
S. furcata	16 (3.5%)	11 (2.4%)	---------