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Echinobothrium joshuai n. sp. (Cestoda: Diphyllidea) from theRoughnose Legskate, Cruriraja hulleyi (Rajiformes: Rajidae),off South AfricaAuthor(s): Natasha Rodriguez, Maria Pickering, and Janine N. CairaSource: Comparative Parasitology, 78(2):306-311. 2011.Published By: The Helminthological Society of WashingtonDOI: http://dx.doi.org/10.1654/4485.1URL: http://www.bioone.org/doi/full/10.1654/4485.1

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Echinobothrium joshuai n. sp. (Cestoda: Diphyllidea) from the RoughnoseLegskate, Cruriraja hulleyi (Rajiformes: Rajidae), off South Africa

NATASHA RODRIGUEZ, MARIA PICKERING, AND JANINE N. CAIRA1

Department of Ecology and Evolutionary Biology, 75 N. Eagleville Road Unit 3043, University of Connecticut, Storrs,

Connecticut 06269-3043, U.S.A.

ABSTRACT: A new species of diphyllidean cestode is described from Cruriraja hulleyi, a recently described species of skate

collected off the coast of South Africa. Echinobothrium joshuai n. sp. differs from all 39 of its valid congeners in its

possession of ‘‘B’’ hooks, each with a knob-like tubercle on its posterior proximal face. The uterus of this species is also

unique in consisting of a meandering tube in mature proglottids rather than a median sac. This species most closely resembles

E. clavatum in hook formula (3–4 8/7 3–4), but differs in possessing fewer cephalic peduncle spines (7–10 vs. 11–16) and a

greater number of testes (23–27 vs. 11–14). This is the first report of a diphyllidean from this genus of skate and also from

South African waters. This is also one of the few diphyllideans known to parasitize deeper-water elasmobranchs.

KEY WORDS: Echinobothrium joshuai, new species, Diphyllidea, Cruriraja hulleyi, roughnose legskate, Cestoda,

tapeworms, South Africa.

Tapeworms of the order Diphyllidea parasitize the

spiral intestines of elasmobranchs, in particular of

batoids (Tyler, 2006). Among the 5 families of

batoids from which diphyllideans have been reported,

12 of the 39 valid species of Echinobothrium have

been reported from hosts belonging to the family

Rajidae (see Kuchta and Caira, 2010). However, of

the 29 genera of rajids (Ebert and Compagno, 2007),

representatives of only 6 have ever been reported to

host diphyllideans. According to Kuchta and Caira

(2010), these are Amblyraja Malm, 1877 (e.g.,

Wojciechowska, 1991), Raja Linnaeus, 1758 (e.g.,

Rees, 1961), Rhinoraja Ishiyama, 1952 (e.g., Camp-

bell and Andrade, 1997), Sympterygia Muller and

Henle, 1837 (e.g., Campbell and Carvajal, 1980), and

Zearaja Whitley, 1939 (e.g., Alexander, 1963). In

addition, diphyllideans have been reported from

Leucoraja Malm, 1877 (e.g., McVicar, 1976).

Collections made on a trawling vessel, fishing off

the coast of South Africa in 2010, provided us with

the opportunity to examine a seventh genus of skate,

Cruriraja Bigelow and Schroeder, 1948, for diphyl-

lideans. The skate in question, Cruriraja hulleyiAschliman, Ebert, and Compagno, 2010, has recently

been determined to represent a new species (Aschli-

man et al., 2010). Thus, not only is this the first report

of a diphyllidean from South Africa, but it is also the

first record of a parasite from this host species.

MATERIAL AND METHODS

Skate specimens were collected off the coast of SouthAfrica (between 34u28.09S to 36u32.89S and 20u15.89E to25u27.579E) in April 2010 as by-catch from a hake surveyconducted by South African Coastal Marine Management onthe FRV Africana using a bottom trawl at depths rangingfrom 82.2 to 344 m. In total, 7 specimens of the roughnoselegskate, Cruriraja hulleyi (3 females 23.5–28.5 cm in discwidth [DW] and 4 males 27–31 cm DW) were examined forcestodes. Images of the 4 specimens (i.e., host Nos. AF-10,AF-17, AF-99, and AF-108) found to host the new speciesof cestode can be accessed via the Global Cestode Databaseat http://tapewormdb.uconn.edu/hosts.php. Each spiral in-testine was removed, opened with a longitudinal midventralincision, and examined under a stereomicroscope fordiphyllideans. A subset of the cestodes found was fixed in95% ethanol for future molecular analysis while theremaining worms were fixed in 4% seawater bufferedformalin. The spiral intestine of each skate was then fixed in4% seawater buffered formalin and taken to the Universityof Connecticut where they were transferred to 70% ethanolfor storage and further examined for additional cestodes.

Whole mounts of the tapeworms were prepared forexamination with light microscopy as follows. The speci-mens were hydrated in a graded ethanol series, stained inDelafield’s hematoxylin, differentiated in tap water, de-stained in acidic 70% ethanol, placed in basic 70% ethanol,and then flattened under small pieces of glass slides; thenthey were dehydrated in a graded ethanol series, cleared inmethyl salicylate, and mounted on glass slides undercoverslips in Canada balsam. A semipermanent mount ofthe rostellar hooks of 1 worm stored in 70% ethanol wasprepared as follows. The scolex was removed and placed inBerlese’s medium overnight. It was subsequently maceratedon a glass slide in Berlese’s medium under coverslippressure until an apical view of the hooks was achieved. Theedges of the coverslip were then sealed with clear nailpolish. Semipermanent mounts of eggs were prepared byclearing macerated gravid proglottids in a 1:1 solution ofglycerin:70% ethanol. Specimens were mounted on glassslides under coverslips sealed with clear nail polish.1 Corresponding author (e-mail: janine.caira@uconn.edu).

Comp. Parasitol.78(2), 2011, pp. 306–311

306

Measurements were taken using a SPOT DiagnosticInstrument digital camera system mounted on a ZeissAxioskop 2 and SPOT software (version 4.5). Theterminology of the hooks and numbering scheme followTyler (2006). The hook formula used follows Neifar et al.(2001) and is as follows: ([LH] AH[A])/AH[B] [LH]),where (LH) presents total number of lateral hooklets on onelateral side and AH(A) and AH(B) are the number of apicaltype A and type B hooks, respectively. Measurements arepresented as the range, followed in parentheses by the mean,standard deviation, the number of worms measured and,when more than one measurement per worm was made, thetotal number of observations. All measurements are inmicrometers unless otherwise indicated. Microthrix termi-nology follows Chervy (2009).

Scanning electron microscopy (SEM) samples wereprepared as follows. Specimens were hydrated in a gradedethanol series, transferred to 1% osmium tetraoxideovernight, dehydrated in a graded ethanol series, transferredto hexamethyldisilizane (HMDS; Ted Pella, Inc., Redding,California), and allowed to air dry. These specimens weresubsequently mounted on aluminum stubs using double-sided carbon tabs (Ted Pella, Inc.), sputter-coated with 30-nm gold/palladium, and examined with a LEO/ZeissDSM982 Gemini field emission scanning electron micro-scope.

Specimens were prepared for histological sectioning asfollows. They were dehydrated in a graded ethanol series,cleared in xylene, embedded in paraffin, and sectioned at 8-mm intervals using an Olympus CUT4060 retracting rotarymicrotome. Sections were placed on glass slides floodedwith 3% sodium silicate and dried on a slide warmerovernight. They were then stained with Delafield’s hema-toxylin, counterstained with eosin, cleared in xylene, andmounted on glass slides under coverslips in Canada balsam.

Museum abbreviations used are as follow: LRP 5Lawrence R. Penner Parasitology Collection, Departmentof Ecology and Evolutionary Biology, University ofConnecticut, Storrs, Connecticut, U.S.A.; SAMCTA 5South African Museum Cape Town Invertebrate Collection,Cape Town, South Africa; USNPC 5 U.S. National ParasiteCollection, Beltsville, Maryland, U.S.A.

Echinobothrium joshuai n. sp.(Figs. 1–12)

Description

Based on: whole mounts of 3 immature worms, 6

mature worms, 3 gravid worms, 4 free gravid

proglottids, and 3 free mature proglottids; 3 scoleces

observed with SEM; 1 semi-permanent Berlese’s

mount of apical hooks; 2 semipermanent mounts of

gravid proglottids; cross sections of 2 mature

proglottids. Worms apolytic; mature worms 2.8–

3.3 mm (3.1 6 0.2; 6) long by 303–406 (356 6 48;

6) wide at level of terminal proglottid; gravid worms

5.2–6.2 mm (5.7 6 0.5; 3) long by 483–821 (648 6

123; 3) wide at level of terminal proglottid;

proglottids acraspedote, 16–17 (16 6 0.6; 3) in

number in gravid worms, 13–15 (14 6 0.8; 6) in

number in mature worms. Scolex consisting of scolex

proper and cephalic peduncle, 561–718 (593 6 47;

11) long. Scolex proper 412–532 (477 6 36; 11) long

by 133–506 (426 6 103; 11) wide, composed of

armed apical rostellum and 1 dorsal and 1 ventral

bothrium; bothria round in form (Figs. 1, 7), 360–490

(445 6 36; 19; 11) long by 385–510 (450 6 33; 20;

11) wide. Rostellum bearing 1 dorsal and 1 ventral

group of solid apical hooks; apical hooks 15 in total

number, with type B symmetry, flanked on each side

by 3–4 (3.8 6 0.4; 11; 29) small, lateral hooklets; A

hooks (i.e., in anterior row) 8 in number, 88–95 (91

6 2; 10; 12) long, spiniform; B hooks (i.e., in

posterior row) 7 in number, 93–106 (100 6 5; 10; 13)

long (Fig. 2); each with tubercle on posterior

proximal face (Fig. 3). Hook formula (3–4 8/7 3–

4). Cephalic peduncle short, 121–207 (154 6 27; 11)

long by 156–202 (178 6 14; 11) wide (including

spines), armed with 8 longitudinal columns of 7–10

(8.3 6 0.9; 12; 88) spines (Fig. 8); spines with

triradiate bases, decreasing in length posteriorly

(Fig. 4); prong of anteriormost spine 47–71 (59 6

6; 12; 36) long; prong of posteriormost spine 14–35

(24 6 6; 12; 36) long.

Distal bothrial surfaces with densely arranged

trifurcate spinitriches bearing extremely long digits

(Fig. 9) interspersed with capilliform filitriches (not

shown), uniform throughout surface. Anterior prox-

imal bothrial surfaces with fairly dense palmate

spinitriches on a surface consisting of anastomosing

ridges and devoid of filitriches; palmate spinitriches

approximately 6–8 long bearing 5 digits (Fig. 10),

more-posterior proximal bothrial surfaces with sparse

palmate spinitriches; palmate spinitriches approxi-

mately 5 long bearing 6 to 8 digits, interspersed with

capilliform and papilliform microtriches. Cephalic

peduncle devoid of microtriches, surface with

anastomosing ridges (Fig. 11). Proglottids with

densely arranged capilliform filitriches (Fig. 12).

Immature proglottids 14–15 (14.3 6 0.6; 3) in

number in gravid worms, 12–14 (13.3 6 0.8; 6) in

number in mature worms, initially wider than long,

becoming longer than wide with maturity (Fig. 6).

Mature proglottids 1 in number, 710–1,342 (975 6

181; 12) long by 300–517 (408 6 77; 12) wide;

length:width ratio 1.7–3.8:1 (2.5:1 6 0.6; 9).

Attached gravid proglottids 1 in number; attached

and free gravid proglottids 931–1,995 (1,445 6 366;

7) long by 483–821 (648 6 123; 7) wide,

length:width ratio 1.7–2.8:1 (2.2:1 6 0.4; 6). Testes

23–27 (25 6 1; 8; 9) in number, 33–80 (66 6 13; 7;

17) long by 75–148 (115 6 19; 8; 19) wide, arranged

RODRIGUEZ ET AL.—ECHINOBOTHRIUM N. SP. FROM SOUTH AFRICA 307

Figures 1–6. Line drawings of Echinobothrium joshuai n. sp. 1. Scolex. 2. Hooks. 3. Lateral view of B hook showingproximal knob-like protrusion. 4. Cephalic peduncle spines; shown in order from anterior to posterior. 5. Gravid proglottid.6. Whole worm. (A, anterior hooks; B, posterior hooks.)

308 COMPARATIVE PARASITOLOGY, 78(2), JULY 2011

Figures 7–12. Scanning electron micrographs of Echinobothrium joshuai n. sp. 7. Scolex. (Small inset numberscorrespond to regions presented under higher magnification in Figs. 9–12.) 8. Cephalic peduncle. 9. Detail of distal bothrialsurface. 10. Detail of proximal bothrial surface. 11. Detail of surface of cephalic peduncle between columns of spines. 12.Detail of surface of strobila.

RODRIGUEZ ET AL.—ECHINOBOTHRIUM N. SP. FROM SOUTH AFRICA 309

in 2–3 irregular columns from anterior margin of

proglottid to anterior margin of cirrus sac, 2–3 rows

deep in cross section. Cirrus sac oval, 152–250 (198

6 35; 7; 13) long by 92–205 (123 6 29; 7; 12) wide,

located between anterior lobes of ovary; cirrus

covered with spinitriches. Internal and external

seminal vesicles absent. Vas deferens extensive.

Ovary posterior in position, H-shaped in frontal

view, bilobed in cross section anterior to isthmus,

312–640 (449 6 121; 7; 11) long by 173–428 (267 6

82; 7; 11) at widest point. Mehlis’ gland posterior to

ovarian bridge. Vagina very sinuous, lined with tiny

microtriches, opening posterior to cirrus sac in

common genital atrium. Genital pore midventral

24–43% (37 6 5; 7; 11) from posterior margin of

gravid proglottid, opening slightly anterior to ovarian

bridge. Vitellarium follicular; follicles in 2 extensive

lateral bands; each band consisting of multiple

relatively large follicles, extending from near anterior

to near posterior margin of proglottid, uninterrupted

by ovary; vitelline fields not confluent in anterior or

posterior extremities of proglottid. Uterus tube-like in

mature proglottids meandering dorsally and ventrally

along length of proglottid, becoming more sacciform

in gravid proglottids, extending approximately two-

thirds length of proglottid. Eggs ovoid, 30–43 (35 6

4; 2; 12) long by 15–20 (18 6 1; 2; 12) wide.

Taxonomic summary

Type and only known host: Cruriraja hulleyi Aschli-

man, Ebert and Compagno, 2010, roughnose legskate

(Rajiformes: Rajidae).

Site of infection: Spiral intestine.

Type locality: Indian Ocean, South Africa

(34u31.119S; 25u24.459E); collected at a depth of

344 m.

Additional localities: 36u23.99S; 20u15.89E at 185 m,

36u32.89S; 20u26.79E at 187.4 m, and 34u28.09S;

25u27.579E at 139.6 m.

Prevalence, intensity, and abundance of infection: Four

of 7 hosts sampled (57%); 2–7 (4.25 6 2.2) worms

per host; 17 worms in 7 skates examined.

Etymology: This species is named in honor of Joshua

Roy for the instrumental role he has played in the

development of the Global Cestode Database (www.

tapeworms.uconn.edu).

Specimens deposited: Holotype (SAMCTA No.

29496); 5 paratypes consisting of 1 immature worm,

2 mature worms, and 2 free gravid proglottids

(SAMCTA Nos. 29497–29501); 8 paratypes consist-

ing of 1 gravid worm, 2 mature worms, 1 immature

worm, 3 free proglottids, and Berlese’s hook medium

preparation (LRP Nos. 7544–7551) and 1 series of

proglottid cross sections (LRP Nos. 7552–7555); 6

paratypes consisting of 1 immature worm, 2 mature

worms, 1 gravid worm, and 2 free mature and 1 free

gravid proglottid (USNPC Nos. 104135–104138),

and cross sections of 1 mature proglottid (USNPC

No. 104136). SEM stub retained in third author’s

personal collection.

Remarks

Among the 39 valid species of Echinobothriumrecognized by Kuchta and Caira (2010), only

Echinobothrium clavatum Probert and Stobart, 1989

resembles E. joshuai n. sp. in its possession of a total

of 8 ‘‘A’’ hooks and 7 ‘‘B’’ hooks in the dorsal and

ventral groups for a total of 15 apical hooks. All of

the other 38 species exhibit either a greater or lesser

number of ‘‘A’’ or ‘‘B’’ hooks and also a greater or

lesser total number of apical hooks (i.e., ‘‘A’’ and

‘‘B’’ hooks combined). However, E. joshuai n. sp.

differs conspicuously from E. clavatum in its

possession of 7–10 versus 11–16 cephalic peduncle

spines in each column and in its possession of 23–27

versus 11–14 testes (see Tyler, 2006). Echinobo-thrium joshuai n. sp. appears to be unique among its

congeners in its possession of B hooks that each bear

a tubercle on the proximal surface, which is posterior

(Fig. 3) rather than more anterior in position. The

condition of the uterus of E. joshuai n. sp. is also

unusual. Rather than being saccate in form in mature

proglottids, as seen in most species of Echinobo-thrium (see Tyler, 2006), the uterus of this new

species consists of a widened tube that meanders back

and forth from dorsal to ventral throughout the length

of the mature proglottid.

DISCUSSION

Echinobothrium joshuai is the first diphyllidean to

be reported from the waters off South Africa. It joins

Echinobothrium reginae Kuchta and Caira, 2010

from Madagascar (see Kuchta and Caira, 2010) as

only the second diphyllidean species known from the

waters of continental Africa, outside of the Mediter-

ranean Sea. This is also the first diphyllidean species

to be described from a skate of the genus Cruriraja,

bringing the total number of skate genera known to

host 1 or more Echinobothrium species to 7. The

randomness of the process by which this host was

selected (i.e., as by-catch from a trawl survey) leads

us to believe that it is likely that more of the 22

genera of rajids that remain to be examined for

310 COMPARATIVE PARASITOLOGY, 78(2), JULY 2011

diphyllideans will be found to host species belonging

to this order.

The summary of type hosts of Echinobothriumspecies presented by Kuchta and Caira (2010)

indicates that members of this genus parasitize

elasmobranchs that occur at a variety of depths.

While a number of species parasitize shallow-water

hosts, others occur in hosts occupying deeper waters.

For example, several species (e.g., Echinobothriumelegans Tyler, 2001, Echinobothrium helmymoha-medi Saoud, Ramadan and Hassan, 1982, Echinobo-thrium heroniense Williams, 1964) parasitize the

bluespotted stingray, Taeniura lymma (Forsskal,

1775), which occurs only in waters up to 20 m in

depth (see Froese and Pauley, 2010); whereas other

species such as Echinobothrium clavatum Probert

and Stobart, 1989 (see Probert and Stobart, 1989)

parasitize skates, such as Raja clavata Linnaeus,

1758, which occurs in waters up to 577 m in depth

(see Froese and Pauley, 2010). The specimens of

Cruriraja hulleyi found here to host E. joshuai were

collected at depths ranging from 139–344 m, sug-

gesting that this is a fairly deepwater species of

diphyllidean. However, to our knowledge, the only

other diphyllidean for which depth of collection was

explicitly provided was E. raschii Campbell and

Andrade, 1997, which was reported by Campbell and

Andrade (1997) to have come from specimens of

Rhinoraja longi Raschi and McEachran, 1991

collected at 100–103 fathoms (i.e., 183–188 m).

ACKNOWLEDGMENTS

We are especially grateful to Tracey Fairweather

and Rob Leslie of the Department of Agriculture,

Forestry and Fisheries, Cape Town, South Africa, for

facilitating J.N.C.’s participation in the April 2010

cruise of the FRV Africana and to the crew and

scientific staff of the FRV Africana for assisting with

the collection of skates. We thank Kirsten Jensen for

her assistance with all aspects of the field work and

Leah Desjardins and Kendra Koch for removal of

worms from spiral intestines. We are especially

indebted to Dave Ebert for alerting us to the novel

nature of the skate from which this worm was collected

and for sharing the manuscript describing that skate

with us prior to its publication. This research was

supported in part by NSF PBI award Nos. 0818823

and 0818696 and an Undergraduate Multicultural

Fellowship to N.R. from the Department of Ecology

and Evolutionary Biology at the University of

Connecticut, provided as a match to the latter award.

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RODRIGUEZ ET AL.—ECHINOBOTHRIUM N. SP. FROM SOUTH AFRICA 311