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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: [email protected]).
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