Redescription of Pseudanthobothrium hanseni Baer, 1956and description of P. purtoni n. sp. (Cestoda: Tetraphyllidea)from different pairs of rajid skate hosts, with commentson the host-specificity of the genus in the northwest Atlantic

H. S. Randhawa Æ G. W. Saunders Æ M. E. Scott ÆM. D. B. Burt

Received: 6 October 2006 / Accepted: 6 June 2007

� Springer Science+Business Media B.V. 2007

Abstract During a parasitological survey of Leuco-

raja erinacea, L. ocellata, Malacoraja senta and

Amblyraja radiata from Passamaquoddy Bay and

waters surrounding the West Isles of the Bay of Fundy,

NB, Canada, seven species of cestodes were recovered.

Examination of these skates revealed the presence of

two distinct species of Pseudanthobothrium Baer,

1956: one was retrieved from M. senta and A. radiata,

identified as P. hanseni Baer, 1956 and redescribed

herein; the other was retrieved from L. erinacea and

L. ocellata and differs from previously described

species. The new species is described herein as

P. purtoni n. sp. on the basis of the degree of apolysis,

the maximum width of the strobila, the length of the

cirrus-sac and the number of testes. Additionally, the

distinctiveness of both species of Pseudanthobothrium

is supported by the characterisation of a 643 base-pair

nuclear marker, which includes most of the D2 variable

region of the large subunit ribosomal DNA (LSU). The

recovery of two different tetraphyllidean species, each

from two different host species, challenges the oioxeny

(strict host-specificity) of echeneibothriine cestodes

and can be explained, at least in part, by the similarities

in diet and substrate preference within each host pair.

Introduction

During a parasitological survey of rajid skates (rays)

from Passamaquoddy Bay and waters surrounding the

West Isles of the Bay of Fundy (New Brunswick,

Canada), seven cestode species were recovered

(Randhawa et al., 2007). Of these, two species with

a morphology consistent with Pseudanthobothrium

Baer, 1956 were identified, including one new to

science. P. hanseni Baer, 1956 was recovered from

Amblyraja radiata (Donovan) and Malacoraja senta

(Garman), while Pseudanthobothrium n. sp. was

recovered from Leucoraja erinacea (Mitchill) and

L. ocellata (Mitchill) (Randhawa et al., 2007). Addi-

tionally, Randhawa et al. (2007) have reported

another echeneibothriine species, Echeneibothrium

vernetae Euzet, 1956, from both L. erinacea and

L. ocellata. These findings question the strictness of

the echeneibothriine parasite and rajid host associa-

tion (Williams, 1966) and may be attributable to

ecological influences as discussed herein.

Although several cestode species have been recov-

ered from the thorny or starry skate A. radiata (Heller,

1949; Myers, 1959; Threlfall, 1969; Margolis &

Arthur, 1979; McDonald & Margolis, 1995; Keeling

& Burt, 1996) and the winter skate L. ocellata (Myers,

1959; Margolis & Arthur, 1979), other than

H. S. Randhawa (&) � G. W. Saunders � M. D. B. Burt

Department of Biology, University of New Brunswick,

Fredericton, NB, Canada E3B 6E1

e-mail: haseeb.randhawa@unb.ca

M. E. Scott

Institute of Parasitology, Macdonald Campus, McGill

University, Ste-Anne de Bellevue, QC, Canada H9X 3V9

123

Syst Parasitol (2008) 70:41–60

DOI 10.1007/s11230-007-9122-6

Randhawa et al. (2007), we are not aware of any

survey of the cestode fauna of the little skate

L. erinacea and the smooth skate M. senta from

Canadian waters. Additionally, some of the identifi-

cations of cestode parasites of skates from Canadian

waters are questionable.

For example, Baer (1956) considered that speci-

mens identified by Heller (1949) as Anthobothrium

cornucopia van Beneden, 1850 from Raja scrabata

Garman were misidentified and were in fact P. hanseni,

and in 1962 he further suggested that Amblyraja

radiata and R. scrabata harbour this same cestode

species. Williams (1966) questioned Baer’s (1956,

1962) conclusion because of the general principle of

host specificity of tetraphyllideans. The skate R. scra-

bata is now considered to be a synonym of A. radiata

(see Scott & Scott, 1988), which supports the consid-

eration proposed by Baer (1956), but nonetheless

sustains the dogma surrounding the specificity of these

cestodes. Examination of Heller’s collection (bor-

rowed from the Canadian Museum of Nature,

Nos CMNP1995-0005, CMNP1995-0006 and

CMNP1995-0007) during the present study supports

Baer’s (1956) suggestion concerning Heller’s mis-

identification. Both Myers (1959) and Threlfall (1969)

also reported Anthobothrium cornucopia from Ambly-

raja radiata collected near the Magdalen Islands and

Newfoundland, respectively. Myers (1959) addition-

ally reported A. cornucopia from L. ocellata collected

near the Magdalen Islands, extending its host range to a

second host species. Unfortunately, Myers’ (1959) and

Threlfall’s (1969) reports were not accompanied by

descriptions or drawings, thus their identifications

should not be accepted without question. Whether the

material collected by Myers (1959) and Threlfall

(1969) is A. cornucopia, P. hanseni or another species

is uncertain without a re-examination of their material.

There are currently three recognised species of

Pseudanthobothrium: P. hanseni, P. minutum

Wojciechowska, 1991 and P. notogeorgianum

Wojciechowska, 1990. The former, P. hanseni, was

originally described by Baer (1956) from Amblyraja

radiata caught in water off the west coast of

Greenland. Williams (1966) identified this parasite

from the same host species caught in the North Sea.

Furthermore, Jarecka & Burt (1984) identified P. han-

seni and Pseudanthobothrium sp. from L. erinacea in

Passamaquoddy Bay, NB, thus extending the host

range of P. hanseni to a second host species. Their

identifications were based on adult worms and

detached proglottides, but no drawings or descriptions

were provided. The presence of P. hanseni in

A. radiata and L. erinacea is, therefore, questionable

and needs to be investigated further. Randhawa et al.

(2007) identified P. hanseni from A. radiata and

M. senta in the northwest Atlantic. Both P. minitum,

and P. notogeorgianum were recovered from Bathy-

raja eatonii (Gunther) and A. georgiana (Norman),

respectively, from Antarctic waters. As mentioned

above, Randhawa et al. (2007) also recovered a

species consistent with the generic description of

Pseudanthobothrium, but different from the three

currently recognised species; this species is described

below as new to science. The identification of this new

species is based on differences in apolysis, maximum

width of the strobila, cirrus-sac length and testes

number, and is confirmed using molecular compari-

sons of partial sequences of a variable area (D2) of the

large subunit of ribosomal DNA (LSU), as discussed

below.

Materials and methods

Collections and examination of material

From May to August, 1997 and between June, 2002

and September, 2004, 208 Leucoraja erinacea, 33

Malacoraja senta, 31 Amblyraja radiata and 11

L. ocellata were collected from Passamaquoddy Bay

and waters surrounding the West Isles of the Bay of

Fundy, NB, Canada (includes the collections reported

in Randhawa et al., 2007). Skates were collected,

identified and processed as described in Randhawa

et al. (2007). Parasites were removed from the spiral

valves and cleaned in fresh saline prior to being

processed. Only scoleces, both attached and detached,

were counted to determine the number of parasites

present in each individual spiral valve and the

attachment-site of those attached was noted.

Some spiral valves, preserved for later examination,

were ligatured with string at both ends and removed

from the body-cavity before 10% formalin was

injected into the pyloric end until fully distended.

The spiral valves were then preserved individually in

plastic bags containing 10% formalin and an identi-

fication tag. Bags were placed in glass jars containing

10% formalin. When these spiral valves were opened

later, using the method described above; parasites

42 Syst Parasitol (2008) 70:41–60

123

from these samples were transferred to vials contain-

ing 70% ethanol.

For light microscopy, worms were fixed in hot,

almost boiling 70% ethanol and stored in fresh 70%

ethanol prior to staining and mounting using routine

histological procedures. Worms used for molecular

analyses were fixed as described for light microscopy,

and immediately transferred to 95% or absolute

ethanol. For whole-mounts, specimens were hydrated

through an ethanol gradient and placed in a 2% Acetic

Acid Alum Carmine stain (AAAC) for 24 hours

before being dehydrated through an ethanol gradient,

cleared in clove oil and mounted in Canada balsam.

Five detached proglottides were prepared for section-

ing, stained as above, dehydrated through an ethanol

gradient, embedded in paraffin, sectioned at 10 lm,

stained in Ehrlich’s haemotoxylin and counter-stained

in eosin, cleared in xylene and mounted in Canada

balsam. The parasites were drawn with the use of a

Wild drawing tube attached to a Wild-M20 compound

microscope and salient features measured and

described. Measurements were taken from 43 mature

P. hanseni specimens and from 40 mature specimens

of the new species (unless specified otherwise). All

measurements for species description are represented

by the range and (mean ± standard deviation), and are

in micrometres unless otherwise indicated. All mea-

surements are compared between species examined

using a two-tail t-test for samples with unequal

variance. Furthermore, the number of testes per

proglottis, cirrus-sac length and maximum width of

the strobila were subjected to Mann-Whitney U-tests

to determine whether measurements came from the

same distribution.

For scanning electron microscopy, worms were

fixed in 4% Lillie’s buffered formalin or 4%

gluteraldehyde, post-fixed in 2% osmium tetraoxide

for 2 hours prior to being dehydrated, through an

ethanol gradient series. Specimens were then critical

point dried in a Balzers Critical Point Drier (CPD

020) in carbon dioxide, and mounted on stubs using

double-sided adhesive carbon tape. Silver paste was

used around the tape in order to provide additional

contact. Specimens were then sputter coated with

gold for 2– 4 minutes in an Edwards S150A sputter

coating unit, before being examined in a JEOL 6400

scanning electron microscope.

Molecular characterisation and analysis

Genomic DNA was extracted using standard tech-

niques (Devlin et al., 2004). The 50 end of the large

subunit ribosomal DNA (LSU) was amplified

(c.1,850 bp), as described in Harper & Saunders

(2001) (Fig. 1), using the Ex-Taq-polymerase PCR

kit (Takara Bio Inc., Otsu, Shiga, Japan). The ampli-

fication protocol consisted of an initial 4 min

denaturation phase (94�C); 38 cycles of denaturation

(30 sec at 94�C), primer annealing (30 sec at 50�C)

and extension (2 min at 72�C), and a 7 min final

extension (72�C). The amplified products were cleaned

on 0.8% electrophoresis grade agarose (MP Biomed-

icals, Aurora, OH, USA) gels as per Saunders (1993).

Single-strand sequencing reactions using the T16

forward primer (Harper & Saunders, 2001) (Fig. 1)

were completed for all specimens using the ‘ABI

PRISM1 Big DyeTM Terminator Cycle Sequencing

Ready Reaction Kit v. 3.10 (Applied Biosystems (ABI),

Foster City, CA, USA) (c.800 bp). Sequences were

obtained using a 16 capillary 3100 Genetic Analyzer

(ABI). Sequence data were edited using Sequencher

4.5 (Gene Codes Corporation, �1991–2005) and

subsequently aligned using MacClade 4.07 (Maddison

& Maddison, 2005). The tranversional model (TVM)

was determined to provide the best fit to our data based

on Modeltest 3.7 (Posada & Crandall, 1998; Posada &

Buckley, 2004). The neighbour-joining clustering

algorithm, implemented in PAUP v.4.0b10 (Swofford,

2002), was used to provide a visual display of the

Pseudanthobothrium spp. distribution. The purpose of

these analyses was not phylogenetic, but rather it

served to assign specimens to one of two species

Fig. 1 Schematic representation of the amplified 50 end of the

LSU ‘‘X’’ fragment (modified from Harper & Saunders, 2001)

with the oligonucleotide sequences of both PCR (T01N and

T13N) and sequencing primers (T16 and T30) provided.

Sequenced region represented in grey, while the region used in

the analyses is represented in white

Syst Parasitol (2008) 70:41–60 43

123

recognised here and to estimate within versus between

species variation for this nuclear marker. Terminal

proglottides were retained for light microscopy and

scoleces were retained for scanning electron micros-

copy as vouchers for each of the sequenced specimens.

These vouchers were deposited in the NB Museum in

Saint John, NB, Canada.

Results

In addition to the two Pseudanthobothrium spp., the

present survey (including data reported in Randhawa

et al., 2007) recovered five additional cestode spe-

cies: Echeneibothrium canadense Keeling & Burt,

1996 (emend.) and E. dubium abyssorum Campbell,

1977 from Amblyraja radiata; E. vernetae Euzet,

1956 from both Leucoraja erinacea and L. ocellata;

Grillotia sp. from all four host-species; and Zyxi-

bothrium kamienae Hayden & Campbell, 1981 from

Malacoraja senta. Excepting the trypanorhynch

Grillotia sp., all other cestodes belong to the

Tetraphyllidea.

Pseudanthobothrium hanseni Baer, 1956

Type-host: Amblyraja radiata (Donovan).

Additional host: Malacoraja senta (Garman).

Type-locality: Off west Greenland.

Other localities: North Sea (Williams, 1966); Upper

Western Passage (West Isles, Bay of Fundy, New

Brunswick, Canada (44�570N, 67�010W).

Sequence: GenBank accession number EF207818.

Site: Throughout spiral valve, preference for anterior

half.

Prevalence: 90.7% (28 of 31 A. radiata); 48.5% (16

of 33 M. senta).

Mean intensity: 19.7 per infected A. radiata (range

1–55); 10.1 per infected M. senta (range 1–73).

Material: Three vouchers are deposited (NBM

002440-002442) in the New Brunswick (NB)

Museum, Saint John, NB; three are deposited (UNBF

Biol HSR 0001-0003) in the Department of Biology,

University of New Brunswick Parasitology Teaching

Collection, Fredericton, NB; and three are deposited

(BMNH 2007.7.31.22-24) in the Natural History

Museum, London, United Kingdom.

Redescription (Figs. 2–10)

[Based on measurements taken from 43 mature indi-

viduals (34 from A. radiata and 9 from M. senta);

number of measurements indicated by ‘‘n’’.] Adult

worms apolytic, 5.1–25.8 (10.4 ± 5.2) mm in length;

maximum width 195–610 (350 ± 101) at the level of

terminal proglottis. Strobila with 39–173 (81 ± 38)

slightly craspedote proglottides, including 4–82

(21 ± 16) immature proglottides with presence of

developing testes (n = 40), 2–21 (8 ± 4) mature pro-

glottides with fully-developed male and female

reproductive organs (n = 41) and 0–9 (3 ± 2) gravid

proglottides characterised by obscuring of testes by

uterus (Figs. 2, 3). Scolex composed of 4 stalked

bothridia (Figs. 4, 5) and retractable myzorhynchus

(Figs. 4, 6). Bothridia non-loculate, cup-shaped, 140–

380 (242 ± 48) long (n = 135), 135–306 (201 ± 41)

wide (n = 61) (Figs. 4, 5); stalks slender, mobile and

extensible, 200–590 (366 ± 70) long (including

bothridia) (n = 125), 65–160 (110 ± 26) wide

(n = 49). Distal bothridial surface covered with fili-

triches, c.4 long (Fig. 7). No microtriches observed on

distal half of proximal bothridial surface; bothridial

margins, stalks and proximal half of proximal bothri-

dial surface covered with blade-like microtriches, c.3

long, 1.5 wide at base (Figs. 8, 9). Myzorhynchus

retractable (Figs. 4, 6), 45–655 (293 ± 164) long

(n = 20), 60–195 (122 ± 41) in diameter (n = 20),

covered with blade-like microtriches (Fig. 10). Neck

60–335 (151 ± 67) long, 40–150 (96 ± 27) wide

(Figs. 2, 4). Immature proglottides at base of neck

c.7 times wider than long, becoming longer than wide

with maturity (Fig. 2). Testes ovoid to round, 45–150

(78 ± 16) long (n = 280 testes), 38–90 (57 ± 10) wide

(n = 280 testes), 19–32 (24 ± 3) (n = 148 proglotti-

des) in number, present anterior to genital atrium

(Fig. 2). Genital atria irregularly alternate (Fig. 2).

Vagina opens anteriorly to cirrus-sac. Cirrus-sac

ovoid, extends posteriorly along mid-line of proglottis,

140–255 (186 ± 30) long (n = 26), 60–105 (81 ± 12)

in diameter (n = 26) (Fig. 2). Ovary bi-lobed (Fig. 2),

X-shaped in cross-section; poral lobe short of cirrus-

sac margin in older (posterior) proglottides (Fig. 2).

Vitelline follicles ovoid, 15–55 (32 ± 11) long

(n = 207), 10–35 (22 ± 5) wide (n = 205), arranged

in 2 paired lateral bands anterior to ovary, extending as

2 single lateral bands posterior to anterior margin of the

ovary along length of proglottis (Fig. 2). Oncospheres

44 Syst Parasitol (2008) 70:41–60

123

Figs. 2–4 Drawings of Pseudanthobothrium hanseni. 2. Complete fixed and stained mature specimen. 3. Terminal gravid

proglottides with uterus fully distended, filled with oncospheres; testes not visible. 4. Scolex with extended bothridia and semi-

retracted myzorhynchus. Scale-bars: 2, 1 mm; 3, 200 lm; 4, 350 lm

Syst Parasitol (2008) 70:41–60 45

123

Figs. 5–10 Scanning electron micrographs of Pseudanthobothrium hanseni recovered from Amblyraja radiata inhabiting waters

surrounding the West Isles of the Bay of Fundy, NB, Canada: 5. Scolex bearing four cup-shaped, stalked bothridia (two of the bothridia

engulfing villi). 6. View of the area between the bothridia showing the site of the myzorhynchus (retracted). 7. Distal bothridial surface

showing filitriches. 8. Bothridial margin showing filitriches. 9. Proximal bothridial surface showing the bothridial margin and naked distal

portion. 10. Surface of the myzorhynchus showing blade-like microtriches. Abbreviations: DHPBS, distal-half of proximal bothridial

surface; RM, retracted myzorhynchus. Scale-bars: 5, 100 lm; 6, 9, 10, 10 lm; 7, 8, 1 lm

46 Syst Parasitol (2008) 70:41–60

123

(‘‘eggs’’ of earlier authors) oval, 19–33 (26 ± 4) long

(n = 25), 16–28 (22 ± 4) wide (n = 25), with polar

filament. Gravid proglottides attached; those where

uterus obscures testes 356–1645 (791 ± 336) long

(n = 63), 205–610 (385 ± 92) wide (n = 63) (Figs. 2, 3).

Remarks

Comparisons of Pseudanthobothrium specimens

recovered from all four host-species are presented in

Table 1. Pseudanthobothrium specimens recovered

from Amblyraja radiata share many characteristics

with those from Malacoraja senta (Table 1). The

range of measurements for all salient features overlap

and are similar. Hence, on the basis of these similar-

ities, Pseudanthobothrium specimens recovered from

the spiral valves of A. radiata and M. senta are

identified as the same species. Moreover, on the basis

of similarities with material described by Baer (1956)

and Williams (1966) (Table 2), specimens recovered

from the spiral valves of A. radiata and M. senta are

identified as P. hanseni.

Pseudanthobothrium specimens recovered from

A. radiata and M. senta are compared with descrip-

tions of P. hanseni from Baer (1956) and Williams

(1966) in Table 2. Specimens used by Baer (1956) to

describe Pseudanthobothrium measured up to 4 mm,

were not gravid and did not have well-developed uteri.

In the present study, many non-gravid specimens,

measuring c.1.8–5.9 mm, were observed. For this

reason, the difference in total length between speci-

mens in Baer’s (1956) description and those from the

present study (Table 2) should not be considered as a

species-defining character. Neither should the number

of proglottides and maximum width of strobila

(Table 2), for the same reason. Baer (1956) did not

provide an accurate count (‘‘few’’; p. 21) or an

objective estimate of the size of the testes (‘‘large’’;

p. 21); therefore, comparisons with present material for

these characters are impossible. Material from both

Baer’s (1956) and the present study were described

from A. radiata, although from different localities

(Table 2). Williams (1966) provided a more detailed

description for P. hanseni in A. radiata from the North

Sea using mature and gravid specimens. The number of

proglottides and measurements for the total length and

maximum width of the strobila for the present material

are consistent with material described by Williams

(1966). The length of the myzorhynchus of present

material is shorter than that reported by Williams

(1966) (Table 2), but this difference may be due to the

degree of contraction of the myzorhynchus at the time

of fixation. The myzorhynchus of live specimens of

this genus observed in elasmobranch saline (recipe in

Laurie, 1961) can extend two to three times the length

of that of fixed specimens (Randhawa, pers. obs.). The

length and width of bothridia reported by Williams

(1966) are greater than those of present material

(Table 2). However, it is assumed that Williams’

(1966) measurements represent maxima, as figures

from his description showed measurements similar to

ours. The scolex has been reported as being highly

polymorphic, the bothridia varying in depth, depend-

ing on the degree of contraction (Baer, 1956), and the

myzorhynchus as an apical organ capable of retraction

and invagination (Caira et al., 1999). Therefore, the

length of the myzorhynchus and the length and width of

the bothridia should not be considered as species-

defining characters. Although the number of testes

reported by Williams (1966) for P. hanseni corre-

sponds to the lower range for present material

(Table 2), the number for Pseudanthobothrium recov-

ered from A. radiata from the North Sea (Randhawa,

pers. obs.) is consistent with those reported herein from

A. radiata and M. senta. The cirrus-sac measurements

described by Williams (1966) are slightly larger than

those reported here (Table 2). However, since

Williams provided no range for his measurements, it

is assumed that these represented maxima. He also

described detached proglottides as being gravid and

having, in some cases, only nine visible testes. In the

present material, no detached gravid proglottides of

P. hanseni in which testes are apparent were found.

Moreover, the present material does not show a single

terminal proglottis of P. hanseni having any visible

testes, as the proglottides are filled with oncospheres.

Williams’ (1966) description of P. hanseni does not

indicate whether worms were recovered from fish

with concurrent infections of similar cestodes, and

thus unequivocal identification of detached proglotti-

des as belonging to P. hanseni cannot be accepted

without question. On the basis of comparisons between

material recovered from A. radiata and M. senta

(Tables 1, 2) and descriptions by Baer (1956) and

Williams (1966), we identify material reported

herein from A. radiata and M. senta as P. hanseni

Baer, 1956.

Syst Parasitol (2008) 70:41–60 47

123

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Syst Parasitol (2008) 70:41–60 49

123

Pseudanthobothrium purtoni n. sp.

Type-host: Leucoraja erinacea (Mitchill).

Additional host: L. ocellata (Mitchill).

Type-locality: Upper Western Passage (West Isles,

Bay of Fundy, New Brunswick, Canada) (44�570N,

67�010W).

Ssequence: GenBank accession number EF207788.

Site: Throughout spiral valve, preference for anterior

half.

Prevalence: 80.8% (168 of 208 L. erinacea); 81.2%

(9 of 11 L. ocellata).

Mean intensity: 13.7 per infected L. erinacea (range

1–96); 10.2 per infected L. ocellata (range 1–26).

Type-material: The holotype (CMNPA 2007-0001)

and two paratypes (CMNPA 2007-0002 and -0003)

are deposited in the Canadian Museum of Nature,

Ottawa, Ontario; four paratypes are deposited (NBM

001997-002000) in the NB Museum, Saint John, NB;

three are deposited (UNBF Biol HSR 0004-0007) in

the Department of Biology, University of NB’s

Parasitology Teaching Collection, Fredericton, NB;

and three are deposited (BMNH 2007.7.31.19-20 ex

L. erinacea; BMNH 2007.7.31.21 ex L. ocellata) at

the Natural History Museum, London, UK.

Etymology: The new species is named in recognition

of the invaluable help and services provided by Mr.

Fred Purton, Huntsman Marine Science Centre, over

the course of this study.

Description (Figs. 11–19)

[Measurements are taken from 40 mature individuals

(34 from L. erinacea and 6 from L. ocellata); number of

measurements indicated by ‘‘n’’.] Adult worms euap-

olytic, 4.3–29.4 (9.9 ± 5.0) mm in length; maximum

width 117–316 (191 ± 42) at level of terminal pro-

glottis (Figs. 11, 12). Strobila with 46–322 (97 ± 58)

slightly craspedote proglottides, including 10–123

(27 ± 23) immature proglottides with presence of

developing testes (n = 37) and 1–31 (7 ± 5) mature

proglottides with fully-developed male and female

reproductive organs (n = 37) (Fig. 11). Scolex com-

posed of 4 stalked bothridia and long and slender

myzorhynchus (Figs. 13, 14). Bothridia non-loculate,

cup-shaped, 186–370 (245 ± 42) long (n = 82), 88–

250 (161 ± 34) wide (n = 80) (Figs. 13, 14, 15); stalks

slender, mobile, extensible, 325–745 (498 ± 103) long

(including bothridia) (n = 83), 36–128 (67 ± 22) wide.

Distal bothridial surface covered with filitriches, c.2.5

long, 0.5 in diameter at base (Fig. 16). No microtriches

were observed on distal half of proximal bothridial

surface; proximal half of proximal bothridial surface

covered with blade-like microtriches, c.3.5 long, 1

wide at base (Figs. 17, 18). Bothridial margin and

stalks covered by blade-like microtriches similar to

those on proximal half of proximal bothridial surface.

Myzorhynchus retractable, 190–455 (297 ± 68) long,

75–175 (110 ± 25) in diameter (Figs. 13, 14, 19). Neck

85–300 (173 ± 78) long (n = 17), 40–100 (70 ± 19)

wide (n = 17) (Figs. 11, 13). Immature proglottides at

base of neck c.5 times wider than long, becoming

longer than wide with maturity (Fig. 11). Testes ovoid

to round, 40–108 (65 ± 13) long (n = 169 testes), 30–

71 (50 ± 9) wide (n = 169 testes), 9–21 (14 ± 2)

(n = 324 proglottides) in number, present anterior to

genital atrium (Figs. 11, 12). Genital atria irregularly

alternate, open 28–50 (43 ± 5)% of proglottis length

from posterior margin (n = 49) in mature proglottides

(Figs. 11, 12). Vagina opens anteriorly to cirrus-sac

(Fig. 12). Cirrus-sac ovoid, extends posteriorly along

mid-line of proglottis, 90–185 (137 ± 21) long

(n = 106), 50–100 (75 ± 9) in diameter (n = 111)

(Figs. 11, 12). Ovary bi-lobed (not typically asym-

metrical, as shown in Fig. 12), X-shaped in cross-

section; poral lobe short of cirrus-sac margin in older

(posterior) proglottides (Figs. 11, 12). Vitelline folli-

cles ovoid, 15–53 (27 ± 8) long (n = 138), 10–40

(19 ± 6) wide (n = 138), arranged in 2 paired lateral

bands anterior to ovary, extending as 2 single lateral

bands beyond ovary (Figs. 11, 12). Oncospheres oval,

16–30 (25 ± 4) long (n = 16), 12–28 (23 ± 4) wide

(n = 16), with polar filament. Detached proglottides

gravid, 920–2125 (1273 ± 293) long (n = 33), 210–

460 (277 ± 51) wide (n = 33); state of uterus variable,

from few oncospheres to full. Genital atria open 40–

50% (47 ± 3) of proglottis length from posterior

margin (n = 27) in gravid proglottides.

Remarks

Except for the size of detached gravid proglottides,

all other measurements taken from specimens recov-

ered from both Leucoraja spp. overlap, are similar, or

may differ due to variations in the state of contraction

at the time of fixation. The lone gravid proglottis,

50 Syst Parasitol (2008) 70:41–60

123

recovered from L. ocellata, was filled with onco-

spheres, without visible testes, and longer and wider

than any detached proglottis found in spiral valves of

L. erinacea. However, additional observations of

detached proglottides in spiral valves of L. ocellata

are required in order to assess whether the larger size

is a true difference. Moreover, terminal proglottides

of Pseudanthobothrium specimens are sometimes

gravid, with testes still intact and visible. Therefore,

on the basis of similarities and of lack of evidence for

unequivocal differences (Table 1), Pseudanthoboth-

rium specimens recovered from spiral valves of

L. erinacea and L. ocellata are identified as the same

species. Furthermore, specimens recovered from the

spiral valves of both host species are distinct from

previously described material (Table 2).

Pseudanthobothrium purtoni n. sp. can be distin-

guished from P. hanseni in four ways: (1) the number

of testes; (2) the length of the cirrus-sac; (3) the

maximum width of the strobila; and (4) the degree of

apolysis (gravid proglottides with few oncospheres

and visible testes vs filled uterus obscuring testes;

Table 2). It should be noted that significant differ-

ences in means for bothridial size (p \ 1 · 10–12),

testicular size (p \ 1 · 10–14), vitelline follicle size

(p \ 1 · 10–6) and cirrus-sac width (p \ 0.031)

between these species were detected using t-tests.

However, due to the overlap in the range of

measurements, these are not deemed as good diag-

nostic features for these species. Even though there is

a slight overlap in testicular number between the two

species, P. hanseni specimens with numbers in the

lower range possessed terminal gravid proglottides

with the uterus filled with oncospheres and obscuring

the testes. The number of testes for P. hanseni

(Williams, 1966) corresponds to the upper range for

P. purtoni n. sp. (Table 2). However, that for

P. hanseni from A. radiata collected from the North

Sea is consistent with the description of P. hanseni

reported herein (Randhawa, pers. obs.). Whereas

P. hanseni possesses gravid terminal proglottides

without visible testes, P. purtoni specimens with the

numbers of testes in the upper range possess mature

or gravid terminal proglottides with visible testes. We

are confident that there is no true overlap in testicular

number between the two species (different means:

p \ 1 · 10–100; different distributions: p \ 1 · 10–5),

as the average number in observed P. hanseni was

24 ± 3 vs 14 ± 2 in P. purtoni (see Fig. 20 for the

frequency distribution of testicular number per pro-

glottis for both species). Williams’ (1966) description

of P. hanseni indicates that, on occasion, only nine

testes are visible in detached gravid proglottides

(p. 254). However, he did not indicate whether these

were recovered from fish with concurrent infections

with similar cestodes and thus an unequivocal

identification of detached proglottides as belonging

to P. hanseni cannot be accepted without question.

Secondly, the cirrus-sacs from material described

from both Leucoraja spp. are shorter than those of

P. hanseni. Although cirrus-sac length measurements

overlap, the means (± standard deviation) of both are

different (136 ± 21 vs 186 ± 30 lm, respectively;

p \ 1 · 10–8) and so are the distributions

(p \ 1 · 10–5). Therefore, we are confident that this

represents a true difference (see Fig. 21 for the

frequency distribution of cirrus-sac length for both

species). Thirdly, the differences in means, for the

maximum width of the strobila (p \ 1 · 10–12) and

in distributions (p \ 1 · 10–5), seem linked to the

difference in apolysis between the two species (see

Fig. 22 for the frequency distribution of the maxi-

mum widths for both species). Specimens recovered

from L. erinacea and L. ocellata are described herein

as euapolytic, whereas P. hanseni was observed to be

apolytic. Detached proglottides belonging unequivo-

cally to P. hanseni were not recovered during the

course of this study. Detached proglottides recovered

from A. radiata were found in concurrent infections

with E. dubium abyssorum and/or E. canadense and

could not be assigned indisputably to P. hanseni.

However, contrary to material from L. erinacea and

L. ocellata, terminal proglottides of mature P. hanseni

specimens are gravid, with the uterus fully distended

and without visible testes. The uteri of the last one to

nine proglottides of P. hanseni are well developed

and filled with oncospheres (Figs. 2, 3), whereas the

uterus in the terminal proglottides of material from

L. erinacea and L. ocellata is not well developed and

the testes are visible (Figs. 11, 12). The difference in

apolysis contributes to the observed difference in the

maximum width of the strobila in both species. The

position of the widest proglottis in both species is

generally the terminal one. Detached gravid proglot-

tides filled with oncospheres in L. erinacea and

L. ocellata are narrower than the terminal proglotti-

des of P. hanseni (Table 2). A width distribution of

gravid proglottides revealed a significant difference

Syst Parasitol (2008) 70:41–60 51

123

52 Syst Parasitol (2008) 70:41–60

123

between the means (p \ 1 · 10–10) and distributions

(p \ 1 · 10–5) for both species. In light of these

differences, we conclude that material from L. erin-

acea and L. ocellata is distinct from P. hanseni.

P. purtoni can also be distinguished from the two

other recognised species of the genus (P. notogeor-

gianum and P. minutum), both from Antarctic skates,

as follows (Table 2). It possesses fewer testes than

both species, and has smaller testes and is narrower in

maximum width than P. notogeorgianum (Table 2).

Furthermore, based on drawings in the original

description of P. notogeorgianum and P. minutum,

the testes are arranged in more numerous columns

than in P. purtoni (four to six vs two). It should be

noted that Table 2 (p. 72) in Wojciechowska (1991)

contradicts Fig. 2 (p. 184) in Wojciechowska (1990)

with respect to the situation (distribution) of the testes.

However, from the figure, it is understood that the

placement of testes in P. notogeorgianum is in the

median part of the proglottis, and that the table

referred to the distribution of vitelline follicles in this

species. The length of the bothridia reported for

P. notogeorgianum is greater than that of P. purtoni

(Table 2), and the widths of the bothridia reported for

P. notogeorgianum and P. minutum are greater than

that of P. purtoni (Table 2). The cirrus-sacs of

P. purtoni are shorter and narrower than those of the

other two species (Table 2). The oncospheres of

P. purtoni are smaller than those of P. notogeorgia-

num (16–30 · 12–28 vs 43–46 · 38–41 lm), but

those of P. minutum were not described in the original

description. P. purtoni differs from the undescribed

Pseudanthobothrium sp. (see Williams, 1966) recov-

ered from R. lintea [now Dipturus linteus (Fries,

1838)] in that it possesses fewer testes (Table 2). On

the basis of comparisons discussed above, P. purtoni

represents a distinct and new species.

Molecular analysis

Using single-strand sequencing of a nuclear marker,

54 adult Pseudanthobothrium specimens were

characterised. Of these, 21 P. purtoni n. sp. speci-

mens were recovered from L. erinacea (GenBank

accession numbers: EF207788 – EF207808) and nine

from L. ocellata (EF207809 – EF207817); 15 P.

hanseni specimens were recovered from A. radiata

(including one specimen from the North Sea donated

by P. Olson of the Natural History Museum, London,

and two specimens from material collected in North

Sea by H. Randhawa in the summer of 2005)

(EF207818 – EF20732) and nine were recovered

from M. senta (EF207833 – EF207841). Double-

strand sequencing reactions adding the T30 reverse

primer of Harper & Saunders (2001) (Fig. 1) were

completed for eight specimens (two for each para-

site-host combination) in order to verify the accuracy

of data generated with only the forward primer. This

region partly includes the D2 domain, one of the

divergent and rapidly evolving regions of the LSU

(Harper & Saunders, 2001). This domain is report-

edly useful for species-level identifications in

cestodes (Mariaux & Olson, 2001; Olson et al.,

2001).

The size of the amplified product was c.1,850

base-pairs (bp). The sequenced region corresponded

to c.800 bp, of which the middle 643 were compared

and analysed. Sequences were resolved as two

distinct clusters (Fig. 23). Of the 54 sequences

determined, 30 were assignable to P. purtoni from

the type-locality, 21 to P. hanseni from the Bay of

Fundy region, and three to P. hanseni from the North

Sea. Of the 30 sequences from P. purtoni isolates, all

were identical in sequence except for four, which

differed by one or two A/G transitions (0.16–0.31%

difference). Of the 24 sequences from P. hanseni

isolates, all were identical in sequence except for

five; one differed by a single A/G transition, one by a

C/T transition and a G/T tranversion (0.16% differ-

ence), and all three specimens from the North Sea

differed by the same A/G transition (0.47% differ-

ence). The genetic distance between isolates of both

species was 2.64–3.42%. Sequence comparison

results are summarised in Fig. 23.

Discussion

Pseudanthobothrium is characterised by: (1) stalked,

unloculate, cup-shaped bothridia and a polymorphic,

pedunculate myzorhynchus (Baer, 1956; Williams,

Figs. 11–13 Drawings of Pseudanthobothrium purtoni n. sp.

11. Complete fixed and stained mature specimen drawn in three

sections. 12. Terminal proglottis with uterus not fully

distended. 13. Scolex with extended bothridia and myzorhyn-

chus. Abbreviations: CP, cirrus-sac; GA, genital atrium; O,

ovary; T, testis; U, uterus; V, vagina; VF, vitelline follicle.

Scale-bars: 11, 1 mm; 12, 300 lm; 13, 200 lm

b

Syst Parasitol (2008) 70:41–60 53

123

54 Syst Parasitol (2008) 70:41–60

123

1966; Schmidt, 1986; Euzet, 1994). The specimens

examined possess these characteristics and were

therefore assigned to this genus. Specimens recov-

ered from Amblyraja radiata and Malacoraja senta

were identified as P. hanseni on the basis of

morphometric and molecular data, whereas speci-

mens recovered from Leucoraja erinacea and

L. ocellata could not be assigned to any known

species of the genus; therefore, described herein as

new to science as P. purtoni n. sp. The recovery of

P. purtoni from L. ocellata suggests that specimens

identified as Anthobothrium cornucopia van Bene-

den, 1850 by Myers (1959) might well have been

P. purtoni

Questionable identifications of hosts and/or para-

sites challenge the validity of studies investigating

host-specificity. Even though it is generally accepted

that tetraphyllideans exhibit strict host-specificity,

there are several records of phyllobothriid tetraphylli-

deans infecting multiple hosts (Schmidt, 1986), such

as Caulobothrium longicolle (Linton, 1890), Myzo-

phyllobothrium rubrum Shipley & Hornell, 1906,

Phormobothrium affine (Olsson, 1867) and Scypho-

phyllidium giganteum (van Beneden, 1858), although

many identifications need to be reconfirmed. Inves-

tigating host-specificity using host records from

published accounts requires verification and confir-

mation of both host and parasite identity. The advent

of molecular tools allows taxonomists to confirm

identifications, thus supplementing character-based

identifications.

That being said, the recovery of P. purtoni from

two species of Leucoraja and of P. hanseni from

A. radiata and M. senta is confirmed using molecular

Figs. 14–19 Scanning electron micrographs of Pseudantho-bothrium purtoni n. sp. recovered from Leucoraja erinaceainhabiting waters surrounding the West Isles of the Bay of

Fundy, NB, Canada: 14. Scolex bearing four stalked bothridia

and a myzorhynchus. 15. Lateral view of a single non-loculate,

cup-shaped bothridium. 16. Distal bothridial surface showing

filitriches. 17. Proximal half of proximal bothridial surface

showing blade-like microtriches. 18. Proximal bothridial

surface showing parts of the naked distal-half and blade-like

microtriches on proximal-half. 19. Lateral view of the

retractable myzorhynchus. Abbreviations: DBS, distal bothri-

dial surface; DHPBS, distal-half of proximal bothridial surface;

M, myzorhynchus; PBS, proximal bothridial surface; PHPBS,

proximal-half of proximal bothridial surface; S, stalk. Scale-bars: 14, 100 lm; 15, 60 lm; 16, 4 lm; 17, 8 lm; 18, 20 lm;

19, 50 lm

Fig. 20 Bar graph showing the frequency distribution of the number of testes per proglottis for Pseudanthobothrium purtoni n. sp.

and P. hanseni specimens examined during this study

b

Syst Parasitol (2008) 70:41–60 55

123

Fig. 21 Bar graph showing the frequency distribution of the length of cirrus-sacs for Pseudanthobothrium purtoni n. sp. and

P. hanseni specimens examined during this study

Fig. 22 Bar graph showing the frequency distribution of the maximum width of the strobila for Pseudanthobothrium purtoni n. sp.

and P. hanseni specimens examined during this study

56 Syst Parasitol (2008) 70:41–60

123

Fig. 23 Phylogram (Neighbour-joining) displaying two clusters: one of the included specimens for Pseudanthobothrium purtoni n. sp.;

and one of the included specimens of P. hanseni (species, ex host, voucher number). Included is a matrix of the number of actual

nucleotide differences (out of 643 bp.) between sequences of Pseudanthobothrium specimens analysed from the different host species:

erinacea, Leucoraja erinacea; ocellata, L. ocellata; radiata, Amblyraja radiata; senta, Malacoraja senta

Syst Parasitol (2008) 70:41–60 57

123

tools and challenges the dogma of the strict specific-

ity of tetraphyllidean tapeworms, thus demonstrating

the need to extend this type of study to other genera

of the group. The low observed intraspecific T16-

sequence (D2 variable domain) variation within

species of Pseudanthobothrium (\0.31% in P. pur-

toni and \0.47% P. hanseni, including isolates from

northeast and northwest Atlantic for the latter) is

consistent with variation observed between individ-

uals of other species of tetraphyllidean (Brickle et al.,

2001; Reyda & Olson, 2003; Agusti et al., 2005;

Randhawa et al., 2007). The 2.64–3.42% sequence

divergence observed between the two Pseudantho-

bothrium spp. is greater than the observed

intraspecific T16-sequence variation. This amount

of difference is greater than expected between two

populations of the same morphological species of

tetraphyllidean (Brickle et al., 2001; Reyda & Olson,

2003; Agusti et al., 2005; Randhawa et al., 2007),

thus supporting our conclusion that these are two

genetically distinct species. Thus, all specimens were

assigned unequivocally to one of the two Pseudan-

thobothrium spp. All specimens of P. purtoni

recovered from L. erinacea and L. ocellata formed

one cluster, while those of P. hanseni recovered from

A. radiata and M. senta formed the second (Fig. 23).

Therefore, P. purtoni seems to be specific to the

L. erinacea and L. ocellata host-pair, whereas

P. hanseni seems to be specific to the A. radiata

and M. senta host-pair.

On the basis of the character-based identifications

and molecular confirmations described in the present

study, it is suggested that at least one of the cercoid

larvae of Pseudanthobothrium spp. (Jarecka & Burt,

1984) may have been misidentified and should now be

recognised as P. purtoni. These larvae were recovered

from harpacticoid copepods experimentally infected

with oncospheres collected from the type-host

(L. erinacea) in the type-locality (Passamaquoddy

Bay, NB) (Jarecka & Burt, 1984) for the new species.

Furthermore, the unidentified Pseudanthobothrium

specimens recorded in Caira et al. (1999, 2001) from

L. erinacea may be tentatively identified as P. purtoni,

as they were also recovered from the type-host and

type-locality, and those reported in Randhawa et al.

(2007) from L. erinacea and L. ocellata are positively

identified as P. purtoni. Additionally, the unidentified

Pseudanthobothrium specimens recorded in Caira

et al. (2001) from M. senta may be tentatively

identified as P. hanseni on the basis of morphological

and molecular similarities noted herein.

The fact that both Pseudanthobothrium spp. are

found respectively in two host species is interesting

(also see Randhawa et al., 2007). While all four hosts

share some environmental affinities, they are dissim-

ilar enough to separate them into two ecological

species pairs (McEachran & Musick, 1975; McEach-

ran et al., 1976). The amphipods Leptocheirus

pinguis and Maera danae were the most prevalent

and abundant prey items recovered from the stomachs

of Leucoraja erinacea and L. ocellata (Randhawa,

unpub. obs.). This is similar to the findings of

McEachran et al. (1976), who identified Leptocheirus

pinguis as the most abundant and prevalent prey item

in the diets of these two skates. On the other hand,

polychaete worms and decapod crustaceans were

identified as the most abundant and prevalent prey

items of A. radiata, whereas those of Malacoraja

senta were decapod and euphausid shrimps (McEach-

ran et al., 1976). To date, no complete life-cycle for a

tetraphyllidean has been elucidated. Preliminary

observations indicate that Leptocheirus pinguis, or

possibly Maera danae, act as potential intermediate

host(s) for P. purtoni. On March 6th 2003, 23

Leucoraja erinacea, measuring 11–14 cm, were

caught and examined for parasites and their stomach

contents were analysed. Specimens measuring 9 and

10 cm were also recovered, but were still feeding on

their yolk sacs (stomach were empty), therefore,

making it improbable for them to acquire the parasite

(cestodes infections are generally acquired via inges-

tion). Of the 23 L. erinacea, 16 were infected with

P. purtoni. Leptocheirus pinguis and Maera danae

were the only prey items recovered from the stom-

achs of these individuals. Increased sampling of the

amphipod community in the northwest Atlantic, or

experimental infections, are necessary before an

unequivocal identification of the intermediate host(s)

for P. purtoni can be confirmed. Additionally,

sampling polychaete worms, decapod crustaceans,

and euphausid shrimp for tetraphyllidean cercoids

and juveniles may enable us to identify the interme-

diate host(s) for P. hanseni.

Although all four skate specie included in this

study are known to be sympatric (McEachran &

Musick, 1975; McEachran et al., 1976), Leucoraja

erinacea and L. ocellata prefer sandy and gravelly

bottoms (Packer et al., 2003a,b), whereas Malacoraja

58 Syst Parasitol (2008) 70:41–60

123

senta shows a strong preference for a soft muddy

substrate (Packer et al., 2003c). Amblyraja radiata

shows little preference for habitat (Packer et al.,

2003d). However, A. radiata and M. senta were

positively associated (McEachran & Musick, 1975;

McEachran et al., 1976), but the difference in habitat

preference may be related to A. radiata being the

more abundant of the two species over their range

(McEachran et al., 1976). L. erinacea and L. ocellata

were positively associated by abundance and were

often negatively associated with A. radiata and

M. senta (see McEachran & Musick, 1975). These

substrate preferences may explain, at least in part,

some of their dietary differences as substrate may

determine the availability of certain prey items.

Therefore, the presence of P. purtoni in L. erinacea

and L. ocellata, and that of P. hanseni in A. radiata

and M. senta, must relate to substrate preference and

its corresponding prey biota.

Currently, there is no skate phylogeny available

at the species level. However, there is a phylogeny

of rajid skates based on 31 taxa (genera) (McEach-

ran & Dunn, 1998). All skates included in our

study belong to the subfamily Rajinae and, with the

exception of M. senta, to the tribe Amblyrajini (see

McEachran & Dunn, 1998). Our preliminary work

indicates that there is no observed pattern of

co-speciation between echeneibothriine tetraphylli-

deans and their rajid hosts; however, this needs to

be investigated further. The ecological separation of

the two rajid skate species-pairs, the lack of strict

host-specificity of P. hanseni and P. purtoni, and

the lack of an observed pattern of co-speciation

indicate that host and/or parasite ecology has a

stronger influence on host-parasite associations than

phylogenetics. This will be investigated in a later

study.

In summary, the morphological and molecular

differences noted above support the recognition of a

fourth species of Pseudanthobothrium, P. purtoni n. sp.

The presence of mature P. purtoni and P. hanseni in

two rajid species pairs, respectively, may be attributed,

at least in part, to similarities in host diet and substrate

preferences for each host-pair, and is evidence that

similar studies are necessary to address the issue of

specificity of tetraphyllidean cestodes.

Acknowledgements Special thanks are due to J. N. Caira,

H. H. Williams and C. P. Keeling for encouraging the senior

author to pursue a career in parasitology and to work on

tetraphyllidean worms, and also for their hospitality and advice.

The authors are grateful to staff of the HMSC: F. Purton and

D. Parker for their technical assistance and availability;

T. Hurley and M. Burgess for assisting with collection of

specimens; and E. Carter who went beyond the call of duty as

Captain of the R/V ‘W. B. Scott’. The help of D. Loveless and W.

Minor Mate/Engineer and Captain of the CCGS ‘Pandalus III’,

respectively, in collecting by Department of Fisheries and

Oceans Canada (DFO) personnel, is also gratefully

acknowledged. We thank S. Belfry of the Electron Microscopy

Unit at the University of New Brunswick, who was instrumental in

teaching the senior author the essentials of scanning electron

microscopy and assisted in the preparation of the material. The

senior author thanks C. E. Lane and L. LeGall as well as T. Moore

and M. Surette for their technical assistance and advice regarding

molecular methodologies. We are most grateful to A. R. Breton and

two anonymous and diligent reviewers for providing useful

comments on this manuscript. The Institute of Parasitology of

McGill University, the HMSC and the Department of Biology of the

University of New Brunswick (UNB) provided lab space and other

research facilities. The following financial assistance is gratefully

acknowledged: Graduate Teaching and Research Assistantships at

UNB, two McGill R. C. Frazee Research Scholarships at HMSC,

one UNB R. C. Frazee Research Scholarship at HMSC, a W. B.

Scott Graduate Research Scholarship in Ichthyology, and two

HMSC Summer Student Assistantships to the senior author. In

addition, the Natural Sciences and Engineering Research Council of

Canada (NSERC) provided assistance through operating/discovery

grants to Drs G. W. Saunders, M. E. Scott and M. D. B. Burt; a

Steacie Fellowship to G. W. Saunders; and a Major Facilities

Access Grant to HMSC. The Canada Research Chairs Program

(G. W. Saunders) also provided financial assistance.

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