Molecules, morphology and morphometrics of Cainocreadium labracis and Cainocreadium dentecis n. sp....

Molecules, morphology and morphometrics of Cainocreadium labracisand Cainocreadium dentecis n. sp.

(Digenea: Opecoelidae) parasitic in marine ®shesq

O. Joussona,b,*, P. Bartolib

aDeÂpartement de Zoologie et Biologie Animale, UniversiteÂ de GeneÁve,154 route de Malagnou, CH-1224 CheÃne-Bougeries/Geneva, SwitzerlandbCentre d'OceÂanologie de Marseille, UMR CNRS 6540, F-13288 Marseille, France

Received 11 December 2000; received in revised form 14 February 2001; accepted 14 February 2001

Abstract

Molecular, morphological and morphometric analyses were conducted on several samples of Cainocreadium labracis (Opecoelidae), a

trematode parasitic in marine teleosts. The samples were isolated from several specimens of Dicentrarchus labrax, the type host, and Dentex

dentex. The molecular analysis of complete Internal Transcribed Spacer sequences of ribosomal DNA revealed that specimens isolated from

each host species form two well-de®ned groups, whose sequence divergence reaches 7.5%. The morphological study showed that the two

groups can be distinguished by several characters, including the level of maximum body breadth, the relative position of the testes, the shape

of the cirrus pouch, and the extent of the uterus. Multivariate analyses of morphometrics demonstrated consistency of most of the characters

for discriminating the two groups. Our results show that C. labracis specimens isolated from D. labrax and D. dentex represent clearly

distinct entities from molecular, morphological and statistical points of view, which has enabled us to describe a new species, Cainocreadium

dentecis n. sp. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Cainocreadium labracis; Cainocreadium dentecis n. sp; Molecules; Morphology; Morphometrics; Host speci®city

1. Introduction

A key problem in evolutionary studies of parasitic organ-

isms lies in the knowledge of their degree of host-speci®-

city, which can be de®ned by the number of host species a

parasite is able to infect (Combes, 1995). Among the

Digenea, the miracidial stage that infects the molluscan

®rst host usually shows a strict speci®city (Gibson and

Bray, 1994; Nunez and De Jong-Brink, 1997). The speci®-

city of the developmental stages infecting second and de®-

nitive host may be less strict. In such cases it is considered

that speci®city is mainly driven by factors associated with

host ecology, such as habitat use or feeding preferences

(Adamson and Caira, 1994; Jousson et al., 2000).

The Digenea lack conserved and hard structures, which

makes the identi®cation of sibling species problematic. As

a consequence, discrete morphological differences observed

between specimens collected from different hosts species

have often been attributed to phenotypic plasticity (Bartoli

et al., 1989a). Given the dif®culties of closely-related species

identi®cation, species diversity and the degree of host-speci-

®city may have been underestimated among these parasites.

Recently, molecular data such as Internal Transcribed

Spacer (ITS) ribosomal DNA sequences have been used to

distinguish among closely related digenean species (Luton

et al., 1992; Adlard et al., 1993; DespreÁs et al., 1995; Jous-

son et al., 1998; Jousson and Bartoli, 2000). Comparative

analyses of morphometrics have been used in studies

attempting the identi®cation of closely related nematodes

(Hugot et al., 1995; Stock and Kaya, 1996) and trematodes

(Fried et al., 1997; Kostadinova et al., 2000) species.

In the present work, we have chosen as a model a well

known species of the family Opecoelidae Ozaki, 1925:

Cainocreadium labracis (Dujardin, 1845) Nicoll, 1909.

This species has been reported from Dicentrarchus labrax

(Moronidae) (in Maillard, 1971), Dicentrarchus punctatus

(in Fischthal, 1982), Dentex dentex (Sparidae) (in Stossich,

1905; Janiszewska, 1953; Bartoli et al., 1989b), Platichthys

¯esus (Pleuronectidae), Scophthalmus rhombus (Scophthal-

midae) (in Stossich, 1905), Labrus bergylta (Labridae) (in

Linstow, 1878), and Gaidropsarus mediterraneus (Gadidae)

(in Osmanov, 1940). We investigated the digenean fauna of

International Journal for Parasitology 31 (2001) 706±714

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(01)00180-1

www.parasitology-online.com

q DNA sequences analysed in this study have been deposited in the

EMBL / GenBank database under accession numbers AJ241795,

AJ241806 and AJ241808.

* Corresponding author. Tel.: 141-22-3498644; fax: 141-22-3492647.

E-mail address: [email protected] (O. Jousson).

D. labrax (the type host of C. labracis), D. dentex, L.

bergylta, and G. mediterraneus. Cainocreadium labracis

specimens were found only in D. labrax and D. dentex.

To test the hypothesis of the existence of a host-associated

species complex within C. labracis, we obtained morpholo-

gical data, as well as ITS ribosomal DNA sequences of

several C. labracis specimens isolated from the two de®ni-

tive host species. These ITS sequences from adults were

compared with opecoelid cercariae sequences obtained in

a previous study (Jousson et al., 1999). Additionally, a

comparative morphometric analysis was conducted on para-

sites isolated from the two host species in order to examine

the degree of the variability in the metrical characteristics of

the adults and to assess their value in discriminating species.

2. Material and methods

2.1. Sample collection

Living adult specimens of C. labraciswere isolated from

the digestive tract of several individuals of D. labrax (40

parasite specimens) and D. dentex (40 specimens). The

hosts were collected from the north-western Mediterranean

(Gulf of Marseille, and Scandola Natural Reserve, France).

2.2. Molecular data

DNA of several C. labracis specimens from both hosts was

extracted in guanidine lysis buffer (Maniatis, 1982), precipi-

tated with isopropanol and dissolved in distilled water. PCR

ampli®cations were performed in a total volume of 50ml with

an ampli®cation pro®le consisting of 40 cycles of 30 s at

948C, 30 s at 528C and 120 s at 728C, followed by 5 min at

728C for ®nal extension. The ITS region of the rDNA was

ampli®ed using a trematode-speci®c primer located 195 bp

from the 3 0 end of the 18S rDNA (S20T2: 5 0-GGTAAGTG-

CAAGTCATAAGC-3 0) and an universal primer at 5 0 end of

the 28S rDNA (L5: 5 0-TTCACTCGCCATTACTT-3 0).Ampli®ed PCR products were puri®ed using High Pure

PCR Puri®cation Kit (Roche Diagnostics), and sequenced

O. Jousson, P. Bartoli / International Journal for Parasitology 31 (2001) 706±714 707

Table 1

Measurements (in micrometres) of ovigerous specimens of the two speciesa

Parasite species Cainocreadium labracis Cainocreadium dentecis n. sp.

Host species Dicentrarchus labrax Dentex dentex

Variable Range Mean SD Range Mean SD

BL 3400±7055 4873 943 2253±4781 3161 697

BWA 1020±2125 1474 339 637±1714 970 262

BWT 829±2125 1302 361 710±1806 1142 296

FO 1063±2318 1700 318 606±1594 1106 277

HI 1636±3910 2452 520 1148±2656 1671 387

OSL 314±666 445 105 189±341 246 38

OSW 336±744 498 133 213±352 270 35

PS 0±107 45 20 0±160 39 37

PPH 16±149 65 37 0±91 20 23

PHL 192±448 283 75 112±240 152 28

PHW 144±480 285 84 107±213 162 25

OE 53±240 125 50 27±267 101 49

PHC 80±246 216 81 64±216 133 40

PHG 240±1065 488 171 123±464 182 92

PHS 496±1396 998 208 197±986 648 220

VSL 437±1009 677 160 213±533 384 67

VSW 544±1275 790 218 304±533 385 56

CPL 533±1450 857 275 373±1103 681 175

CPW 107±400 190 72 107±240 156 36

SVL 139±1226 481 265 160±573 361 106

VSO 53±490 275 111 53±464 218 106

OL 235±586 350 86 176±480 320 79

OW 213±666 373 109 181±480 296 73

ATL 373±640 462 76 320±746 442 115

ATW 346±906 528 145 267±650 443 100

PTL 405±746 503 75 373±800 528 120

PTW 267±906 504 124 277±692 438 101

TTL 680±1296 905 146 637±1207 737 156

PTS 533±1934 986 303 320±906 565 132

SRL 133±533 252 88 107±426 245 83

SRW 107±320 187 51 69±240 146 40

EL 65±87 76 5 59±74 66 2.7

EW 33±48 40 3 29±43 36 2.5

a (n � 27). SD: Standard deviation.

directly using an ABI-377 DNA automated sequencer

(Perkin±Elmer), all according to the instructions of the

manufacturers. For DNA sequencing, the ampli®cation

primers were used, together with an internal reverse primer

located at 5 0 end of the 5.8S rDNA (5.8S1: 5 0-GCTGCGCTCTTCATCGACA-3 0). The sequences were

aligned manually using the GDE 2.2 (Larsen et al., 1993)

and their divergence was analysed using the Phylo-win

program (Galtier and Gouy, 1996).

2.3. Morphological data

Hosts (D. labrax and D. dentex) were necropsied imme-

diately after death for parasite studies. The digeneans

collected were studied while still living and later in perma-

nent preparations. Only ovigerous specimens have been

used in the descriptions. Cainocreadium labracis specimens

from each host were ®xed in Bouin's ¯uid between a slide

and a coverglass without pressure, coloured in acetic

carmine and mounted in Canada balsam. Studies were

conducted using a differential interference contrast micro-

scope and illustrations were made with a camera lucida.

Measurements are given in micrometres.

2.4. Variables

The following metrical features (variables) were

subjected to analyses (see Table 1): (1) body length (BL);

(2) body width at acetabulum level (BWA); (3) body width

at anterior testis level (BWT); (4) forebody (FO); (5) hind-

body (HI); (6) oral sucker length (OSL); (7) oral sucker

width (OSW); (8) preoral space (PS); (9) prepharynx

(PPH); (10) pharynx length (PHL); (11) pharynx width

(PHW); (12) oesophagus (OE); (13) pharynx to caecal bifur-

cation (PHC); (14) pharynx to genital aperture (PHG); (15)

pharynx to ventral sucker (PHS); (16) ventral sucker length

(VSL); (17) ventral sucker width (VSW); (18) cirrus pouch

length (CPL); (19) cirrus pouch width (CPW); (20) seminal

vesicle length (SVL); (21) ventral sucker to ovary (VSO);

(22) ovary length (OL); (23) ovary width (OW); (24) ante-

rior testis length (ATL); (25) anterior testis width (ATW);

(26) posterior testis length (PTL); (27) posterior testis width

(PTW); (28) two testes length (TTL); (29) post-testicular

space (PTS); (30) seminal receptacle length (SRL); (31)

seminal receptacle width (SRW); (32) egg length (EL);

(33) egg width (EW).

2.5. Statistical analyses

Initially, the descriptive univariate statistics (means and

SD) were calculated from original data. Statistical analyses

were performed on 33 metric characters (Table 1) using the

ADE-4 package (Thioulousse et al., 1997). An initial prin-

O. Jousson, P. Bartoli / International Journal for Parasitology 31 (2001) 706±714708

Fig. 1. Complete alignment of the internal transcribed spacer 1 ribosomal DNA for adults and cercariae of Cainocreadium labracis and C. dentecis n. sp. DIC1-

3: adults from Dicentrarchus labrax; CAL1: cercaria from Calliostoma striatum; DEN1-3: adults from Dentex dentex; HAL1: cercaria from Haliotis

tuberculata.

cipal component analysis (PCA) was performed using

normalised data. The size effect was controlled by measur-

ing only ovigerous specimens. A discriminant analysis was

performed to determine which part of the variance can be

explained by the differences between the two groups (i.e. the

specimens isolated from both hosts) and to verify results

obtained by PCA. A variance analysis (ANOVA) was

performed to determine which characters signi®cantly

discriminate the two groups.

3. Results

3.1. Molecular data

The analysed data set of the ITS region (ITS1 1 5.8S 1ITS2) and part of the ¯anking genes coding for 18S rRNA

and 28S rRNA of Cainocreadium specimens comprises

1164 sites. When all gaps-containing sites are removed,

there remain 1125 sites, of which 84 are variable (7.5%).

Two types of ITS sequences were found; one type includes

all adult specimens isolated from D. labrax and the cercaria

found in Calliostoma striatum. A second type includes all

adult specimens isolated from D. dentex and the cercaria

found in Haliotis tuberculata (Fig. 1). The ITS sequence

divergence between the two types reaches 7.5% (84 variable

sites), whereas the divergence within each type does not

exceed 0.8% (®ve variables sites). Both sequence types do

not differ signi®cantly regarding GC content (51.4±51.8%)

and complete ITS length (938±945 nucleotides). The

presence of a particular ITS type in parasites of each de®-

nitive host species suggests the occurrence of two species of

Cainocreadium: C. labracis from D. labrax and C. dentecis

n. sp. from D. dentex.

3.2. Morphological data

A complete description of C. labracis from D. dentex was

given by Bartoli et al. (1989b). Common features of adults

C. labracisfrom D. labrax and C. dentecis n. sp. from D.

dentex can be summarised as follows: Body elongate-oval to

fusiform (Figs. 2 and 3). Tegument unarmed. Ventral sucker

slightly protuberant to sessile. Prepharynx absent. Pharynx

large. Oesophagus short. Caeca end blindly near posterior

extremity. Testes two, entire, oblique to tandem, near

middle of hindbody. Cirrus-sac claviform (Figs. 4 and 5).

Internal seminal vesicle, wide, coiled. Pars prostatica short.

Ejaculatory duct long; may avert to form unarmed cirrus.


Fig. 2. Cainocreadium labracis(Dujardin, 1845). Slightly ¯attened adult

specimens from the duodenum of Dicentrarchus labrax. (A) Dorsal view.

(B) Ventral view.

Fig. 3. Cainocreadium dentecis n. sp. Slightly ¯attened adult specimens

from the duodenum of Dentex dentex. (A) Ventral view. (B) Dorsal view.

Genital atrium small. Genital pore median, post-bifurcal.

Ovary lobed; immediately anterior or antero-lateral to ante-

rior testis. Canalicular seminal receptacle and Laurer's

canal present (Figs. 6 and 7). Uterine ®eld pre-ovarian or

pre-testicular, usually restricted to hindbody. Vitellarium

follicular; lateral ®elds extend into forebody, only con¯uent

in post-testicular ®eld and dorsally in forebody. Excretory

vesicle tubular, reaches to level of ovary.

Cainocreadium dentecis n. sp. is distinguished from C.

labracisin its smaller measurements (Table 1, Fig. 8), the

level of maximum body breadth, sessile ventral sucker,

diagonally organised testes, subrectilinear cirrus pouch,

dextral ovary, pretesticular uterus, and S-shaped excretory

vesicle (Table 2, Fig. 8).

3.3. Taxonomic summary

Cainocreadium dentecis n. sp.

Synonym: Cainocreadium labracis (Dujardin, 1845)

Nicoll, 1909 of Bartoli et al. (1989b); Jousson et al. (1999).

Material studied: 40 adults from the pyloric caeca and

anterior intestine of Dentex dentex from off Scandola

Natural Reserve, Corsica, France. Specimens deposited in

1988 by Bartoli et al. (1989b): BM(NH) Reg. No.

1988.4.22.9±11.

New specimens: Type BM(NH) Reg. No. 2000.12.1.1;

Paratypes: BM(NH) Reg. No. 2000.12.1.2±6.

3.4. Statistical analyses

The results of PCA of the normalised measurements of

Cainocreadium specimens are given in Fig. 9. Axis 1

explained 61.5%, axis 2, 10.3%, and axis 3, 5.9% of the

variation (axes 1, 2 and 3 combined, 77.7%). A discriminant

analysis was performed on the set of mensural data. The

between- and within-groups inertia represent 30.8 and

69.2% of the total variation, respectively. We computed the

total between-groups inertia for each random distribution


Fig. 4. Cainocreadium labracis (Dujardin, 1845). Slightly ¯attened adult

specimen from the duodenum of Dicentrarchus labrax. Terminal genitalia.

Ventral view. Anterior part of ventral sucker omitted for clarity.

Fig. 5. Cainocreadium dentecis n. sp. Slightly ¯attened adult specimen

from the duodenum of Dentex dentex. Terminal genitalia. Ventral view.

Left side of ventral sucker omitted for clarity.

(1000 permutations) of individuals within groups. The sum

of inertia between groups is signi®cantly high (frequency, 1),

indicating that the groups resulting from our hypothesis

(specimens from different hosts represent distinct entities)

are signi®cantly discriminated. The between-groups analysis

shows that the discrimination between the two groups can be

explained in totality along the ®rst axis. Thus, to obtain a

bidimensional representation of the discriminant analysis,

morphometric separation was expressed using the values of

between-groups analysis plotted against the most discrimi-

nant variable, which is the length of eggs (EL) (Fig. 10). A

variance analysis (ANOVA) was performed to determine

which characters most contribute to discriminate the two

groups. The analysis showed that the mean value of most

of the variables were signi®cantly different between the

two groups (P , 0:05). The characters that allow the best

discrimination between groups (P � 0) are EL, VSW,

OSL, OSW, FO, VSL, and PHL. The characters which

means did not show signi®cant differences (P . 0:05) are:

PS, CPW, SVL, VSO, PTL, and SRL (Fig. 11).

4. Discussion

The analysis of independent data sets consisting of ITS

rDNA sequences, the description of morphological struc-


Fig. 6. Cainocreadium labracis (Dujardin, 1845). Slightly ¯attened adult

specimen from the duodenum of Dicentrarchus labrax. Proximal region of

female reproductive system. Dorsal view.

Fig. 7. Cainocreadium dentecis n. sp. Slightly ¯attened adult specimen

from the duodenum of Dentex dentex. Proximal region of female reproduc-

tive system. Ventral view.

Fig. 8. Habitus of the two adult species reconstructed from mean data of the

Table 1. (A) Cainocreadium labracis. (B) Cainocreadium dentecis n. sp.

Squared area represent the uterine loops; stippled area represent the vitel-

line follicles.

tures and multivariate analyses of morphometrics clearly

showed a high degree of clustering between adults of C.

labracisisolated from D. labrax and D. dentex, demonstrat-

ing the occurrence of distinct species. Cainocreadium

labracis occurs in D. labrax, whereas C. dentecis n. sp.

occurs in D. dentex.

The amount of genetic divergence between the two

species is quite high (7.5%), and greatly exceeds the intras-

peci®c variation (0.8%). This degree of variability is much

higher than values of interspeci®c ITS sequence divergence

observed for other congeneric opecoelids (Jousson et al.,

1999) and among other digenean families (Adlard et al.,

1993). The occurrence of a host-associated species complex

within C. labracis is reinforced by the fact that correspond-

ing cercariae were isolated from two species of gastropods

belonging to different families of the order Archaeogastro-

poda: Haliotis tuberculata (Haliotidae) and Calliostoma

striatum (Trochidae) (Jousson et al., 1999). These cercariae

were found to correspond to C. labracis and C. dentecis,

respectively (Fig. 1). The more obvious non-metrical char-

acters that allow to distinguish the two adult species are the

disposition of the testes, the position of the uterus, and the

shape of the cirrus pouch (Fig. 8). Discriminant statistical

analyses showed that the differences between the two

groups (i.e. specimens isolated from C. labracis and from

C. dentecis) are highly signi®cant. The variance analysis

(ANOVA) demonstrated that most of the metrical charac-

ters show signi®cant differences in mean between C. labra-

cis and C. dentecis and thus allow a good discrimination

between the two species. The characters with the best scores


Table 2

Main non-metrical features differentiating the two species

Parasite species Cainocreadium labracis Cainocreadium dentecisn.sp.

Host species Dicentrarchus labrax Dentex dentex

Level of maximum body width Ventral sucker Anterior testis

Ventral sucker Prominent; often with peripheral folds Sessile; peripheral folds absent

Testes Rounded, tandem Somewhat triangular, diagonal

Anterior testis Median, smaller than acetabulum Sinistral, larger than acetabulum

Cirrus pouch S-shape Rectilinear

Cirrus Heavily sinuous Rectilinear

Ovary Submedian, in front of anterior testis Dextral, at level of anterior testis

Number of ovarian lobes 3±5 3

Uterus Preovarian Pretesticular

Vitelline follicles Very abundant Less abundant

Excretory vesicle Sinuous S-shaped

Fig. 9. Principal component analysis of C. labracis (1) and C. dentecis n. sp.

(2). Star-representation of individuals scores in the plane F1 £ F2 using

normalised variables.

Fig. 10. Morphometric separation of C. labracis (1) and C. dentecis n. sp.

(2). First axis of the between-groups discriminant analysis plotted against

eggs length (EL).

(P � 0) are the eggs length (EL), the dimensions of suckers

(OSL, OSW, VSL, and VSW), the forebody (FO), and the

pharynx length (PHL) (Fig. 11). The application of indepen-

dent methods and data sets enabled us to distinguish species

unambiguously. Therefore, future taxonomic studies of

digeneans at the species or genera level should include

detailed morphological, morphometric and molecular

analyses to provide accurate results. Several host-associated

factors have been shown to have an in¯uence on parasite

morphology and development (e.g. Machado-Silva et al.,

1994). The amount of genetic variation occurring between

C. labracis and C. dentecis removes the possibility that the

morphological differences we observed represent phenoty-

pic plasticity due to the nature of the host. Further work will

show if the other ®sh hosts listed for C. labracis (D. punc-

tatus, P. ¯esus, S. rhombus, L. bergylta, and G. mediterra-

neus) are also infected by a speci®c parasite species. Our

results, together with those obtained in a previous study

(Jousson and Bartoli, 2000) con®rm that the species diver-

sity has been underestimated among digenean trematodes

parasitic in marine ®shes. Further studies will show if such a

restriction of the de®nitive host spectrum may be related to

®sh habitat and/or feeding preferences.

Acknowledgements

We are indebted to Emmanuel Castella for his help in

performing and interpreting statistical analyses, and to

JoseÂ Fahrni for sequencing assistance. We also thank Alan

Johnson and two anonymous referees for their suggestions

which improved the manuscript.

References

Adamson, M.L., Caira, J.N., 1994. Evolutionary factors in¯uencing the

nature of parasite speci®city. Parasitology 109, S85±95.

Adlard, R.D., Barker, S.C., Blair, D., Cribb, T.H., 1993. Comparison of the

second internal transcribed spacer (ribosomal DNA) from populations

and species of Fasciolidae (Digenea). Int. J. Parasitol. 23, 423±5.

Bartoli, P., Bray, R.A., Gibson, D.I., 1989a. The Opecoelidae (Digenea) of

sparid ®shes of the western Mediterranean. III. Macvicaria Gibson and

Bray, 1982. Syst. Parasitol. 13, 167±92.

Bartoli, P., Gibson, D.I., Bray, R.A., 1989b. The Opecoelidae (Digenea) of

sparid ®shes of the western Mediterranean. IV. Peracreadium Nicoll,

1909 and Cainocreadium Nicoll, 1909. Syst. Parasitol. 14, 53±67.

Combes, C., 1995. Interactions Durables. Masson, Paris.

DespreÁs, L., Kruger, F.J., Imbert-Establet, D., Adamson, M.L., 1995. ITS2

ribosomal RNA indicates Schistosoma hippopotami is a distinct species.

Int. J. Parasitol. 25, 1509±14.

Fischthal, J.H., 1982. Additional records of digenetic trematodes of marine

®shes from Israel's Mediterranean coast. Proc. Hel. Soc. Wash. 49, 34±

44.

Fried, B., Mueller, T.J., Frazer, B.A., 1997. Observations on Echinostoma

revolutum and Echinostoma trivolvis in single and concurrent infections

in domestic chicks. Int. J. Parasitol. 27, 1319±22.

Galtier, N., Gouy, M., 1996. SEAVIEW and PHYLO_WIN: two graphic

tools for sequence alignment and molecular phylogeny. Comp. App.

Biosci. 12, 543±8.

Gibson, D.I., Bray, R.A., 1994. The evolutionary expansion and host-para-

site relationships of the Digenea. Int. J. Parasitol. 24, 1213±26.

Hugot, J.P., Morand, S., Gardner, S.L., 1995. Morphology and morpho-

metrics of three oxyurids parasitic in primates with a description of

Lemuricola microcebi n. sp. Int. J. Parasitol. 25 (9), 1065±75.

Janiszewska, J., 1953. Some adriatic sea ®sh trematodes. Zool. Pol. 6, 20±

48.

Jousson, O., Bartoli, P., 2000. The life-cycle of Opecoeloides columbellae

(Pagenstecher, 1863) n. comb. (Digenea, Opecoelidae): evidence from

molecules and morphology. Int. J. Parasitol. 30, 747±60.

Jousson, O., Bartoli, P., Pawlowski, J., 1998. Molecular phylogeny of

Mesometridae (Trematoda, Digenea) with its relation to morphological

changes in parasites. Parasite 5, 365±9.

Jousson, O., Bartoli, P., Pawlowski, J., 1999. Molecular identi®cation of

developmental stages in Opecoelidae (Digenea). Int. J. Parasitol. 29

(11), 1853±8.

Jousson, O., Bartoli, P., Pawlowski, J., 2000. Cryptic speciation among

intestinal parasites (Trematoda: Digenea) infecting sympatric host

®shes (Sparidae). J. Evol. Biol. 13 (5), 778±85.

Kostadinova, A., Gibson, D.I., Biserkov, V., Ivanova, R., 2000. A quanti-

tative approach to the evaluation of the morphological variability of two

echinostomes, Echinostoma miyagawai Ishii, 1932 and E. revolutum

(Frolich, 1802), from Europe. Syst. Parasitol. 45 (1), 1±15.

Larsen, N., Osen, G.J., Maidak, B.L., Mc Caughey, M.J., Overbeek, R.,


Fig. 11. Variance analysis (ANOVA) showing the capacity of the metric characters to distinguish the two species. Signi®cant differences in means are

represented by values lower than 0.05.

Macke, T.J., Marsh, T.L., Woese, C.R., 1993. The ribosomal database

project. Nucleic Acids Res. 21, 3021±3.

Linstow, O.F.B., 1878. Compendium der Helminthologie. Hahn'sche

Buchhandlung, Hannover, p. 382.

Luton, K., Walker, D., Blair, D., 1992. Comparisons of ribosomal internal

transcribed spacer from two congeneric species of ¯ukes (Platyhel-

minthes: Trematoda: Digenea). Mol. Biochem. Parasitol. 56, 323±8.

Machado-Silva, J.R., Galvao, C., Presgrave, O.A., Rey, L., Gomes, D.C.,

1994. Host-induced morphological changes of Schistosoma mansoni

Sambon, 1907 male worms. Mem. Inst. Oswaldo Cruz. 89 (3), 411±6.

Maillard, C., 1971. Cycle eÂvolutif de Cainocreadium labracis (Dujardin,

1845) (Trematoda, Allocreadiidae). C. R. Acad. Sci. 272, 3303±6.

Maniatis, T., 1982. Molecular Cloning, Cold Spring Harbor Laboratory,

New York.

Nunez, P.E., De Jong-Brink, M., 1997. The suppressive excretory-secretory

product of Trichobilharzia ocellata: a possible factor for determining

compatibility in parasite-host interactions. Parasitology 115, 193±203.

Osmanov, S.U., 1940. Studies on the parasite fauna of ®sh from the Black

Sea. Uchenye Zapiski Leningradskogo Gosudarstwennogo Pedagogi-

cheskogo Instituta im Gertsena 30, 187±265 (In Russian).

Ozaki, 1925; queried.

Stock, S.P., Kaya, H.K., 1996. A multivariate analysis of morphometric

characters of Heterorhabditis species (Nemata: Heterorhabditidae) and

the role of morphometrics in the taxonomy of species of the genus. J.

Parasitol. 82 (5), 806±13.

Stossich, M., 1905. Note distomologiche III-IV-V. Bolletino della SocietaÁ

Adriatica di Scienze Naturali in Trieste 22, 211±27.

Thioulousse, J., Chessel, D., DoleÂdec, S., Olivier, J.M., 1997. ADE-4: a

multivariate analysis and graphical display software. Stat. Comput. 7

(1), 75±83.


Molecules, morphology and morphometrics of Cainocreadium labracis and Cainocreadium dentecis n. sp....

Documents

Transcript of Molecules, morphology and morphometrics of Cainocreadium labracis and Cainocreadium dentecis n. sp....