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M o l e c u l a r p h y l o g e n y o f M e s o m e t r id a e (T r e m a t o d a , D ig e n e a )

Summary : R é s u m é : P h ylog én ie
d es M esom etrid ae ( T rem atoda , D ig enea )
Complete ITS (Internal Transcribed Spacer) ribosomal DNA
sequences were obtained for the six species know at present time Les séquences complètes de la région ITS (Internal Transcribed
within the Mesometridae Poche, 1926. The adult stages are Spacer) de l'ADN ribosomique ont été obtenues pour les six
intestinal parasites of herbivorous sparid teleosts. Aligned espèces actuellement connues de la famille des Mesometridae
sequences were analysed with Maximum Parsimony, Maximum Poche, 1926. Les stades adultes sont des parasites intestinaux des
Likelihood and Neighbor-Joining phylogenetic methods to infer sparidés herbivores. Les séquences alignées des six espèces ont
evolutionary relationships among mesometrid species. The ITS- été analysées avec les méthodes de reconstruction phylogénétique
based phylogeny obtained showed the two Wardula species as a Maximum Parsimony, Maximum Likelihood et Neighbor-Joining, en
sister group to other Mesometridae, and as compared to vue d'établir leurs liens de parenté. La phylogénie ainsi obtenue a
morphological data, suggest some general tendencies in the montré que les deux espèces du genre Wardula constituent un
morphological evolution of this group. It consists mainly in changes groupe frère par rapport aux autres espèces de cette famille. La
from elongated to subcircular forms, regression of the pharynx, comparaison des analyses de séquences et des structures
and the development of a strong accessory attachment organ. anatomiques de chaque espèce suggère une tendance évolutive
caractérisée par le passage d'une morphologie allongée à une
KEY W ORDS : phylogeny, Mesometridae, Digenea, rDNA, Internal Transcribed forme subcirculaire, une régression du pharynx ainsi que le
Spacer, specialization. développement d'un puissant organe accessoire servant à

MOTS CLÉS : phylogénie, Mesometridae, Digenea, ADN ribosomique, Internal

Transcribed Spacer, spécialisation.

three species o f the M esometridae ( W a rd u la ca p itel­

he M esom etridae P o ch e, 1926 form a very
h om ogeneous digenean family, consisting in lata, E lstia sto ss ich ia n u m and C en trod erm a sp in osis­
only four genera and six species: M esom etra sim a), using ITS rDNA sequences analysis (Jousson et
o r b ic u la r is (Rudolphi, 1819), M. b r a c h y c o e lia Lühe, a l , 1998). The M esometridae are mainly characterized
1901, C en trod errn a sp in osissim a Stossich, 1883, Elstia by the absence o f a ventral sucker, the developm ent
sto ss ich ia n u m (Monticelli, 1892), W a rd u la c a p itella ta o f an accessory attachm ent organ, and a reticular
(Rudolphi, 1819), and W. s a r g u ic o la Bartoli & Gibson, excretory system. The taxonomy of this family has been
1989. The adult stages inhabit the digestive tract o f her­ notably revised by Paggi & O recchia (1964). These
bivorous sparid teleosts. The first five previous species authors divided the M esometridae into Mesometrinae
occur in the intestine o f S a rp a s a lp a (L., 1758), w he­ and Wardulinae subfamilies on the basis o f vitellaria
reas W. s a r g u ic o la is found in the rectum o f D iplod u s distribution and testes position. Recent descriptions of
sa rg u s (L., 1758). The encysted metacercariae occur on the mesometrid adults (Bartoli, 1987; Bartoli & Gibson,
different substrates as algae or marine flowering plants. 1989) show ed the presence o f a strong pharynx in
In a previous study, w e elucidated the life cycles o f W a rd u la species only. This character, added to those
retained by Paggi & Orecchia (1964), supports the divi­
sion o f mesometrids into Wardulinae and M esom e­
*Département de Zoologie et Biologie Animale, Université de trinae.
Genève, CH-1224 Chêne-Bougeries, Suisse.
Recently, several PCR-based methods o f genotype ana­
** Centre d’Océanologie de Marseille, U.M.R. CNRS 6540 Dimar, F-
13288 Marseille, France.
lysis have b een developed for the purposes o f taxo­
*Muséum d’Histoire Naturelle, CH-1211 Genève 6, Suisse. nomic identification o f parasites (Mac Manus & Bowles,
Correspondence: Olivier Jousson, Département de Zoologie et B io­ 1996). Internal Transcribed Spacer (ITS) o f the nuclear
logie Animale, Université de Genève, Station de Zoologie, 154, ribosom al DNA (rDNA) region sequencing has been
route de Malagnou, CH-1224 Chênes-Bougeries, Suisse.
Tel.: 004122/3498644 - Fax: 004122/3492647 -
used for species distinction and phylogenetic analyses
E-mail: jousson2@sc2a.unige.ch within m any digenean genera, e.g. D o lic h o s a c c u s

Parasite, 1998, 5, 36 5 -3 6 9
Note de recherche 365

(Luton et al., 1992), F a s c io la (Adlard et al., 1993), M esometridae available in the EMBL/GenBank data-
E c h in o sto m a (Morgan & Blair, 1995) and S ch istoso m a base, which are the two echinostom atids E ch in o sto m a
(D esprès et al., 1995). These studies show ed that the trivolvis and E. revolutum . Comple te ITS sequences
ITS rDNA is characterized by a high nucleotide sub­ w ere aligned manually using the GDE 2.2 (Larsen et
stitution rate, particularly in ITS1, demonstrating its abi- al., 1993) and analysed using the follow ing methods:
lity to distinguish betw een closely related species. the neighbor-joining (NJ) m ethod (Saitou & Nei, 1987)
Another reason for choosing the ITS region is its acces- applied to distances corrected for multiple hits, and for
sibility. It is flanked by conserved sequences which can unequal transition and transversion rates using Kimura’s
be used in the design o f primers for amplification by two-parameter model (Kimura, 1980), the maximum
the polymerase chain reaction (PCR). Furthermore, the parsimony (MP) method, using heuristic search with
presence o f multiple copies in each cell provides large the branch swapping option included in PAUP 3-1-1
num ber o f target sequences for PCR. (Swofford, 1993) and the maximum likelihood (ML)
In this study, the com plete ITS rDNA sequences from m ethod with a transitions/transversions ratio o f 2, as
the six species constituting the M esometridae were im plem ented in the fast DNAml program (O lsen et
used to elucidate phylogenetic relationships among this a l.,1994). The reliability o f internai branches in the NJ,
group. MP and ML trees was assessed using the bootstrap
method (Felsenstein, 1988), with 500 replicates for NJ
and 100 replicates for MP and ML trees. The PHYLO-
WIN program (Galtier & Gouy, 1996) was used for dis­
tance com putations, NJ and ML trees building and

iving adult specim ens o f the six mesometrid spe­ RESULTS

L cies: M e s o m e tr a o r b ic u la r is (196 sp ecim en s),

M. b r a c h y c o e lia (78), C e n tro d en n a sp in osissim a

CR amplification o f the ITS rDNA and part o f the
(72), E lstia sto ssich ia n u m (176), W a rd u la c a p itella ta
18S and 28S rDNA region from the Mesometridae
(6) and W. sa rg u ico la (4) w ere isolated from the diges­
give a single product for w hich size varies from
tive tract o f their de finitive hosts, 37 individuals o f
1550 to 1800 bp, depending on the species. The PCR
S a rp a s a lp a for the five first species and 10 individuals
products averaged 1550 bp in M esom etra orbicu laris,
o f D ip lod u s sa rg u s (Sparidae) for the last one. The
M. b ra c h y c o e lia , W a rd u la ca p ite lla ta and W. sa r g u i­
fishes w ere collected from the north-western Mediter­
cola, 1670 bp in C entroderrna spinosissim a, and 1800 bp
ranean coast (Marseille, France).
in E lstia stossich ia n u m , respectively.
DNA from the six species o f M esometridae was extra-
The subsequent sequen cing o f am plified fragment
cted in guanidin lysis buffer, precipitated with isopro-
shows that these differences are due mainly to the pre­
panol and disolved in distilled water. PCR amplifica­
sence o f a 85 bp long domain near 5’ end o f the ITS1,
tions w ere performed in a total volume o f 50 pl with
w hich is repeated twice in C. sp in osissim a, and four
an amplification profile consisting o f 40 cycles o f 30 s
times in E. sto ssich ia n u m . The com plete sequences of
at 93.5 °C, 30 s at 50 °C and 120 s at 72 °C, followed
PCR products, including ITS1 + 5.8S + ITS2 w ere
by five min at 72 °C for final extension. The ITS1 +
obtained for all specim ens. The length o f the ITS
5,8S + ITS2 re gion o f the rDNA was amplified using
region reaches up to 1119 bp in M esom etra orbicu laris,
universal primers localized in conserved regions o f the
1129 bp in M. b ra c h y c o e lia , 1218 bp in C en trod errn a
18S rDNA (5 ’-TAACAGGTCTGTGAT-3’) and 28S rDNA
sp in osissim a, 1352 bp in E lstia stossich ia n u m , 1078 bp
(5 ’-TTCACTCGCCATTACT-3’). Amplified PCR products
in W a rd u la c a p itella ta , 1125 bp in W. s a rg u ic o la ,
w ere purified using High Pure PCR Purification Kit
1046 bp in E ch in o sto m a revolu tu m , and 1047 bp in E.
(Boehringer), and w ere either ligated in the p-GEM-T
trivolvis, respectively. The interspecific variability within
Vector (Prom ega) and cloned in the XL-2 Ultracom-
the M esometridae ranges from 6.6 % to 19.1 % in ITS1,
petent Cells (Stratagene) or sequenced directly with the
w hich is slightly higher than in ITS2 (3.4-15.1% ).
fmol DNA Sequencing System (Prom ega) using a -S 35
isotope, all according to the instructions o f the manu- The phylogenetic analysis o f M esometridae inferred
facturers. from ITS rDNA seq u e n ces using NJ, MP and ML
methods gives identical results (Fig. 1). The trees were
rooted with ITS sequences o f both echinostom atids
E ch in o sto m a trivolvis and E. rev olu tu m (see Morgan &
As an outgroup taxa, w e ch o o sed the ITS rDNA Blair, 1995). All analyses show the group com posed
sequences o f the closely related digenean species to o f the two species o f the genus W a rd u la as a sister

Parasite, 1998, 5, 365-369

366 Note de recherche
ITS rD N A - b a s e d ph ylogen y o f M e s o m e t r id a e

Fig. 1. - Phylogeny o f Mesometridae inferred from ITS rDNA sequences using the Neighbor-Joining method (pairwise gap removal option),
including schematic morphology of adult specimens. Bootstrap values are given for NJ/MP/ML trees along branches. ML tree: ln (L) =
- 2764,437. MP tree: length = 566; consistency index = 0,922; rétention index = 0,871.

Parasite, 1998, 5, 3 6 5 -3 6 9
Note de ‘recherche 367

group to other M esometridae. The relationships bet­ exists a relationship betw een the specialization o f the
w een other four species are not well resolved. Tw o trematoda found in S. salp a, and the abundance o f this
M esom etra species branch together, but seem to b e as host, according to Basset (1992) w ho suggested that
closely related to each other as they are to C entro- specialized parasites occur preferentially in predictible
d e r m a spin osissim a. Elstia sto ssich ia n u m appears as a hosts. In fact, all mesometrids are oioxenous parasites,
sister group to these three species. i.e. they infect a single host species (Sey, 1968 and
1970; Papoutsoglou, 1976; O recchia & Paggi, 1978;
Fischthal, 1980; Bray, 1984; Bartoli, 1987; Bartoli &
DISCUSSION Gibson, 1989), possibly due to the m ode o f definitive’s
host infection o f the Mesometridae. Indeed, there are

he current repon was designed to establish the very few herbivorous fish species in the Mediterranean
phylogenetic relationships among the M esom e­ (Verlaque, 1990); this could explain the oioxeny o f the
tridae, w hich are intestinal parasites o f herbi- M esometridae, in contrast with the quite low defini-
vorous sparid teleosts. Relative to Echinostomatidae, the tive-host specificity noticed am ong the D igenea (Lym-
ITS rDNA-based sequences analyses o f all species o f bery, 1989).
Mesometridae show ed with 100 % bootstrap support Our results confirm ed the suitability o f the ITS rDNA
that both W a rd u la species constitute a sister group to for studying phylogenetic relationships betw een clo­
other Mesometridae, thus supporting their division into sely related species o f digenetic flukes and suggested
subfamilies M esometrinae and Wardulinae based on a high specialization level o f subcircular M esom etra
morphological and anatomical criteria, according to species to the smooth intestinal e pithe lium o f their her­
Paggi & O recchia (1964). However, such tree topology bivorous de finitive host.
should be confirmed by analyses using a closely related
outgroup taxa, as repre sentatives o f Microscaphidiidae
Looss, 1900 or Notocotylidae Lühe, 1909 (see Brooks
et al., 1985). The ITS1 region is the most variable in
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