Published by A.P.E.
Researe/¡ and Reviews in Parasilology, 55 (3): 149·154 (1995)
© 1995 Asociación de Parasitólogos Españoles (A. P.E.)
Printed in Barcelona. Spain
CHROMOSOME ANALYSIS OF TWO DIGENEAN SPECIES
OF THE FAMILIES HETEROPHYIDAE AND MONORCHIIDAE (TREMATODA)
J. B ARSIENÉ I, V. ROCA\ G. TAPIA 2 & J.E. MARTIN 2
' /nSlilUle of Ecology, Akademijos 2, 2600 Vi/nius, Lithuania
2Deparramenr de Biologia Animal, Facu/tal de Ciencies Biologiques, Universital de Valencia,
Dr. Moliner 50, 46 / 00 Burjassol . Valencia, Spain
Recei ved 8 May 1995; accepted 20 luir 1995
REFERE CE: BARSIENÉ (J. ), ROCA (V.), TAPIA (G.) & MARTIN (J.E.), 1995.- Chromosome analysis of two digenean species of the families Heterophyidae and Monorchiidae (Trematoda). Research alld Reviews in Parasilology, 55 (3): 149-154.
ABSTRACT: The karyotypes of two non-identified di genean species, belonging to the families Heterophyidae and Monorchiidae, have been determined in embryonic cells of their larval stages found parasitising the intermediate molluscan host species Melanopsis duJou,.i , collected from environs
of Lake Albufera (Va lencia province), from Gaibiel and Palancia rivers (Castell ón province) and from some springs in these provinces of Spain. [n
the heterophyid species, the dipl oid set is 14 chromosomes; the 1st, 2nd and 7th pairs were composed of metacentric, all others of submetacentric
chromosomes; the relative length of the two biggest chromosome pairs reaches 59,38% of the total hapl oid set length. The taxonomic status of this
non-identified heterophyid species and its phylogenetic relati onships with digeneans from closely related families are discussed. In the monorchiid
species, the chromosome complement consists of 20 elements: 1st, 2nd, 8th- [Oth pairs of metacentrics, 3rd, 5th-7 th of submetacentrics; the subterminal centromere position was found in the 4th chromosome pair. The comparati ve karyometrical analys is suggests !hat this monorchiid species is
close to the genus Asymphylodora.
KEy WORDS: Karyotypes, Digenea, Heterophyidae, Monorchiidae.
INTRODUCTION
The classification of organi s m s into sep arate groups
accordi ng to their rese mbl a nce to m orpho logical features
was a lo ng process in s pecies and hi g he r taxon forma li zatio n (GOSUNG, 1994). Controvers ial asp ects in the
description of species us ua ll y remain in the group s of organisms whic h undergo the complex of metamorphos is,
e. g. paras itic worm s s uch as those of the the class Digenea. The identification of their species s hould include
morpholog ical, biochemical , ph ys io logical, karyological
a nd other criteria. Nevertheless, o nl y a little inform atio n
has been publi shed on c hromoso m es of digenetic flukes.
In the present stud y we report the k aryo logical data of
two non- ide ntified trematode spec ies b e lon g ing to the
families Heterophyidae a nd Monorchiidae. Karyolog ica l
methods were earlier u sed in the study of o the r three H eterophyidae and two Monorchiidae s pecies (CABLE,
1931; BRITT, 1947 ; DHlNGRA, 1955 ; P ERKINS, 1956;
BARSIENÉ, 1993). Karyosys tematic and phylogenetic aspects of investigated digenean s are di scu ssed .
MATERIAL AND METHODS
Samples of trematodes were collected from environs of Lake
Albufera (Val encia province), fro m Gaibie l and Palancia rivers
(Castell ón prov in ce) and [rom so rne sp rings in these provinces of
Spain . A total o[ 60 specimens of intermediate hosts, the molluscan species Melanopsis dufouri with larva l stages of one di genean
species belonging to the family Heterophyidae (see cercari a coming [rom sporocysts in Fig. 1) and 37 snails of the same species
presenting larval stages of another digenean species belonging to
the fami ly Monorchiidae (see cercaria in Fig. 2) were used. The
material was collected and prepared according to the modified
method of BARSIE É & GRABDA-KAZUBSKA ( 1988). After stainin g
with 4% Giemsa in phosphate buffer (pH=6,8), the slides were airdried. Chromosomes were observed and well-spread metaphase
pi ates were photographed on Micrat 300 film usi ng a l ena Med cytology microscope. The karyometric analysis was carried out on 10
karyotypes of each species measured. The fo ll ow ing parameters
were measured: L" = abso lute length of chromosomes; L' = relative
length; le = centromere index o The classification of chromosomes
follow s that of LEY AN, FREDGA & SANDBERG ( 1964).
RESULTS
The majority (93,8%) of mitotic meta phase pI ates
analysed in the ti ssues of the heterophyid species contained 14 c hromoso m es (Fig. 3). The absolute length of
chromosomes ranged from 2,47 to 11 ,70 flill. The first
and second pair of e lements comprised about 60% of the
total hapl o id set length . Al! remainin g chromosomes
con stituted a gradually dimini shing roW. Their absolute
le ng th ranged from 2,47 to 3,43 !lll1 and relative sizes
from 6,85 and to 9 ,59% of the total haploid complement
length, respecti vely. Al! chromosomes of thi s H eterophyidae species were biarmed, metacentric and submetacentric. The most variable localization of centromeres
was found in the 1st and 5th chromosome p airs (Table 1).
Mod al karyotypes in so matic cells of digeneans belonging to the family Monorchiidae were found to conta in
20 c hrom oso mes (Fig. 4) . The a b solute chromosome
length ranged from 1,94 to 9,45 !lll1. According to the
absolute le ngth, the 1st and 2nd chromosome pairs were
distinct, including 20,76 and 19, 17 % of the total h aploid
set length , res pectivel y. E lements from the 3 rd a nd 4th
pairs were comparatively bigger. Chromosomes of the
5th-10th pairs formed a row of units gradually decreasing in size. Al! chromoso mes were biarmed, except for
150
J. BARSIENE et al.
I
.1
!i
1/& illJ
(i
-,
Fig.3.- Chromosomes of the digenean Heterophyidae species.
Scale bar= I0 urn.
Fig.I.- Cercaria of the family Heterophyidae found in Melanopsis
dufouri. Scale bar=IOO urn.
Chromosome
pair o.
I
2
3
4
5
6
7
Chromosome
length (urn)
Relative length
of chromosomes
(%)
Centromere Chromoindex
some
type
M
SO
M
SO
M
SO
11,70
9,73
3,43
3,13
2,93
2,63
2,47
2,91
1,30
0,41
0,49
0,49
0,48
0,46
32,22
27,16
9,59
8,73
8,15
7,30
6,85
2,13
1,79
0,51
0,56
0,20
0,18
0,29
45,50
44,90
30,90
30,86
27,23
28,70
49,49
3,33
1,51
1,07
1,37
3,41
2,01
0,48
m
m
sm
sm
sm
sm
m
Table 1.- Measurements of the chromosomes of the digenean Heterophyidae species. m=metacentric; smesubmeracentric; Mernean;
SD=standard deviation.
the 4th pair. The centromeres were often variably localized in chromosomes of the 5th pair, including elements
of a transitional submeta-subtelocentric type of structure
(Table 2).
DISCUSSION
Fig.2.- Cercaria of the family Monorchiidae found in Melanopsis
dufouri. Scale bar= I00 urn.
Previous reports gave a haploid number n=6 for other
members of the family Heterophyidae Cryptocotyle linqua (CABLE, 1931; BRITI, 1947) and Acetodextra amiuri
(PERKINS, 1956). Working with C. linqua material of
low mitotic index from the White Sea, it was difficult to
obtain sufficient well-defined metaphase plates. In a few
embryonic nuclei 14 chromosomes were marked (BARSIENE,unpublished data).
Chromosomes
in digenean species of Heterophyidae
and Monorchiidae
~_
"
,II
"
li
-,
B
1-
,)
Chromosomes
Scale bar: I0 urn.
FigA.-
Chrorno- Chromosome
length (urn)
some
pair No.
I
2
3
4
5
6
7
8
9
10
of the digenean Monorchiidae species.
Relative length
of chromosomes
(%)
Centromere Chrornoindex
some
type
M
SO
M
SO
M
SO
9,45
8,76
5,87
5,46
3,40
3,14
2,80
2,44
2,25
1,94
1,32
1,47
0,89
0,94
0,35
0,36
0,27
0,26
0,31
0,21
20,76
19,17
12,87
11,95
7,5 I
6,92
6,18
5,39
4,97
4,28
1,07
0,86
0,70
0,79
0,53
0,38
0,41
0,50
0,60
0,37
44,36
41,73
30,52
14,53
27,73
29,20
31,40
48,46
48,82
49,50
1,93
2,56
3,41
1,63
4,26
1,48
1,08
1,56
2,13
0,70
m
m
srn
st
sm-st
srn
srn
m
m
m
Table 2.- Measurements of the chromosomes of the digenean Monorchiidae
species.
memetacentric;
srnesubrnetacentric;
Mernean; SO=standard deviation.
The diploid chromosome number 2n=14 has been observed in the sets of another Heterophyidae species,
Apophallus miiehlingii, living in snails of the species Lithoglyphus naticoides in the Nemunas river (Lithuania).
The karyotype was found to include two pairs of large
biarmed chromosomes and five pairs of small (from 1,92
to 3,04 urn) metacentric and submetacentric elements
(BARSIENE, 1993). The present study has shown that the
151
non-identified heterophyid species possesses a karyotype very similar to that of A. muehlingii and also to
that of the representatives of the family Opisthorchiidae.
Both Heterophyidae species as well as Opisthorchis felineus and Metorchis intermedius (Opisthorchiidae) have
14 chromosomes in their somatic cells. Two pairs of the
biggest elements in all the above mentioned species included about 60% of the total haploid complement
length (BARSIENE, 1993). The comparative karyornetrical analysis of representatives of Heterophyidae and
Opisthorchiidae allow us to state that representatives of
both mentioned families of digeneans belong to the karyologically monotypic group of digeneans (Fig. 5). Karyological differentiation between families or between
genera is practically on the same level. Moreover, the
chromosome complement of Atrophecaecum burminis
(Cryptogonimidae)
(MAOHAVI & RAMANJANEULU,
1988) greatly resembles the digeneans of the families
Heterophyidae and Opisthorchiidae.
Concerning the systematic status of the Spanish Heterophyidae species here in question, we conclude that we
are describing the karyotype of a digenean species belonging to the genus Massaliatrema. Massaliatrema sp.
aff. gyrinicola is a widely distributed parasite of Rana
perezi (Arnphibia: Ranidae) inhabiting Lake Albufera
and various rivers in the province of Valencia (LLUCH,
ROCA & NAVARRO,1986). Although not much is known
about the life-cycle of this parasite species, it seems that
the first intermediate host is the molluscan species M.
dufouri. Other Heterophyidae species have also been reported from amphibians in Spain, but not in Valencia
province: Euryhelmis squamula found at metacercarial
stage in Rana temporaria (LLUCH,ROCA & MAS-COMA,
1987) and at adult stage in Mustela putorius (Carnivora:
Mustelidae) (FELIU, TORRES& MOTJE, 1989). Previous
observations have shown that the snail Lithoglyphus naticoides, first intermediate host of the heterophyid digenean species A. muehlingii, is karyologically similar to
M. dufouri (BARSIENE, unpublished data). It is known
that closely related trematode species are common in the
tissues of congeneric first intermediate hosts (BARSIENE,
1993).
The family Monorchiidae has not a clear position in
the system of the Digenea. This family was placed on a
branch, on a top of which Dicrocoelidae and Gorgoderidae are located (CABLE, 1974). BROOKS et at. (1985)
have proposed a phylogenetic tree of Trematodes based
on 113 features of adults and 90 larval characters. However, these authors did not determine the phylogenetic relationships between Monorchiidae and other families of
Digenea, monorchiids being a very heterogenic group.
The karyological heterogeneity of them have also been
demonstrated. Diploid chromosome sets of digenean
species of the genus Asymphylodora comprise 20 or 22
elements. The karyotype of Paleorchis sp. includes 16
chromosomes and do not show a similarity to the chromosome sets of Asymphylodora representatives (BARSIE E, 1993).
J,
152
BARSIE
E et al.
1
2
•
;!
•
':
'.
..
"
,
Lr
"
-
:
'!
4
3
5
4
6
7
"
;;
2
•
0
2
•
"
•
::
••
"
"
•
4
,
•..
·
;\
.
"
•
"
a
"
b
~
d
:
\
c
Fig.5.- Comparative ideograms of relative chromosome lengths of digeneans from the families Heterophyidae (Apophallus miiehlingii
and Heterophyidae species from Spain) and Opisthorchiidae (Opisthorchis felineus and Metorchis intermedius): a=O. felineus (Kazakhstan; BARS lE E, 1993); b=A. miiehlingii (Lithuania; BARSlENE, 1993); c=M. intermedius (Lithuania; BARSlE E, 1993); d=Heterophidae species (Spain; present paper).
Comparing the relative chromosome lengths of the
monorchiid species from Spain and the two Asymphylodora species from Poland and Lithuania, it can be concluded that the species from Spain is very similar to the
species belonging to the genus Asymphylodora (Fig, 6),
Karyological differences between these three species are
found at the same level. The morphological features of
the cercaria are also very close to those of the species of
Chromosomes
in digenean
species
of Heterophyidae
153
and Monorchiidae
2
1
Lr
3
5
4
4
2
0
2
4
•
•
.'
7
8
9
10
•
•
•
•
•
·
•
. :~
· ~.
6
.
~
.
\'
c
.\
a
b
Fig.6.- Comparative
ideograms of relative chromosome
(Spain; present paper); b=Asymphylodora
tineae (Poland;
Asymphylodora
(TAPIA
sion
is reached
when
xies
(BA YSSADE-DuFOUR,
studies
could
on the life cycle
furnish
taxonomic
mainly
final
et al., 1993). A
comparing
more
hosts
CASTANON
of
this
REGUERA,
on the
ACK
CORDERO
Further
peculiarities
so far
digenean
fish
concluchaetota-
1990).
TAPIA,
Spanish
(see
similar
cercarial
biological
for the freshwater
of this parasite
&
1979;
and other
information
status
the search
the
lengths of digeneans from the family Monorchiidae:
a=Monorchiidae
BARSIENE:,1993); c=Asymphylodora
sp. (Lithuania; BARSIENE:,1993).
which
unknown
species,
act as the
DEL CAMPILLO,
1994).
OWLEDGEMENTS
This research was supported by a grant of Valencia Government
(Conselleria de Cultura, Educacio i Ciencia, Orden de 15/4/1993)
to Janina Barsiene, Authors would like to thank colleagues from
Valencia University, Animal Biology Departament for the discussion on various aspects of this study.
REFERENCES
BARSIENE: (1.), 1993.- The karyotypes of trematodes.
Academia,
Vilnius, 370 pp. (in Russian).
BARSIENE:(1.) & GRABDA-KAZUBSKA (B.), 1988.- A comparative
study on chromosomes
in plagiorchiid trematodes.
I. Karyotypes of Opisthioglyphe
ranae (Frolich, 1791), Haplometra cylindracea (Zeder, 1800) and Leptophallus
nigrovenosus
(Bellingham, 1844). Acta Parasitologica
Polonica, 33: 249-257.
BAYSSADE-DuFOUR (Ch.), 1979.- L'appareil
sensoriel des cercai-
species
res et la systematique des Trernatodes Digenetiques, Memoires
du Museum Nationale d'Histoire Naturelle de Paris, Serie A,
Zoologie, 113: 1-81.
BRIIT (H.G), 1947.- Chromosomes
of digenetic trematodes. American Naturalist, 81: 276-296.
BROOKS (D.R.), BANDONI (S.M.), MAcDoNALD (C.A.) & 0'GRADY (R.T.), 1985.- Aspects of phylogeny of the Trematoda
Rudolphi, 1808 (Platyhelminthes:
Cercomeria). Canadian Journal of Zoology, 67: 2609-2624.
CABLE (R.M.), 1931.- Studies on the germ-cell cycle of Cryptocotyle linqua Creplin. I. Gametogenesis
in the adult. Quarterly
Journal of Microscopic Science, 74: 563-589.
CABLE (R.M.), 1974.- Phylogeny and taxonomy of trematodes
with special reference to marine species. In: Symbiosis in the sea
(W.B. Vernberg edit.), Carolina Press, Columbia: 173-194.
CORDERO DEL CAMPlLLO (M.), CASTANON (L.) & REGUERA (A.),
1994.- lndice-Catdlogo
de Zoopardsitos
Ibericos. Secretariado
de Publicaciones,
Universidad de Lean, Lean, 686 pp.
DHINGRA (O.P.), 1955.- Gametogenesis,
fertilization and cleavage
in Asymphylodora
sp. Research Bulletin of the Panjab University, 66: 19-29.
FELlU (C.), TORRES (1.) & MOTJE (M.), 1989.- Primera cita en Espaiia del adulto de Euryhelmis squamula (Rudolphi, 1819) (Trematoda: Heterophyidae).
Revista lberica de Parasitologia, 49:
215-216.
GOSLING (E.M.), 1994.- Speciation and wide-scale genetic differentiation. In: Genetics and Evolution of Aquatic Organisms
(A.R. Beaumont edit.), Chapman and Hall, London: 1-15.
LEVAN (A.), FREDGA (K.) & SANDBERG (A.), 1964.- Nomenclature for centromere
position on chromosomes.
Hereditas, 52:
201-220.
154
LLUCH (J.) , ROCA (V.) & NAVARRO (P.), 1986.- Helmintofauna de
los herpetos ibéricos. VII. Metacercarias de Rana perezi Seoane,
1885 (Amphibia: Ranidae) . Miscel.li1l1ia Zoológica, 10: 55-60.
LLUCH (J.), ROCA (V .) & MAS-COMA (S.), 1987.- Helmintofauna
de los herpetos ibéricos: estado actual de conocimientos, consideraciones ecológicas y estimaciones corológicas. In: Mamife-
ros y Helmintos. Volumen Homenaje al Pro! Dr. Dr. Herman
Kahmann en su 81 Aniversario (V. San s-Coma, S. Mas-Coma &
J. Gosálbez edit.), Ketrés Editora S.A., Barcelona : 143-16 1.
MADHAVI (R.) & RAMA JANEULU (J.V.), 1988.- Chromosomes of
Atrophecaecum burminÍ5 (Bhalerao, 1926) (Trematoda: Acanthostomidae). Caryologia, 41: 341-346.
J. BARSIENÉ el al.
PERKI NS (K.W.), 1956.- Studies on morphology and biology of
AcetodeXlra amiuri (S tafford) (Trematoda: Heterophyidae).
American Midland Naturalist, 55: 139- 161.
TAPIA (G.), 1990.- Nuevas aportaciones al conocimiento de las fa-
ses larvarias de trema todos digenea en Melanopsis dufouri
(Mol/usca, Prosobranchia, Thiaridae) como hospedador intermediario. Tesis de Licenciatura, Facultad de Ciencias Biológicas, Uni vers idad de Valencia , Valenci a, 196 pp.
T APIA (G.), CARBONELL (E.), MARTINEZ (F.) & PUJANTE (A .),
1993.- Multiple infections by digenean trematodes in the freshwater snail Melanopsis dufouri Ferussac. Journal of Medical
and Applied Malacology, 5: in press.