ORIGINAL ARTICLE /ARTÍCULO ORIGINAL
KARYOTYPE OF ADENOSCOLEX OREINI: THE FIRST CYTOGENETIC DATA WITHIN
THE ORDER CARYOPHYLLIDEA (CESTODA) FROM SCHIZOTHORAX SPECIES OF
KASHMIR VALLEY, INDIA
CARIOTIPO DE ADENOSCOLEX OREINI: PRIMER DATO CITOGENÉTICO DEL
ORDEN CARYOPHYLLIDEA (CESTODA) DE ESPECIES DE SCHIZOTHORAX DEL
VALLE DE KASHMIR, INDIA
1 1 1 2 3
Tanveer A. Sofi *, Fayaz Ahmad , Bashir A. Sheikh , Omer Mohi ud Din Sofi & Khalid M. Fazili
1 2
Department of Zoology, University of Kashmir, Srinagar – 1900 06, India; SK University of Agricultural Sciences and
3
Technology, Shuhama, Aluestang Srinagar, 1900 06, India; Department of Biotechnology, University of Kashmir, Srinagar –
1900 06, India. *stanveer96@gmail.com Mob. 09797127214
Neotropical Helminthology, 2015, 9(1), jan-jun: 21-28.
ABSTRACT
Keywords: Adenoscolex oreini - Chromosomes - India - Kashmir - Schizothorax.
A karyotype of Adenoscolex oreini Fotedar, 1958 (Capingentidae), a cestode parasite of the
cyprinid fish, Kashir Gaad, (Cypriniformes) was studied for the first time. A
chromosome set consisted of 20 pairs of metacentric, submetacentric and acrocentric
chromosomes (2n=20; n= 8m+1sm+1a). All pairs are small, measuring 1.86-7.22 µm. It seems
that the chromosome number of A. oreini is higher than those of the members of family
Caryophyllaeidae. The variation in size of smallest bivalents and other bivalents indicates that
the somatic chromosomes will show a marked variation in the length of longest and shortest
chromosome in A. oreini. So far no one has undertaken the cytological studies of this parasite; its
chromosome number is being reported for the first time.
Schizothorax niger
21
RESUMEN
Palabras clave: Adenoscolex oreini- Los cromosomas- India- Kashmir- Schizothorax.
Un cariotipo de Adenoscolex oreini Fotedar, 1958 (Capingentidae), un parásito céstodo de
carpas, Kashir Gaad, Schizothorax niger (Cypriniformes) ha sido estudiado por primera vez.
Consiste en un conjunto de cromosomas de 20 pares de cromosomas metacéntricos,
submetacéntricos y acrocéntricos (2n = 20; n = 8m+1sm+1a). Todos los pares son pequeños,
miden 1,86-7,22 µm. Parece que el número de cromosomas de A. oreini es mayor en comparación
con la de los miembros de la familia Caryophyllaeidae. La variación en el tamaño de los más
pequeños bivalentes y otros bivalentes indican que los cromosomas somáticos mostrarán una
marcada variación en la longitud del cromosoma más largo y más corto en A. oreini. Hasta ahora
nadie ha emprendido los estudios citológicos de este parásito y en la presente investigación se
está divulgando su número cromosómico por primera vez.
ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043
INTRODUCTION
22
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015
Caryophyllids are unique among Eucestoda in
having a monopleuroid body plan that is they
have neither internal proglottidization nor
external segmentation and have a single set of
reproductive organs. The embryo has six
hooks; scolex is highly variable, with
acetabula, loculi, bothria, folds, fimbriae,
terminal introvert or without special
attachment structures (Mackiewicz, 1972,
1994). They are intestinal parasites of
cypriniform and siluriform freshwater fishes;
intermediate hosts are aquatic annelids
(Mackiewicz, 1982). Some progenetic
representatives, such as Archigetes, may also
have a complete life cycle with aquatic
annelids. Phylogenetically, Caryophyllidea
and Diphyllobothriidea are regarded as sister
lineages; both belong to basal tapeworm
groups (Olson et al., 2008). Knowledge of
chromosome sets of caryophyllidean
tapeworms has increased within the past 40
years (Table 1). To date, 23 species of all four
existing families have been studied
cytogenetically and karyotypes of 14 species
have been completed (Table 1). The diploid
chromosome number of all, but one species
ranges from 14 to 20; Caryoaustralus sprenti
Mackiewicz et Blair, 1980, represents the
exception, having only six chromosomes
(Grey, 1979: listed as ''gen. et sp. n.'').
Congeners may have constant chromosome
numbers as, for example, four species of the
genus Khawia with 2n = 16 (Grey, 1979;
Petkeviciute, 1998; Mutafova & Nedeva,
1999; Orosova et al., 2010b); or they may
differ in diploid number as in Glaridacris, with
diploid number 2n =16 in two species and 20 in
two others (Grey, 1979; Grey & Mackiewicz,
1974, 1980). This order has the greatest range
in chromosome numbers, 6–30, if one includes
triploids (Table 1). Adenoscolex oreini
Fotedar, 1958 is an intestinal parasite of
Schizothorax niger (Ale gad), a fresh-water
fish in Kashmir, India. So far no body has
undertaken the cytological studies of this
parasite and in the present investigation its
chromosome number is being reported for the
first time.
The objective of the present study is to describe
the karyotype of A. oreini, a cestode parasite of
Cyprinid fish, Kashir Gaad, Schizothorax
niger (Ale Gad) (Cypriniformes) of Kashmir.
Next, these results will be compared with
published data obtained by molecular studies
of different species and with cytogenetic
in f o r m a t i o n a v a i l a ble f o r o t h e r
Caryophyllaeidae species.
Specimens of A. oreini of different sizes and
maturity were obtained by the dissection of
Schizothorax niger (Ale Gad), caught in Dal
Lake of Kashmir, India in the year 2014.
Living specimens were incubated for 3–4 h in
0.01% colchicines in physiological solution at
room temperature and were then transferred to
distilled water for 1 h for hypotony. Fixation
was in a freshly prepared mixture of ethanol
and glacial acetic acid (3:1). Specimens were
kept refrigerated until they could be processed
in the laboratory. Slides were made from cell-
suspensions using an air-drying technique
(Petkeviciute & Ieshko, 1991), stained with
4% Giemsa, pH 6.8, rinsed in tap water and
allowed to dry.
Suitable mitotic metaphases were
photographed with a 100X objective under oil
immersion using Mikrat-300 film. For
karyotyping, chromosomes were cut out of the
photographs and paired on the basis of size and
centromeric position. Means and standard
deviations of the absolute length in
micrometres, relative length (100x absolute
chromosome pair length/total length of
haploid complement) and the centromeric
index (100x length of short arm/ total length of
MATERIAL AND METHODS
Sofi et al.
23
the remaining chromosomes and contributed
48.62% to the total chromosome length. The
karyotype formula of A. oreini can be
summarized as 2n=20=8m+1sm+1a. A
summary of the results obtained after
measuring the Giemsa-stained chromosomes
of ten complete metaphase plates is given in
Table 2. The chromosomes are middle sized;
the largest measured 7.22 µm and the smallest
were 1.86 µm. The total chromosome length of
the haploid complement was 39.88 µm. The
homologues of pairs 5 and 6 could not be
distinguished clearly. There are no statistically
significant differences in their sizes and
centromeric indexes.
In order to better visualize the existing
differences in chromosome morphology,
ideograms were constructed using the
centromere indexes and relative length values
(Fig. 2).
chromosome) were calculated for each
chromosome pair. The centromere position on
the chromosomes was classified according to
the nomenclature of Levan et al. (1964). When
a centromere position was on the borderline
between two categories, the confidence limits
of the means were calculated and two
chromosome categories are reported.
Analysis of 57 mitotic metaphase spreads from
seven specimens showed that the modal
diploid complement of A. oreini contains 20
chromosomes (2n=20). The karyotype (Fig.
1a, b) included eight metacentric; one
submetacentric and one acrocentric
chromosome pair. First three pairs of
metacentric elements are distinctly larger than
RESULTS
Table 1. Measurements (means + SD) and classification of chromosomes of Adenoscolex oreini.
Chromosome
Number
Absolute
length (µm)
Relative
length (%) Centromeric
Index Classification
1 7.22+1.35 26.09+2.06 46.13+1.31 Metacentric
2
6.63 + 0.98
26.77 + 1.43 44.31 + 1.71 Metacentric
3
5.42 + 0.82
21.85 + 0.89 45.67 + 1.38 Metacentric
4
3.45 + 0.44
13.95 + 0.56 46.51 + 1.54 Metacentric
5
2.93 + 0.37
11.87 + 0.89 44.46 + 1.29 Metacentric
6
2.68 + 0.30
10.87 + 0.49 44.68 + 2.30 Metacentric
7
1.77 + 0.22
7.15 + 0.27 43.79 + 2.98 Metacentric
8
5.73+1.08
20.70+1.05 42.24+3.66 Metacentric
9
1.86 + 0.22
7.52 + 0.35 37.69 + 3.74 Submetacentric
10 2.19+0.53 7.83+0.58 3.14+2.88 Acrocentric
a
1
2
3 4
5
6 7 8 9 10
b
Figure 1. a–b Mitotic chromosomes of Adenoscolex oreini. Scale Bar 10 µm.
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Karyotype of Adenoscolex oreini
24
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Sofi et al.
Table 2. Summary of chromosomes and karyotype data of Caryophyllidea Cestodes (Tapeworms) (1929–till Date).
Order/ Family
Number
2n [3n] Morphology
TCL
(mm) Absolute
length (mm) References
CARYOPHYLLIDEA
Balanotaeniidae
Balanotaenia bancrofti
Johnston, 1924
14 Grey (1979)
Capingentidae
Capingens singularis Hunter,
1927
14 Grey (1979)
Adenoscolex oreini
Fotedar,
1958 20 8m+1sm+1a
39.88
µm
Present Study
Caryophyllaeidae
14
3m + 1sm
+3a
2.5–8.0
Mackiewicz &
Jones (1969)
Hunterella nodulosa
Mackiewicz et McCrae, 1962
14
3m + 4a 56.6 1.9–7.0 Grey (1979)
Archigetes sp. (=appendiculatus)
18
<4 Motomura
(1929)
Biacetabulum biloculoides
Mackiewicz et McCrae, 1965 20
Grey (1979)
[30]
Grey (1979)
20 [30] 10m
87.8
3.0–12.9
Petkeviciute &
Kuperman
(1992)
Caryophyllaeus laticeps (Pallas,
1781)
20
10m
Bombarova et
al. (2009)
Glaridacris laruei Lamont, 1921
16
3m + 1sm +
4a
37.7
2.0–12.0
Grey
&Mackiewicz
(1974), Grey
(1979)
Glaridacris confusus Hunter,
1927 16
Grey (1979)
Glaridacris catostomi Cooper,
1920 20 [30]
8m + 2sm
55.7
3.2–7.6
Grey (1979),
Grey &
Mackiewicz
(1980)
Glaridacris vogei Mackiewicz,
1976 20
8m + 1sm +
1a
60.5
3.0–8.6
Grey (1979)
Monobothrium hunter
Mackiewicz, 1963
20
9m + 1a
61.2
2.1–8.7
Grey (1979)
Isoglaridacris folius Fredrickson
et Ulmer, 1965 18 1m + 8a 24.1 1.5–3.8 Grey (1979)
Isoglaridacris jonesi
Mackiewicz, 1972 18 2m + 7a 25.9 1.7–4.3 Grey (1979)
Isoglaridacris bulbocirrus
Mackiewicz, 1965 18 [27] Grey (1979)
Lytocestidae
Atractolytocestus huronensis
Order/ Family
Number
2n [3n] Morphology
TCL
(mm) Absolute
length (mm) References
[24]
4m + 3a+ 1
minute
24.9
0.8–7.2
Jones &
Mackiewicz
(1969)
Anthony, 1958
[24]
4m + 3a + 1
37.0
0.9–9.6
Kralova-
Hromadova et al.
(1979)
Caryoaustralus sprenti
Mackiewicz et Blair, 1980
6
Khawia iowensis Calentine et
Ulmer, 1961 16
5m + 3a
35.5
3.0–6.1
Grey (1979)
Khawia rossittensis (Szidat,
1937) 16
Grey (1979)
16
3m + 5a
59.3
5.2–10.8
Petkeviciute
(1998)
Khawia sinensis Hsu, 1935
16
3m + 5a
41.2
3.8–6.8
Mutafova &
Nedeva (1999)
Khawia saurogobii Xi et al.,
2008 16
3m + 5a
56.3
3.9–10.0
Orosova et al.
(2010b)
Lytocestus indicus (Moghe,
1925) 16 Vijayaraghavan
&
Subramanyam
(1977)
20 Bombarova et
al. (2009)
Caryophyllaeides fennica
(Schneider, 1902) 20 10m 58.6 2.8 – 8.2 Orosova et al.
(2010a)
Notolytocestus minor Johnston et
Muirhead, 1950 12 6a 17.0 1.7–4.2 Grey (1979)
Figure 2. Idiogram of Adenoscolex oreini.
TLC = Total Chromosome Length; m = Metacentric; sm=Submetacentric; t=Telocentric; a=Acrocentric.
25
Table 2. Continuation
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Karyotype of Adenoscolex oreini
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Sofi et al.
DISCUSSION
It may be concluded that the diploid
chromosome number of A. oreini is 20 and it is
perhaps the first report of chromosome number
of a genus from family Capingentidae of order
Caryophyllidea. However, in the family
Caryophyllaeidae the chromosome number
has been reported by Motomura (1929);
Mackiewicz & Jones (1969), Grey &
Mackiewicz (1974); Vijayaraghavan &
Subramanyam (1977); Grey (1979); Grey &
Mackiewicz (1980); Petkeviciute &
Kuperman (1992); Petkeviciute (1998);
Bombarova et al. (2009) and Orosova et al.
(2010a,b) for Archigetes appendiculatus
(2n=18); Hunterella nodulosa (2n=14);
Glaridacis laruei (2n=16); Lytocestus indicus
(2n=16); Capingens singularis (2n=14);
G l a r i d a c r i s c a t o s t o m i ( 2 n = 2 0 ) ;
Caryophyllaeus laticeps (2n=20); Khawia
sinensis (2n=16); Caryophyllaeides fennica
(2n=20) and Khawia saurogobii (2n=16),
respectively. It seems that the chromosome
number of A. oreini, a member of family
Capingentidae is higher as compared to that of
the members of family Caryophyllaeidae. The
variation in size of smallest bivalent and other
bivalents indicates that the somatic
chromosomes will show a marked variation in
the length of longest and shortest chromosome
in A. oreini. The same has been reported by
Motomura ( 1 9 2 9 ) i n A rc h i g e t e s
appendiculatus and Mackiewicz & Jones
(1969) in Hunterella nodulosa. Therefore, this
type of chromosome complement may be
characteristic of Caryophyllidea in general.
Chromosomes of 14 caryophyllidean species
are of mid or large length, up to 12.9 mm long
in C. laticeps (Table 2). They are among the
largest of the Cestoda, a feature they share with
Eubothrium crassum Bloch, 1779, of the
Bothriocephalidea. The mean TCL in
Caryophyllidea is 47.8 mm, with a high of 87.8
for C. laticeps, the highest yet recorded from
any tapeworm. Satellites (i.e. secondary
constrictions), rare with cestode
chromosomes, have been found in H.
nodulosa, A. huronensis and K. saurogobii
(Grey, 1979; Kralova-Hromadova et al., 2010;
Orosova et al., 2010b).
In individual species, chromosome
morphology differs significantly; a
predominance of bi-armed elements
(symmetrical karyotypes) was detected in four
species of the family Caryophyllaeidae and
one lytocestid species (Table 2), one-armed
acrocentric chromosomes (asymmetrical
karyotypes) prevailed in four species of
Caryophyllaeidae and Lytocestidae and the
rest of five karyotypes with rather balanced
chromosome morphology occurs in both
families. Because of this variation, no relevant
hypothesis on karyotype evolution can be
made on basis of traditional karyological
analyses among the four families of the
Caryophyllidea. It is noteworthy that recent
cladistic analysis by Oros et al. (2008), based
on unweighted morphological characters, are
only partly congruent with the existing
classification into four families that is based on
the placement of the internal, longitudinal
musculature. However, paraphyly of
Caryophyllidean families should be
corroborated by molecular studies.
It is apparent that classical karyological data
are hardly sufficient to resolve phylogenetic
and systematic relationships within
insufficiently investigated animal groups such
as the Caryophyllidea. However, the recent
inputs of molecular approaches into the
cytogenetics of Caryophyllidea (Kralova-
Hromadova et al., 2010; Orosova et al.,
2010a,b) raises hopes that new data will
substantially help in elucidating the
phylogenetic relationships within this unique
group of monozoic tapeworms.
26
Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Karyotype of Adenoscolex oreini
ACKNOWLEDGMENT
The authors extend their thanks to the
authorities of the Department of Zoology,
University of Kashmir for the facilities
provided. TAS is also highly thankful to Fayaz
Ahmad for compiling the paper.
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