New CHAPTER 03shodhganga.inflibnet.ac.in/bitstream/10603/11838/8/08... · 2015. 12. 4. · index (MI) in the slides, which were used for the analysis ofthe chromosomes is also included

CHAPTER 03

03. Mitotic Chromosome Analysis

03. MITOTIC CHROMOSOME ANALYSIS

3.1. Introduction

In spite of several studies on morphology, allozyme and morphometric parameters, the taxonomic sta-

tus of the mussels remains obscure (Martanez-Lage et al. 1997). Perna viridis is distinct from the rest

of the two species belonging to the same genus and other closely related genera of the order mytilidae.

The characterization of the chromosomes in the genus Perna is scare in order mytilidae. The earlier

works provide only fragmentary information, since those studies were aimed at establishing the chro-

mosome counts. The objective of the present study is to describe the karyotype of the green mussel in

detail, since chromosome number alone is insufficient to describe the cytotaxonomic relationships. The

cytogenetic studies of this species will provide a framework for the better understanding ofthe evolution

of the molluscs in general and bivalves in particular. Apart from the significance in the evolutionary

systematics, chromosomes data in this species has great consequence in the studies concerning aquac-

ulture and mutations as Perna viridis is having immense aquacultural and genotoxic studies. Since the

proper examination ofthe chromosomes is dependent partially upon the number of cells in the metaphase,

a study incorporating the number of cells in the mitosis in two tissue types. In the present study mitotic

index (MI) in the slides, which were used for the analysis ofthe chromosomes is also included. This data

in turn will also provide some information on the rate of cell division in these tissues. The rate of cell

division depends upon cell loss, apoptosis and to some extent the prevailing conditions to compensate

the cell loss. Generally cell division is a complicated process and involves number ofprecise events, to

maintain the genetic integrity ofthe cell.

A complete somatic cell division cycle comprise of S — synthetic, G1 — gap 1, G2 — gap2 and m —

mitosis. In the S —phase the DNA replication occurs, so it is called as the synthesis phase after pro-

gressing through the G2 phase, cells enter the complicated M — phase. Postmitotic cells in multicellular

Molecular Cytogenetic Studies in Perna viridis (Bivalvia:Mollusca) from Goa, West Coast of India. 48


organisms can exit the cell cycle and remain for days, weeks or in some cases like nerve cells even the

lifetime of the organism without proliferating further. Most postmitotic cells enter the G1 phase, which

will eventually begin new cell cycle (Lodish et al. 1999). During Mitosis the multiplication of the cells

takes place to increase the number of cells and replenish the lost or dead cells. Mitosis further com-

prises of prophase, metaphae, anaphase and telophase. Chromosomes begin to condense by tightly

folding loops of the 30-nm chromatin fiber attached to the chromosome scaffold (Nelson and Cox,

1997). The condensation of the chromatin material continues throughout the prometaphase stage. Dur-

ing metaphase the nuclear membrane disintegrates and highly condensed chromosomes arrange them-

selves on the equator plane. Each chromosome at this stage comprises of two sister chromatids which

were produced by DNA replication during the S phase. They attach to the spindle fibers at the point of

centromeres and align themselves. During anaphase sister chromatids get segregated and move to the

opposite poles of the mitotic spindle. Thus two new daughter cells are formed each carrying one sister

chromatid of the parent cell.

Depending upon the requirement of a tissue and the intrinsic cellular environmental conditions, the

details of mitosis pertaining to the abundance of certain stages can differ from one tissue to another. The

intensity of cell division is of great interest in any cytogenetic studies, as the possibility of getting good

metaphase plate depends partially on the number of cells in mitosis and also due to the fact that some

cells after mitosis exits the cell cycle (Lodish et al. 1999). With some chemical treatments cells can be

arrested at the metaphase stages of the mitosis, the chromosomes in this stage are most suitable for

analyzing karyotypic details of chromosomes

Advent of sophisticated cytogenetic techniques and improved transmitted light microscopes gave new

dimensions to the studies of chromosomes in the last two decades. Due to the small size of the pelecy-

pod chromosomes and difficulty in the cytoplasmic clearance in the solid tissues, it is difficult to prepare

good metaphase plates for detailed karyotypic analysis (Ramammorthy, 1958; Burch, 1968.)

Cytogenetic information is available on species belonging to some orders of Pelecypods. For example

in the order Unionoida the genera like Anodontoides, Gonidea, Inversidens, lampsilis, Lasmigona,



Potamilus, Alasmidonta Anodonta Pseudodon, Ptychobranchus, Dudrula, Toxolasma, Unio,

Villosa were described A survey of the literature suggested a conserved diploid chromosome number

of 38 (2n=38) in all the members of this order (Jenkinson, 1976; 1984; Van Griethuysen et al. 1969;

Vitturi, 1982). In Order Mactridae species like Labiosa plicatella and Mulinia lateralis showed

2n=36 (Menzel 1968a; 1968b). Mactra chinensis however, showed 211=38 (Wada and Akira 1993).

Donax variabilis and Donax trunculus of the order Tellinacea had 2n=38, (Menzel 1968a; Cornet

and Soulard, 1990). In order veneroida cytogenetic characterization has been done in Chione cancellata,

Circi scripta, Irus mitis, Paphia vernicosa, Pitaria chione, Ruditapes decussates, Tapes

philippinarum, Venerupis decussata. The diploid number in all these species is 38 (Menzel 1968a;

Ieyama 1980; Rasotto et al. 1981; Gerard 1978; Nishikawa and Hisatomi, 1959) except for Tapes

philippinarum which had 2n=28 (Nishikawa and Hisatomi, 1959). In order Myoida Barnea truncata

and Cyrtopleura costata have diploid number of 34 whereas Teredo utriculus had diploid number of

chromosomes of 38 (Menzel, 1968a; Vitturi et al. 1983). Ostrea denselamellosa order Ostreidae ,

2n=20 (Insua and Thiriot-Quievreux, 1991), Acharax japonicus and Petrasma pusilla of the order

Solemyoida both were reported to have a diploid number of 22 chromosomes (Ieyama, 1982). Only

some information was available on the karyological aspects ofIndian species viz: Crassostrea gryphoides

, Saccostrea cucullata and Perna viridis (Goswami, 1991; 1992; Goswami and Femnandes,1993).

Chromosome number and karyotype in the different families ofthe pelecypoda vary considerably. The

first authentic report on the pelecypod cytogenetics was published on the clams Mercenaria mercenaria,

M campechiensis and their hybrids (Menzel and Menzel, 1965) and they reported the diploid number

(2n) of chromosome in this species as 38. The behavior of chromosomes, their gross morphology and

chiasma frequencies were outlined to some extent. The first thorough paper on the pelecypod karyo-

type was published on the Olympic Oyster Ostrea luridae and Pacific Oyster Crassostrea gigas, both

of which were shown to have n=10 (2n=20) (Ahmed and Sparks, 1967). Longwell and co-workers

(1967) studied the karyotype of Crassostrea virginica and reported some interesting chromosomic

features such as secondary constrictions. The same authors subsequently found the same number in



Crassostrea virginica, Crassostrea rhizophorae, 0. edulis and 0. eques tris . Later chromosome num-

bers in the species belonging to nine families of pelecypoda were reported (Menzel et al. 1968a). The

diploid chromosome number in pelecypods range from 14 to 46

The sub class Pteriomorphia consists of five orders Arcoida, limoida, Mytiloida, ostreoida and Pterioida

and is accepted by many paleontologists as a phylogenetic unit (Newell, 1965). Out of these five orders

chromosomes of orders arcoida and limoida are poorly understood. In order Arcoida karyotypic work

has been carried out to inArca barbata (n=19), A. bourcardi (2n=36), Arcopsis symmetrica (2n=38),

Barbatia velata (2n=38), B. virescens (2n=28), Didimacar tenebricum (2n=38), Porterius dalli

(2n=38) and Scapharca subcrenata (2n=38). (Rasotto et al. 1981; Ieyama 1975; 1983; 1984a;

1984b) In order limoida chromosome studies have been restricted to Limaria hakodatensis with

2n=32 (Ieyama, 1984b). In order Ostreoida, family Pectinidae, is cytogenetically known to some ex-

tent in this order, which includes about 360 living species of which only 16 species have been studied

cytogenetically (Beaumont and Gruffydd, 1974; Komaru and Wada, 1985; Insua et al. 1998; Pauls and

Affonso, 2000). Chromosome number and karyotype vary considerably in the family pectinidae. While

most of the species have a haploid number of 19 chromosomes, some have 16 (Komaru and Wada,

1985), and one species, Aequipecten opercularis, has 13 (Beaumont and Gruffydd, 1974). Even among

species with 19 pairs of chromosomes, variation in karyotype is apparent, and the number of telocentric

chromosomes varies from zero in Chlamys farreri to 14 in Pecten maximus and P albicans (Beau-

mont and Gruffydd, 1974; Komaru and Wada, 1985). Families Ostreidae is the best studied family in

the order Ostreida, in which several species belonging to family Ostroidae especially in the genera

Crassostrea, Ostrea, Saccostrea have been studied, all the species in this order have a conserved

diploid chromosome number of 20 (2n=20) (Menzel 1968a; 1968b; Kobayashi 1954; Ieyama and

Inaba, 1974; Ieyama 1975; 1990; Rodriguez-Romero et al. 1978; 1979; Nadamitsu and Shinkawa

1973; Ahmed and Spark 1967; Thiriot-Quievreux and Ayraud 1982; Thiriot-Quievreux, 1984b;

Kobayashi 1954; Longwell et aL 1967; Lapegue et aL 2002 ). Recently chromosomes of Dendostrea

folium of this family and Hyotissa imbricata belonging to family Gryphaeidae have been described



(Ieyama, 1990).

In order mytiloida several species were cytogenetically characterized and karyological data is available

for about 30 mytilidae species (Nakamura, 1985), but the work along the Indopacific coast is inad-

equate, especially along coastline of India.

Some cytogenetic work has been carried out in some of the genera of family mytilidae for example

Brachidontes recurvus and Brachidontes minimus have diploid chromosome of about 30 (Rasotto

et al. 1981), whereas Brachidontes rodriguezi d'Orb (Genus Brachidontes), 2n=32(Torreiro et al.

1999). Crenomytilus grayanus has diploid number as 28 (Ieyamam, 1984). Lithophaga curata,

Modiolus auriculatus, Modiolus barbatus have 2n=32 (Ieyamam, 1984; Rasotto et al. 1981). In the

genera Musculus, Musculus cupreus and Musculus senhousia have diploid number of 30 (Ieyamam,

1975, 1984a), whereas Musculus laevigatus has 28 as diploid chromosome number (Ieyama, 1984).

Family mytilidae has four closely related genera i.e. Mytilus, Aulacomya, Choromomytilus, and Perna.

Cytogenetic studies in the Genus Choromytilus, Choromytilus chorus (Palma-Rojas, 1997) Out of

these four genus mytilus is well studied cytogenetically

A detailed account of Chromosome number and autosomal polymorphism of the marine mussels Mytilus

eudulis and Mytilus californianus was first reported (Ahmed and Sparks 1970). This is the only study

of its kind that has presented some evidence of chromosome polymorphism in pelecypods. Chromo-

some number and morphology have also been described in Mytilus trossulus, Mytilus edulis and

Mytilus galloprovincialis, Mytilus coruscus M desolationis (Ahmed and Sparks 1970; Ieyama, 1983;

1984a; Moyniha and Mahon 1983; Thiriot-Quievreux, 1984a; Dixon and Flavel 1986; Pasantes et al.

1990; Insua et a/. 1994).

The genus Perna includes three species Perna perna, Perna canaliculus, Perna viridis (Siddal,

1980). Diploid chromosome number 28 was also observed in Perna canaliculus and Perna Perna of

Venezuela coast (Ahmed 1974) but not in Perna viridis (Ahmed 1974; Goswami and Fernandis,

1993), this species is exceptional in having diploid chromosome as 30. 3 5 9 Variation in the chromosome number in the related species has also been reported earlier in some



species of order artidactyla (Wurster and Benirschke, 1970; Lin et al. 1991; Nils Hartmann and Harry

Scherthan, 2004), in some fishes belonging to groups Blenniidae, Gobiidae and Scorpaenidae (Cataudella

and Capanna, 1973; Sola et al. 1978; Thode et al. 1983; Amores et al. 1990; Yokoyama et al. 1992;

Caputo et al. 1996, 1997; Correaand Galetti, 1997), and also in the veneroida, Tapes phili• iiinarum

(Nishikawa et al. 1959). This has been attributed to the chromosomal rearrangements like Robertsonian

translocation and perecentric inversion during the course of evolution (Nadler, 1969; Wallace, 1959,

White 1969; 1973; Mayr 1969a; 1970; Brinkley et al. 1984; Elder and Hsu, 1988) between closely

related species but is only rarely found.

Note: Materials and methods are described in chapter 2 section I.

3.2 Results

Rate of mitosis was evaluated in branchial roots of the gill and diffused gonads from male and female

animals. As many as 72094 total cells from male and 53526 total cells from female animals in the

branchial roots of gill and 50948 cells from male and 38799 cells from female animals in mantle tissue

with diffused gonads were scored.

3.2.1 Mitotic Index

The mitotic index (MI) (Bin Cong, et al. 2002) of the gill and gonad tissues was calculated from the

data obtained from ten male and ten female animals and tabulated in table 1 and table 2.

Mitotic index of the branchial roots of gill tissue in the male and female animals were compared from the

data of table 1, the average mitotic index was found to be 0.578 ± 0.03 and 0.549 ± 0.02 respectively

in these tissues. The Figure 1 and 2 clearly shows overlapping in the mitotic index of the branchial roots

of the gill and the mantle with diffused gonads.

It has been observed that, there is no significant difference (P > 0.05) in the % MI of the branchial roots

of gill tissue in both male and the female animals. The average % MI in gonads of male and female from


12 0.15

0.10

0.05

0.00

5 0.25

e 0.20


Table 1. Percent Mitotic index and t - test values in the branchial root cells of gills.

Animals Male Female

P - value TC CM %Ml TC CM %Ml 1 10250 57 0.55 7945 39 0.49 2 6238 32 0.51 2361 17 0.72 3 8472 41 0.48 14562 74 0.5 4 4581 30 0.65 2106 11 0.52 5 1678 11 0.65 7415 41 0.55 6 7531 39 0.51 3411 19 0.55 7 5624 38 0.67 5008 25 0.49 8 13545 96 0.7 7235 37 0.51 9 8692 42 0.48 1370 8 0.58 10 5483 32 _ 0.58 2113 11 0.52

Avg % Ml ± SE 0.578 ± 0.03 0.549 ± 0.02 0.4 TC- Total number of cells, CM- Cells in mitosis, MI - Mitotic index, P >0.05

Figure 1. Percent Mitotic Index in branchial root cells of male and female gills

-0-- Male -0- Female

0.75 -

0.70 -

0.85 -

0.80

0.55 - 0.50-

In

•

0.45

u 0.40

O 0.35

2 0.30

1 2 3 4 5 8 7 8 9 10

Number of Animals

the table 2, being 0.745 and 0.69 and shows no significant difference (P > 0.05).

When the % MI in male and female branchial roots of gill tissue against gonad tissue was analysed

and the data depicted in table 3 and the results are interpolated in the figure 3 and 4 , it has been

observed that there is a very high significant difference (P < 0.000) in the rate of cell proleferation in

the branchial roots of gills and gonads in both males and the females.

0



Table 2. Percent Mitotic index and t - test values in the mantle with diffused gonads.

Animals

Male Female

P - value TC CM % MI TC CM % MI 1 4356 28 0.64 4280 32 0.74 2 12533 112 0.89 3689 25 0.67 3 3286 24 0.73 1842 13 0.7 4 6172 41 0.66 2603 21 0.8 5 9641 68 0.7 5317 35 0.65 6 3950 30 0.75 1432 10 0.69 7 5142 34 0.66 8259 61 0.73 8 1830 14 0.76 2155 14 0.64 9 3147 28 0.88 1571 10 0.63 10 891 7 0.78 7651 57 _ 0.74

Avg. % MI ± SE 0.745 ± 0.027 0.69 ± 0.017 0.172 TC- Total number of cells, CM- Cells in mitosis, MI - Mitotic index, P >0.05

Figure 2. Percent Mitotic Index in mantle with diffused gonads of males and females

-0- Male -0- Female

0.90 -0.85 -

0.80 -0.75 0.70 - s 0.85 -

lir, 0.80 -

2 0.55 0.50 -

0.45 0.40

0.35

001 32°- 5 -

it 0.20 -

0.15 -0.10

0.05 - 0.00

1 2 3 4 5 8 7 8 9 10

Number of Animals

Table 3. Comparison of percent Mitotic Index and t - test values in branchial root cells of gill and mantle with diffused

gonads in males. Animal % MI in gills ( X ± SE)/e MI in gonad ( X ± SE P- value

Male 0.578 ± 0.03 0.745 ± 0.02 0.000m Female 0.549 ± 0.02 0.699 ± 0.01 0.000"*

*** P <0.001



Figure 3. Comparison of percent Mitotic Index in branchial root cells of gill and mantle with diffused

0.90 -

0.85 -

0.80 -

0.75 -

0.70

0.65 0 60 _

12 0.55 -

• 0.50-

.2 0.45

0.4°- 0.35 -

c O 0.30

0.25 -

13. 0.20 -

0.15 -

0.10 -

0.05 -

0.00

Gill Tissue —A— Gonads

0 1 2 3 4 5 6 7 8 9 10

Number of Animals

Figure 4. Comparison of percent Mitotic Index in branchial root cells of gill and mantle with diffused

—*— Gill Tissue —A— Gonad

V

2

0.80-

0.75-

0.10-

0.65-

060-

0.55-

0.50-

0.45 -0.40-

0.35-

0.30- C 8 0.25 -

8 0.20 -

0.15 -

0.10 -

0.05 -

0.00

1 2 3 4 5 • 6 7 8 • 9 10

Number of Animals

0

3.2.2 Diploid Chromosome number

For frequency distribution of diploid number of chromosomes in Perna viridis, as many as 301 cells in

metaphase phase stages obtained from somatic cell preparation from branchial roots of the gill were

screened in ten males and ten females and the data is depicted in table 4 and figure 5. The results show

that in 198 cells (65.78 %) accounted with 30 number of chromosomes and in rest of the metaphase


30 31>

• Metaphase Cells

MEM AN= MB

>20 21 22 23 24 25 26 27 28 29

Diploid Chromosome Number

200 -

180 -

160 -

140 -

120 -

100 -

80-

60 -

Num

ber

of M

etap

ha

se C

ells

40

20

0


cells accounted with variable number of chromosomes, which range between minimum of 0.99 % to

maximum of 7.97 %. No metaphase cell was reported to have more than 30 number of chromosomes.

The results confirmed the diploid complement of 30 chromosomes in Perna viridis

Table 4. Frequency distribution of diploid chromosomes in Perna viridis No. of

anim

Shell lengt

h Number of chromosomes Total

<20 21 22 23 24 25 26 27 28 29 30 <31 1 7.6 1 1 2 0 1 0 0 0 1 6 16 0 28 2 9.1 2 0 0 0 0 0 1 0 1 0 12 0 16 3 9.2 9 0 0 2 1 0 0 0 0 2 12 0 26 4 8 1 2 0 , 0 1 2 1 0 0 1 8 0 16 5 7.9 3 4 5 0 2 0 3 0 0 0 31 0 48 6 8.6 5 1 1 1 0 1 0 0 3 2 9 0 23 7 8.1 , 0 1 5 1 0 0 0 1 1 3 38 0 50 8 10.2 0 0 6 0 0 0 0 3 0 1 25 0 35 9 8.8 1 2 1 2 0 0 0 0 0 1 17 0 24 10 8.7 2 0 0 3 0 0 0 0 0 0 30 0 35

24 11 20 9 5 3 5 4 6 16 198 0 301

Figure 5. Frequency distribution of diploid chromosome number in Perna viridis



3.2.3 Chromosome structure Analysis.

The morphometric data of the chromosomes and the means of the relative lengths of the chromosomes,

arm ratios and the centromeric index are depicted in the table 5 and table 6 The karyotype showed ten

metacentric and five submetacentric chromosomes. The total mean length of the haploid complement

was 61.12 ± 0.556 um (SE). The mean length ranged from 2.49 to 6.03 micrometers. Ten chromo-

some pairs that are 1, 2,3,4,5,8,9,10,14 and 15 with arm ratio 1.00 to 1.69 were classified as meta-

centric and the five chromosomes that are 6,7,11,12 and 13 classified as submetacentric (Levan et al.

1964) in 86 % of metaphase cells, rest 14 % of metaphases were observed to have eight metacentric

that is 1, 2,3,4,5,8,9,10 and seven submetacentric that is 6,7,1011,12,14 chromosome pairs. The

chromosome pair 10 and 14 have been measured with arm ratio of 1.73 and 1.78 respectively.

Karyogram (Figure 6) and the ideogram (Figure 7) was prepared according to Levan and co workers,

based on the relative lengths, arm ratio and centromeric index.

Table 5. Mean short arm, long arm and total chromosome lengths of the Perna viridis chromosomes

Chromos ome Pair

_ p (X ± SD) - q (X ± SD) TCL ('"R ± SD)

1 2.32 ± 0.048 3.70 ± 0.148 6.03 ± 0.156

2 2.41 ±0.130 3.00 ±0.094 5.41 ±0.154 3 2.09 ± 0.046 3.16 ± 0.115 5.25 ± 0.116

4 2.28 ± 0.059 2.78 ± 0.088 5.06 ± 0.097 5 2.075 ± 0.082 2.60 ± 0.098 4.68 ± 0.079

6 1.505 ± 0.086 2.92 ± 0.059 4.43 ± 0.118 7 1.52 ± 0.059 2.80 ± 0.085 4.32 ± 0.079 8 1.80 ± 0.050 2.26 ± 0.110 4.07 ± 0.086

g 1.56 ±0.084 2.15 ± 0.071 3.71 ± 0.144

10 1.34 ±0.046 2.17 ±0.092 3.51 ±0.088

11 1.16 ± 0.057 2.12 ± 0.100 3.26 ± 0.135

12 1.10 ± 0.068 2.03 ± 0.094 3.14 ± 0.099

13 1.07 ± 0.054 1.86 ± 0.110 2.93 ± 0.157

14 1.06 ± 0.097 1.76 ± 0.097 2.84 ± 0.117

15 1.00 ± 0.047 _ 1.49 ± 0.110 2.49 ± 0.137

p - Short chromosomal arm, q - Long chromosomal arm, TCL - Total chromosomal length



Mitotic karyotypes of all the populations of the Perna viridis were found to be symmetrical and out-

wardly identical. Specimens collected from different intertidal zones along the coast of Goa exhibited

marginal change in the total lengths and arm ratios. There are no discreet sex chromosomes observed in

all the populations of Perna viridis studied.

Table 6. Mean relative length, arm ratio, centromeric index and centromeric Position

Chromosome Pair

RI, (X ± SE)

AR (X ± SE)

CI (TC. ± SE)

Centromere Position

1 9.85 ±0.266 1.59 ±0.070 0.38 ±0.010 M 2 8.84 ±0.252 1.25 ±0.083 0.44 ±0.016 M 3 8.59 ±0.190 1.51 ±0.069 0.40 ±0.017 M 4 8.28 ±0.159 1.22 ±0.054 0.44 ±0.011 M 5 7.65 ±0.129 1.26 ±0.090 0.44 ±0.173 M 6 7.24 ±0.193 1.95 ±0.103 0.34 ±0.019 sM

7 7.06 ±0.129 1.84 ±0.107 0.35 ±0.013 sM

8 6.66 ±0.140 1.25 ±0.086 0.44 ±0.017 M 9 6.06 ±0.237 1.38 ±0.053 0.42 ±0.009 M

10 5.74 ±0.144 1.62 ±0.101 0.38 ±0.146 M 11 5.36 ±0.219 1.83 ±0.103 0.35 ±0.012 sM 12 5.13 ±0.162 1.85 ±0.164 0.35 ±0.019 sM 13 4.79 ±0.256 1.72 ±0.058 0.36 ±0.008 sM 14 4.64 ±0.191 1.67 ±0.167 0.37 ±0.028 M 15 4.07 ±0.224 1.49 ±0.100 0.40 ±0.017 M

RI., - Relative length, CI - Centromeric index, M - Metacentric, sM -submetacentric


Fig. 6 Metaphase spread of Perna viridis

R

Karyogram of Perna viridis

) 1( 1! it os so os is

3 4 5 6 7 8

If SO II SI it so 9 10 11 12 13 14 15

%-• N N-

I

Lo


co .

Fig

. 7.

Idi

ogra

m o

f Pe r

na v

irid

is



3.3. Discussion

The diploid chromosome number of30 chromosomes encountered during the present studies is consistnt

with the earlier results of Ahmed, 1974 and Goswami, 1993. Metaphase cells with chromosme number

less than 30 appeared in an insignificant proportion and were ranged from minimum of 0.99 % to

maximum of 7.97 %. The chromosome number in the family mytilidae appears to be highly conserved,

in all the genera studied so far. particularly the genera Mytilus and Perna (Ahmed and Sparks 1970;

Ieyama, 1983; 1984a; Moyniha and Mahon 1983; Thiriot-Quievreux, 1984; Dixon and Flavel, 1986;

Pasantes et al. 1990; Insua et al. 1994). Only Perna viridis is unique in having diploid chromosome

number as 30 (Ahmed 1974; Goswami and Fernandis, 1993).

On the basis of euthyneuran studies, it has been remarked on the general tendency among the gastro-

pods for the more specialized species to have higher chromosome numbers (Burch, 1965). Even in

pelecypods it has been maintained that the " the specialized " eulamellibranchs possess high chromo-

some numbers than " primitive" filibranchs based on the available data (Patterson, 1970; Inaba, 1959).

On the contrary to this theory, some workers proposed that the chromosome numbers have in fact

decreased from primitive to specialized molluscs (Ahmed, 1976) based on the amount of DNA in the

cell (Hinegardner, 1974), as in some higher groups like pisces, insects and amphibians where the spe-

cialization is accompanied by loss of DNA (Ahmed, 1976; Vitturi, 1982). The implication of chromo-

some numbers in the evolutionary studies must be intrepretated with caution. It has been observed that

the there is a tendency for chromosome number to increase with phylogenetic step from Arcoida to

Pteroida in the Pteriomorphia, but in many species of Arcidae higher chromosome number were en-

countered, which goes against the primary hypothesis (Ieyama, 1975; 1983). Even in some pulmonate

gastropods there have been evolutionary trend towards lower chromosome numbers with specializa-

tion (Butot and Kiauta, 1969; Kiauta and Butot, 1969).

The relative lengths of the chromosomes range at maximum being very close to ten micrometers in

chromosome 1 and the minimum being just above four micrometers in chromosome 30 observed. The



values obtained for the relative length of individual chromosomes one to nine shows a similarity with the

corresponding chromosomes in the most of the species of the genus Mytilus especially Mytilus trossulus

and Mytilus californianus, but rest of the chromosomes in the Perna viridis show divergence from the

chromosomes in all the species of the genus Mytilis (Ieyama, 1983; Moynihan and Mahon, 1983;

Dixon and Flavell, 1986; Martinez-Lage et al. 1997). The relative lengths of the chromosomes ten to

fourteen in the Perna viridis are marginally smaller when compared to the relative lengths of the many

species belonging to the genus Mytilus. Chromosome pair fifteen of the Perna viridis with a relative

length of just 4.07 is the smallest chromosome in the Perna viridis and this chromosome shows a

remarkable size disparity from the rest of the chromosomes in Perna viridis and other Mytilus specieses.

The chromosome classification shows a sharp variation from the types of chromosomes in several

species in the genus Mytilus (Ieyama, 1983; Moynihan and Mahon, 1983; Dixon and Flavell, 1986;

Martinez-Lage et al. 1997).

In Perna viridis ten metacentric and five submetacentric chromosome have been found. In Mytilus

edulis and Mytilus galloprovincialis have six metacentric and eight submetacentric chromosomes

(Ahmed et al. 1970; Ieyama, 1983; 1984 Moynihan et al. 1983; Thiriot-Quievreux, 1984; Dixon and

Flavell. 1986; Pasantes et al. 1990; Insua et al. 1994) where as Mytilus californianus and Mytilus

trossulus have seven metacentric and seven submetacentric chromosomes. This reflects that the chro-

mosomes in the genera Mytilus and Perna not just vary in number but also in the morphology of the

chromosomes. Family mytilidaf show a sharp diversity in morphology of chromosome at inter and intra

species level. The chromosomes of Mytilus californianus and Mytilus trossulus show considerable

similarity (Martinez-Lage et al. 1997). The chromosomes of the Mytilus edulis and Mytilus

galloprovincialis share the chromosomal similarity to some extent (Dixon and Flavell, 1986). Many

species of the genus Mytilus also exhibit inter and intra population differences in the chromosome

morphology (Dixon and Flavell, 1986) where as in the Perna viridis only small percentage of metaphases

were found to have a karyotype with eight metacentric and seven submetacentric chromosomes. The

phenomena of variation in the number of submetacentrics have also been reported in other animals like



rat, human embryos Australian flat oyster ostrea angasi and Mytilus edulis (Sasaki, 1961; Thiriot-

Quievreux, 1984; Moynihan and Mahon, 1983; Li and Havenhand, 1997). Such variations were at-

tributed to differential contraction of chromosomes. Chromosomes of the Perna viridis are very much

dissimilar with the chromosomes of the members belonging to Mytilus and the data in genus Perna is

insufficient for any comparision, since the literature on the genus Perna is only limited to chromosome

counts.

Chromosomal rearrangement might be the possible reason for such diversity in the chromosome num-

ber and morphology. It is very likely that genera, species and subspecies in the family mytilidae might

have differentiated through mechanisms like pericentric inversion and Robertsonian translocation of the

chromosomal fragments. It has generally been believed that the intensity of cytokinesis is low in most of

the invertebrates. Comparative studies of cytokinesis and cytokinesis-related phenomena in marine

invertebrates in a model organism like Perna viridis is of great interest to the toxocologic studies

especially in cellular and cytokinesis responses upon exposure to cytotoxic agents. The rate of cytoki-

nesis varies not just from one animal to the other but also from one tissue to other. There is absolutely no

information on the mitotic index in molluscs. Most ofthe literature published deals with the mitotic index

are on the marine dinoflagellates (Weiler and Chisholm, 1976; Wilkerson et al. 1983; 1988). In bran-

chial roots of the gills and the gonads, with possible highest rate of cell division in the mussels reflect that

the mitotic index in these tissues is very low. Mitotic index is not uniform in the same tissue. Average %

mitotic index is negatively correlated with the sex of the animal and positively correlated with the tissue

type. The mitotic index in gonads is high probably because the animals have to replenish the spent

gonads.

3.4. Conclusion

The mitotic index in branchial gills and the gonads of the Perna viridis is low. There is a high significant

difference in the % mitotic index in branchial roots of the gills and gonads. Specimens collected from

several intertidal regions along the coast of Goa were examined for the chromosomal studies. The

diploid chromosome complement in Perna viridis is 30. The chromosomes are classified according to



Levan and co workers as ten metacentric and five submetacentric. Subtelocentric and telocentric chro-

mosomes were never observed. The karyotype formulae for this species is 20M + 10sM. Chromo-

somal length variations were observed in different the specimens collected from different sites. of the

green mussel. There are no discrete sex chromosomes in Perna viridis

Chromosome number and karyological studies are essential but not sufficient for the proper under-

standing and implication of chromosomes in establishing phylogenetic relationship between the genera

of the family mytilidae. It appears that chromosomal rearrangements might have played a significant role

in the evolution of the Pelecypods in general and order mytilidae in particular.


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New CHAPTER 03shodhganga.inflibnet.ac.in/bitstream/10603/11838/8/08... · 2015. 12. 4. · index (MI) in the slides, which were used for the analysis ofthe chromosomes is also included