Indian Journal of Marine Sciences

Vol. II, December 1982, pp. 303-310

Gametogenesis & Somatic versus Gonadal Growth in the Oyster Crassostreamadrasensis (Preston)

M MOHAN JOSEPH* & M N MADHYSTHA

Department of Postgraduate Studies and Research in Bio-Sciences, University of Mysore, Konaje, Mang<llore 575002

Received 15 Oc.wber 1981; revised received 29 October 1982

Histological changes in gonad during the breeding season, gametogenic pattern, gonadal and somatic growth and factorsaffecting gonadal cycle of C. madrasensis inhabiting a tropical estuary, were studied for 2 yr. Gametogenic activity was noticed

twice in a breeding season (October and February - March). Increase in ambient salinity frgm 0.3 to 17"/00 induced

gametogenesis in oysters which had stock piled reserve carbohydrates. Relationship between gon~d thickness and gonadalcycle was distinct and the indeterminate gonads were the thickest. Development of testis was fa~ler.Jhan that of the ovary inoysters < 105 mm shell-height. Spawning was slow but nearly complete in females while in males itw.lis rapid and incomplete.Temporal variations in wet weights of tissue in different sexes indicated that the bulkiest oysters were the indeterminates, which

constituted as much as 100'l~ of the population during southwest monsoon (July - September) period when the salinity was< 1"/00'

Crassostrea madrasensis (Preston) is widely distributedin the estuaries and backwaters of the east andsouthwest coasts of India. Sexual maturation, breedingseason and factors affecting spawning of this speciesare reported from the east coast of India 1 - 7.

Reproductive biology and neurendocrine control ofreproduction in this and related species from thesouthwest coast are also reported 8-10. Bivalvemolluscs are known to show considerable variations in

their reproductive mechanisms depending on the·environment. In this paper gametogenesis and factorsaffecting gonadal maturation, somatic and gonadalgrowth of the species inhabiting a tropical.estuary onthe west coast of India are presented.

Materials and Methods

Random samples of oysters covering all size groupswere collected from the oyster bed at Mulki estuary(lat. 13°5'N; long. 75°47'E) at fortnightly intervals for 2yr from Jan. 1976. The size of each sample was c 50.SheIl-height of individual oyster was determined with avernier callipers. Wet weight of oyster meat wasdetermined after blotting to remove excess moisture.The macroscopic appearance of individual gonad wasrecorded. Sex and arbitary stage of maturity wereascertained from fresh smears of gonad from individualoysters. A total of 2277 oysters were examined duringthe course of the study.

In order to study cytological and histologicalcharacteristics of the gonad and to ascertain the actualstage of maturity, samples of gonad tissue (1 cm2) wereexcised from the right and left lobes, fixed in alcoholicBouin's solution, processed and sectioned at 7 11m.

* Present address: University. of Agricultural Sciences, College of

Fisheries, Mangalore 575002.

These were stained either with Heidenhain's orDelafield's haematoxylin and" (counterstained witheosin. For thestudy of the relation, between the stage ofmaturity and gonad thickness, oysters of 50-80 mmshell-height belonging to various ~tages of maturitywere sectioned across the soft body at the region of thedigestive gland and gonad thickness measured under amIcroscope.

Temperature, salinity, pH, dissolved.; oxygen andturbidity were recorded' from the same station alongwith the collection of oysters. Data on ra\nfall wereprovided by--the Agricultural Research Statiq,n, VAS,Mangalore.

Results

Environmental features

Seasonal changes in different parameters studied areshown in Fig.I. Salinity varied virtually fromfreshwater to typical marine condition. Variations in.salinity were related to the total rainfall and heavyfreshwater influx during the southwest monsoon (July­September). Equally pronounced were variations indissolved oxygen, pH, turbidity and temperatureduring the monsoon. The oysters inhabiting theestuary were subjected to an annual variation insalinity from 0.29%0to 36.98%0'temperature from 24°Cto 31.8°C, paJrom 7.3 to 8.6, dissolved oxygen from2:87 ml/l t~ 5.19 mill and turbidity from 12.5 ppm to95.5 ppm.

Gametogenesis

Based on cell size, nuclear features aq~i staining.characteristics, 4 types of female (PI. Ia) and 5 types ofmale (PI. Ib)germ cells were recognised. The female

303

INDIAN J. MAR. SCI., VOL. 11, DECEMBER 1982

30

,.....

2:")20;j!!«<

III 10

o

Fig. I-Seasonal vaflatlOn in salinity, dissolved oxygen, pH

turbidity, temperature and rainfall at the station

germ cells are the oogonia, primary oocyte, secondary

oocyte and free oocyte. Oogonia are relatively small (6­

10 tLm diam.) with darkly stained nucleus and thin layer

of clear cytoplasm. Dense chromatin granules arepresent. In the primary oocyte (12-18 pm diam.),cytoplasm is diffuse and partly granular. Oocytes areround to elliptical in shape. Chromatin granules andnucleolus are distinct. Secondary oocytes are bigger(20-25 tLm diam.) and polygonal, rectangular orsuboval in shape. Nucleolus is darkly stained anddistinct. Dense chromatin granules are present. Manyoocytes are amphinucleolated. Free oocytes are large(35-45 JLm diam.), spherical or oval in shape andcontain large nuclei. Free oocytes occupy the centre ofthe follicular lumen.

The male germ cells are the spermatogonia, primaryspermatocytes, secondary spermatocytes, spermatidsand spermatozoa (Pl. Ib). Spermatogonia appearduring the commencement of the breeding season andare found attached to the follicular wall. They are verysmall (5 JLm diam.) with a distinct nucleus enveloped bya thin, clear layer of cytoplasm. Chromatin strands andnucleolus are distinct. Primary spermatocytes (2-3 JLm

diam.) are found along the inner margin of the follicles.nucleus is spherical and cytoplasm very thin. Scatteredchromatin granules are distinct within the nucleus.Secondary spermatocytes too are characteristicallysmall (2 JLm diam.) and are found inner to the primaryspermatocyte layer. Cytoplasm is not distinct. Nucleusis darkly stained, spherical. Spermatids (1 JLm diam.)have darkly stained nuclei and are found trailingtowards the centre of the follicular lumen.

Spermatozoa have darkly stained nuclei. The

304

Plate I -(a) CS through ovary of C. madrasensis showing femalegerm cells, x 1200 [PO-primary oocyte; SO-secondary oocyte; FO­free oocyte] and (b) CS through testis showing male germ cells,x 3000 [PS-primary spermatocyte; SS-secondary spermatocyte; ST-

spermatid; SZ-spermatozoa]

cephalosome is about 1 JLm in diam., mesosome is notdistinct.

Gonadal cycle

The scheme of classification of maturity stages usedin the present study is that proposed by Joseph8.Characteristics of various maturity stages of gonadsare given in Table 1 and PI. II and III.

Gonadal versus somatic growth

Relation between gonad maturity and gonadthickness-Although gonad thickness may vary

,f f

{

n'

_____ - __ u _

JOSEPH & MADHYSTHA: GAMETOGENESIS & GROWTH OF OYSTER

Table I-Characteristics of Female, Male and Indeterminate Gonads in Different Stages of Maturity

Maturitystage

Macroscopic appearance of gonad Microscopic features of fresh smear Histological characteristics in permanentpreparation

Very few residual oocytes. Large Follicles appear shrunken and collapsed.quantity of fluid in the gonad Relict ova very few. Phagocytes in large

number. Interfollicular tissue developed

Gonad thicker. Genital ductules visible Secondary spermatocytes and spercthrough body wall matids appear as spherical bodies.

Motile spermatozoa rare

Gonad bulky, cream coloured. Genital Large number of free motileductules very prominent. Germ cells ooze spermatozoa visiblefreely when gonad is punctured

FOl

FD2

FD3

FD4

FRI

FR2

MOl

MD2

MD3

Gonad thin, whitish

Gonad thicker. Underlying genital duc"tules visible through body wallGonad bulky_ Ramifications of genitaltubes clearly visible. Germ cellsooze whengonad is punctured

Gonad thick and massive. Colour creamy.Extends over whole of visceral mass.Oocytes ooze freely when gonad ispunctured

Gonad and mantle dull brown in colour.Mantle thick. Fluid accumulates at theanterior part of body near labial palps.Oocytes do not ooze freely when gonad ispunctured. Underlying digestive glandpartly visibleGonad and mantle brownish, flaccid.Tissue overlying gonad very thin. Visceralmass and labial palps with accumulatedfluid. Digestive gland clearly visiblethrough overlying tissue. Residual germcells visible as white patches in gonad

Gonad thin, whitish

FEMALE

Oogonia and primary oocytesdistinct

Primaryoocytes with distinct nucleinumerousSecondary oocytes 33-35 /lm diam.with nuclei of 25 pm appear in largenumber. Oocytes elongated withnarrow stalksLarge number of free oocytes (45-70pm) with spherical nuclei (25-30pm). Oocytes conical or spherical inshape. Secondary oocytes few

Good number of free oocytesappear, but not densely packed

MALE

Spermatocytes as spherical bodies(3 pm). Rest of tissue mostlyconnective

Follicles appear as scattered patches in thegonad. Follicular wall lined with oogonia,primary oocytes and secondary oocytes.Follicles small in size

Follicles appear larger in size. Follicularwalls lined with secondary oocytesFollicles enlarged in size and extent.Lumen filled with secondary oocytes.Shape pedunculate ot suboval. Size 2~25pm. Primary oocytes and free oocytes fewFollicles appear thickly packed in gonadwith interfollicular tissue. Free oocytes(3~45 pm) with nuclei (19-22 pm) filllumen of follicles. Pedunculated secon­dary oocytes few, attached to follicularwallFollicles appear partly shrunken.Follicular lumen empty except for a fewunspent residual ova. Sometimes secondserieses of secondary oocytes seen on thefollicular wall

Follicles appear as small round or ovalpatches. Size of follicles small. Darklystained spermatogonia and few sperma­tocytes visible along periphery of thefollicular wallFollicles appear as large irregular patcheswith anastomosing branches. Secondaryspermatocytes and spermatids form awide band along follicular wall. A fewspermatozoa appear in lumenFollicles fill entire area of gonad withanastomosing branches. Large number offree spermatozoa occupy entire lumen offollicles while a few spermatids trailbetween centre and follicular wall. Band ofspermatocytes along follicular wallnarrow

MRI

MR2

Gonad and mantle dull brown in colour, Good number of motile sperma­flaccid. Fluid accumulates near the labial tozoa appear, but less denselypalps. Germ cells do not ooze freely when packedgonad is punctured. Underlying digestivegland partly visibleGonadal wall brownish, flaccid. Large Motile spermatozoa very fewquantities of fluid in the body. Unspentgerm cells visible as white patches.Digestive gland clearly visible

INDETERMINATE

Normal site of gonad white, massive and No gonadal matter is discerniblehealthy in appearance. Tissues thicklypacked and break easily at slight pressure.Does not ooze when punctured. Labialpalps thick, whitish

Follicles appear partly empty with centralportion devoid of spermatozoa.Connective tissue starts developing inbetween the follicles

Follicles appear empty except for patchesof unspent spermatozoa. Interfollicularconnective tissue well developed.Phagocytes 'few

Follicles are not discernible. Whole area inbetween body wall and digestive glandfilled with connective tissue formed bylarge lightly stained leydig cells

305

---~ ~fit*"IiI,iij_dliWl,IiIII"»~ -- --

INDIAN 1. MAR. SCL VOL. 11, DECEMBER 1982

"

Plate II -CS through ovary of C. madrasensis in different stages of maturity, x 140[I-stage FDI; 2-stage FD2; 3-stage FD3;'4-stage FD4; 5­stage FRI; 6-stage FR2 (FW-follicular wall; SO-secondary oocyte; Fa-free oocyte; IF-interfollicular tissue; RO-residual oocyte)]

306

"

JOSEPH & MADHYSTHA: GAMETOGENESIS & GROWTH OF OYSTER

Plate III -CS through testis and indeterminate gonad of C. madrasensis in different stages of maturity, x 140[1-stage MO 1;2-stage M02; 3­stage MD3; 4-stage MR J; 5-stage MR2; 6-stage I (FW-follicular wall; ST-sperma!id; SZ-spermatozoa; IF-interfoJlicular tissue; RS-residual

spermatozoa; CT -connective tissue)]

307

./

INDIAN J, MAR. SCI., VOL. II, DECEMBER 1982

Table 2-Relation between Stages of Maturity andGonadal Thickness

[Figures in parentheses indicate range in gonadal thickness]

depending on the size of the oyster, the gonad thickness

of oysters of comparable size during a season reflectsthe condition of the gonad in a marked way (Table 2).As gonad development progresses from stage M D1tostage M D3, the mean thickness of right and left lobe ofthe gonad increases followed by a rapid regressionthrough stage M R 1and M R2. It appears that in malethe left lobe is thicker than the right lobe and thedecrease in bulkiness suffered by the latter is

considerably greater than that by the former. A similar

trend is observed in the female oyster also (Table 2).Comparing the gonad thicknesses of both sexes,

females appear to be bulkier than males at full maturity

while at fully spent stage the condition is reversed. The

indeterminate oyster has the thickest 'gonad'. This maybe due to rapid development of interfollicularconnective tissue during the regression taking placetowards the end of the breeding season as in most otherbivalves.

Sex-based allometric gonadal growth-Earlier

studies have revealed that in C. madrasensis the

somatic and gonadal growth take place duringdifferent seasons of the year and the population as a

whole does not present any clear relationship between

Mean gonadal thickness (/lro)

~.

jod" ••••/••'Y10

20Yv ...,. ~_ 10

.. -------•...0J:

~ 25~ /~~:yvV0o

20

o I· I I I I I I 1 I I r-I I I I I , •• A •..

J( F •• A •• ~9~ SO" D)_t_F_M_A_M_J'19fr SO" r

Fig, 2-Seasonal variation in the meat weight of male, female andindeterminate oysters

size and gonadal or somatic growth8• Therefore, data

on wet weights were segregated into 3 categories: (1)

weights of males and females undergoing activegonadal development, (2)weights of males and femalesundergoing gonadal regression and (3) weights ofindividuals in the sexually indeterminate stage. Curvesof the form Y =bxa were fitted to the observed data

and F test applied to test the significance of differences

between sexes and between above 3 categories (Table

3). Comparison of males in the gonadal developmentstage with those in the gonadal regression stage

indicated that the difference in the relationship is also

significant. However, a comparison of females in the 2separate stages indicated no significant difference.Therefore, females in both stages are comparable and

may be treated together to arrive at a generalregression equation (W =0.0001589 HZ,3466).

Seasonal variation in meat weights-Variations in

the meat weights showed the same trend (Fig.2) aswould be shown by the ratio meat to body weight.

Moderate mean weights were recorded during Jan.1976 and the highest value was recorded in both sexes

during April. This increase was primarily owing to the

development and proliferation of gonad during thisperiod. As a result of intense spawning, the meanweights decreased to the lowest levels during May.June to September witnessed not only a gradual and

left lobe

512( 417- 666)1064( 500-1583)

1697( 916-2083)

886( 749-1083)

525( 333- 749)

375( 250- 583)

546( 333- 666)

1101( 666-2166)

1735( 833-2582)

760( 583-1166)

365( 250- 500)3310(1749-5415)

right lobe

369( 250- 500)

657( 417- 833)

968( 666-1166)

295( 166- 417)

225( 166- 333)

308( 166- 500)

426( 250- 583)

592( 417- 999)

972( 500-1583)

260( 166- 333)

229( 83- 333)2575(1250-4165)

\ADI 7

MD2 9

MD3 8

MRI 11

MR2 10

FDI 10FD2 9

FD3 9FD4 12FRI 8

FlU 81 10

Stage of No, ofmaturity obser-

vations

Table 3-Relationship betweenSexesand DifferentWeight Categories in C. madrasensis

[Wt=weight, H=sheJl height, NS=not significant]

l \

(i) Gonadal development

(ii) Gonadal regression

Male (a)

W =0,007921 HI.SS02

W=0.001765 H1.7613

Female (b)

W =0,0001477 H2.400S

W=0.0001597 H2.2911

P value

<0.05 (between a & b)

<0,05 ( do )

<0.05 (between a in (i) & (ii)

NS {between b in (i) & (ii)

(iii) Indeterminate W=O.00049 H2,lS08

During gonadal growth and regression periods, the weight (W) to shell-height

(H) relationships between sexes are significant

(

308

11 "II

JOSEPH & MADHYSTHA:GAMETOGENESIS & GROWTH OF OYSTER

/

Fig. 3-Seasonal variation in average weight of oysters in relationto maturing, spawning and indeterminate gonadal condition in the

population

gametogenesis in C. gryphoides14• The present datashow that during Oct. 1976 and Sept. 1977 the salinityincreased from nearly fresh (O.3roo) to brackish ( '" 17roo)

water. The onset of gametogenesis during both theyears synchronized with this rapid increase in salinity.

A clear-cut relationship between relative gonadthickness and reproductive cycle has been revealed bythe present study. Loosanoff26 used such a relationshipto study the spawning in C. virginica. The present studyindicates the possibility of using gonad thickness as anindex of gonadal condition in routine analysis andunder field conditions.

The allometry exhibited by oysters during gonaddevelopment is an aspect which has received littleattention hitherto. Increase in weight with size is morerapid in male than in female in the present study whenthe size of th;; oyster is < 105 mm shell height.Therefore, in young- oysters the testis of the maledevelops faster and increases in bulk than the ovary inthe female. During the gonadal regression {5hase,thefemales > 90 mm shell-height are heavier than themales of equal size. This probably indicates slow orincomplete spawning or accumulation of fluids in thetissue during the gonadal regression phase. In contrastto these, oysters in. the indeterminate stage are, ingeneral, heavier than males and females.

The value of the equilibrium constant ex in theequation Y=bxll indicates the relative growth inweight as compared with length. In the present studythe ex values in the case of males in the gonadaldevelopment and regression phases are 1.5502 and1.7613 respecPively,while in all the other cases they arebetween 2.1 and 2.4.When the value of IX is other than 1the growth is considered allometric27• However, the ~values reported here are lower than the range from 2.5

Discussion

Tropical and subtropical invertebrates mostly havesemiannual or annual breeding seasons and thecontinuous breeding seasons reported in tropicalspecies are really not continuousll. The present studyshows that C. madrasensis has gametogenic activityat least twice in a breeding season, 1st du~ing Octoberand 2nd'during February - March. Variability ingametogenic activity· of Indian oysters has beenreport~d4.S.12-16 which indicates that initiation ofgametogenic activity in the same or related species varygreatly depending on the habitat and intrinsic orextrinsic factors.

When individual gametogenic cycles are synchro­nised resulting in simultaneous breeding within. aspecies population, exogenous factors such as light,temperature and salinity act in combination and exertproximate control over breeding periods. Althoughthere is evidence to show that Orton's rule17 (criticalbreeding temperature) is applicable to a few tropicalspecies subjected to narrow variations in -te~pera­ture18, in shallow marine areas and estuaries variations

in the salinity appear to be a possible factor regulatingreproduction3.13.19 -24. Under low salinity conditions,gametogenesis is inhibited in c. virginica2S whiledecrease in salinity from 40 to 32/~o incites

marked increase in the mean weight of oyster, but alsoa transition of male and female oysters toindeterminate sex. Mean tissue weights as high as 16 gwere recorded during this period. This was evidentlythe result of development of considerable quantity of. ~vesicular connective tissue during the indeterminate ~stage as well as storage of large quantities of reservecarbohydrates in the tissues as reported earlier8. ByOctober, the majority of oysters had initiatedgametogenesis and sexes were discernible.

The general trend that emerges from the study of thegonadal and somatic growth is presented in Fig.3. Itindicated that during 1976 and 1977, maximumsomatic growth was during June - September when theoysters were in the sexually indeterminate stage. Themeat was bulky and the mantle thickly laden withconnective tissue formed by Leydig cells. It has alsobeen reported that during this period the indices ofcondition and edibility were the highest and the level ofcarbohydrates which formed toe major reserve fuelreached the yearly peaks. This period alsosynchronised with the southwest monsoon when thesalinity of the estuarine water was the lowest. Gonadalgrowth commenced during October. The sexual phasecontinued up to the end of Mayor the early half ofJune. Variations in the mean weight of the oystersduring this period reflected the development andsubsequent shedding of gametes.

309

INDIAN J. MAR. seI.. VOL J 1, DECEMBER 1982

to 4.5 generally accepted for bivalves27• Similar low

values have been reported in C. gigas from differentgeographical areas28 - 30.

A distinct correlation between the variations in themeat weight and annual gonadal cycle was evident inthe present study. The somatic growth takes placeduring the period when the salinity is on the decline.During late September - early October as the salinity inthe estuary builds up, gametogenesis commences andas a result the mean weight shows an initial decline.Depending, on the degree of gonadal development,fluctuations in mean weights are noticed during thisseason. Spawning results in drastic reduction in wetweights. The relative changes in weight with respect togonadal cycle reported in the present study support thefindings of Durve14 in C. gryphoides, but are not inagreement with those of Walne31 in Ostrea edulis.

References

1 Hornell J, Madras Fish Bull, 4 (1910) 25.

2 Hornell J, Madras Fish Bull, II (1917) 1.

3 Panikkar N K & Aiyar R G, Proc Indian Acad Sci, 9 (1939) 343.

4 Rao K V, Proc Indian Acad Sci, 33 (1951) 231.

5 Rao K V, Proc Indian Acad Sci, 44 (1956) 332.

6 .Agastheesapillai A & Subramoniam T, in Proceedings symposiumcoastal aquaculture (MBAI, Cochin) (in press).

7 Rajapandian M E & Rajan C T, in Proceedings symposium

coastal aquaculture (MBAI, Cochin) (in press) .

310

8 Joseph M M, Studies on the biology of the Indian backwater oyster

Crassostrea madrasensis (Preston), Ph.D. thesis, University

of Mysore, 1979.

9 Nagabhushanam R, in Proceedings symposium coastal

aquaculture (MBAI, Cochin) (in press).

10 Joseph M M & Madhystha N M, Aquaculture (1982) (in press).

11 Giese A C & Pearse J S, in Reproduction of marine invertebrates,

edited by.A C Giese and J S Pearse (Academic Press, New

York) 1974, 1.

12 Rao K S, in Molluscs of India, edited by R V Nair and K S Rao

(CMFRI Bu1/15) 1974,4.

13 Paul M D, Proc Indian Acad Sci, 15 (1942) 1.

14 Durve V S, J mar Bioi Ass India, 7 (1965) 328.

15 Nagabhushanam R & Bidarkar D S,IndianJ Fish, 24 (1977) 135.

16 Ansari F & Ahmed M, PakistanJ Zool, 4 (1972) 35.

17 Thorson G, Medd Greenland, 100 (1946) 1.

18 Yonge eM, Great Barrier Reef Exped 1928-29 Sci Rep, (1940) 1.19 Pearse J S, Proc Indian Acad Sci, 68 (1968) 247.

20 Schlieper C, Annee Bioi, 33 (1957) 117.

21 Sandison E E, J Anim £col, 35 (1966) 363.

22 Sandison E E, J Anim Ecol, 350966) 379.

23 Hill M B, J Anim Ecol, 36 (1967) 303.

24 Wilson B R, J Natur Hist, 3 (1969) 93.

25 Butler P A, Bioi Bull, 96 (1949) 263.

26 Loosanoff V L, Bioi Bull, 129 (1965) 546.

27 Wilber K M & Owen G, in Physiology of Mollusca, edited by KM Wilber and C M Yonge (Academic Press, New York)

1964,211.28 Lee C K & Yoo S K, Bull Natl Fish Univ Busan, 14 (1975) 41.

29 Hughes-games W L, Aquaculture, 11 (1977) 217.

30 King M G, Aquaculture, 11 (l977) 123.31 Walne P R,J mar Bioi Assoc UK, 44 (1964) 293.

\ .

.\ (

\ I ~ II