ON THE USE OF DETERMINATION OF

ANGUILLA NEBULOSA

OTOLITHS FOR THE AGE AND GROWTH OF NEBULOSA McCLELLAND

BY V. RAYAPPA PANTULU AND VIJAI DEC S ~ a a (Central Inland Fisheries Research Institute, Barrackpore, India)

Received Janualy 23, 1962 (C3,-nnunicated by Dr. B. S. B'aimachar, r.A.SC.)

THE use of periodic markings on otoliths and other hard parts of th ~. body, for the estimation of age and growth of fishes, was established early in the present century. Though otoliths were used widely for ageing fishes from temperate zones (Roleffson, 1933, 1935, Parrish and Sharman, 1958, etc.), their utility in the determination of age and growth of tropical fishes, parti- cularly from Indian waters, has not been adequately investigated. Rao (1935), Nair (1949) and Radhakrishnan (1954)observed rings on the otoliths of some Indian fishes and indicated the possibility of their utility in age studies and Radhakrishnan (1957) estimated the length~ of sand-whiting (Sillago sihama) for the first four years of its life based on the rings on otoliths. Since studies on age and growth are of supreme importance in following the changes in abundance of fish populations and as the utility of hard parts of the body in ageing tropical fishes is still a matter of con- troversy (Pantulu, 1961), this investigation, which is essentially concerned with technique, is yet one more contribution towards the solution of an important problem.

MATERIAL AND METHODS

The Eels were collected from an escape channel conveying waste water from the Calcutta Water Works (at Palta near Barrackpore) to the river Hooghly. Though situated at a distance of about I00 miles fi'om the sea, this channel is subject to tidal influence and maintains its connection with the river, perennially. Smaller individuals of the eel were collected with lhe help of small-meshed drag nets and the adults by groping at the bottom of the channel with hands (Job and Pantulu, 1953). Collections were made duriiag all the monlhs of the calendar year, 1953. Sex was determined by the examination of a squashed preparation of the gonad under a microscope. Both the saccular otoliths were removed from each fish and stored in a labelled tube of rectified spirit. Examination of otoliths, after initial cleaning

263

264 V. RAYAPPA PANTULU AND VIJAI DEV SINGH

by rubbing between the finger and thumb, was made in pure beechwood- creosote, Xylol and clove oil under reflected light against a black background. In all these media alternating opaque and translucent zones were clearly visible only in the smaller otoliths. Bigger otoliths were ground on a glass plate in carborundum or alundum powder to enable the rings to be clearly seen. For final examination, all the otoliths (smaller, unground and bigger, ground) were mouvted on glass slides in canada balsam. About 120 otoliths were examined in all, of which only 8 were those of females and the rest of males and immature individuals. Since the sample of otoliths of females was very small, studies were confined to the determination of age and growth of only immature and male eels. About 3070 of the otoliths were unreadable in the sense that the demarcation, between the opaque and translucent zones, was not clear. These were discarded, and the studies described below are based on the examination of 76 clear otoliths.

AGE DETERMINATION

Otoliths of glass eels (freshly metamorphosed leptocephali, 40--60 mm. long) had a small opaque centre (nucleus) surrounded first by a translucent and then by an opaque zone, and with an outer border of translucent margin (Plate, Fig. 1). These obviously were the "seewasserringe" of Hornyold (1922) which, according to the customary interpretation (Hornyold, op. cit. ; Cairns, 1941), indicate that prior to their entry into the estuaries the lepto- cer, hali of A. nebulosa spent over one and a half years in the sea. Though the annual nature of translucent zones in the case of bigger eels was estab- lished (vide infa) no categorical statemeent regarding the validity of the above inference was possible in leptocephali in the absence of definite information regarding the pattern of growth-zone formation in them.

Otoliths of bigger eels showed a similar centre with alternating trans- lucent and opaque hands, the number of bands increasing with the size of the fish. ~Ihe margins of otoliths of eels captured during different months of the year were carefully examined and it was observed that, generally, a predominantly high percentage of otoliths had translucent margins during the months, September-December (Text-Fig. 1 a). Further, the width of opaque bands, wherever they constituted the margin, was measured during all the months. Text-Fig. 1 b illustrates the mean width of the marginal opaque bands, expressed as percentage of the total radius of the respective otolilhs, during different months of the year. It is evident from the figure that the opaque bands ~are broadest in August and narrowest in September, progressively becoming broader till August of the subsequent year. This disposition of the opaque band tends to confirm the above con-

Otoliths for Determination of Age & Growth of A. nebulosa 265

clusion regarding the time of formation of the translucent band. Hence the age of the fish was assessed by the number of translucent bands on the o toh t~ .

I0" . b

IO

N

$

I 0 0 -

).. U Z

SO- w u .

Q

I l i i i

J F M A

I ; i i i

M J d A

I -LA-

I i i 1 S 0 N D

T~xT-F]o. 1. (a) A histogram showing the percentage frequency distril~ution of otolitks with transparent margins, during differet:t mor, tt:s of the year. (b) Illustration of the average thickness of opaque margins during different months of the year.

Since the growth of any structure to be used in ageing has to be pro- portional to the growth of the fish, the relationship between otolith radius and fish length was estimated. The otoliths were measured at right angles to a perpendicular line passing through the nucleus. Measurements were taken with the aid of an ocular micrometer, from the nucleus to the margin of the otolith, for total radius, and up to the point of termination of the respective translucent zones, for radii at different ages (Text-Fig. 2). A scatter diagram of total radius of otoliths (Y) on the corresponding total lengths of fish (X) indicated a linear relationship, in the logarithmic form (Text-Fig. 3). The regression equation was estimated to be

logY = -- 0.5893 ÷ 0.8781 logX.

A coefficient of correlation, r = 0.9025, was obtained, indicating the high level of significance of this correlation. In deriving a formula for back calculation it was considered more appropriate to take into consideration

266 V. RAYAPPA PANTULU AND VIJAI DEV SINGH

the regression of total length (L) on otolith radius (R) and not v i ce v e r s a

(Pantulu, in press) since the object of the study was to predict intermediate values of length for known intermediate values of otolith radius. Such a regression equation was found to be

log L = 1.0181 + 0.9278 log R.

TExT-FIG. 2. I 'bxstra t ion o f o to l i th g r o w t h - z o r . c m e a s u r e m e ~ t s , n = nucleus , R = t6tal rad ius , r x to r~ = rad ius o f respect ive annua l f l ags f rom tt-,e cer:tre o f growth .

Based on this relationship, the following formula for back calculation of lengths, at the times of formation of various translucent bands on the otoUth, was derived.

log l~ = log L T -]- b (log r n - - log RT)

where In = length at age n; LT = length at the time of capture; r n =

radius of the otolith at age n; R,r the total radius of the otolith at the time of capture; and b = slope of the regression line (0.9278).

One particular merit of this equation is that the estimates of length at a given age depend on the ratio of the total radius of the otolith to the

Otoliths for Determination of Age & Growth of A. nebulosa 267

radius at" age n, and back calculation are made along the estimated slope, b, of the regression line (Smith, 1955, Pantulu, 1961) and no assumption of a direct proportional relationship is made.

17-

,,.¢

,& ~5-

13-

22 2!3 2:4 2'5 2'e 2'7

TE×T-FIG. 3. Regression of otolilh radius on tolaI length of the eel

Intermediate lengths were calculated by the use of the above formula, individually in all cases and the mean back-calculated lengths at various ages are given in Table I. Growth rate appeared to be fastest during the third year of the life of the eel and a decline thereafter in annual increments of the linear dimension with increase in age was generally noticed. The observed range of fluctuations, on either side of the mean values, of lengths at ages and the remarkable facility with which the smaller eels, particularly, escape through the mesh of the drag net, lead us to the conclusion that the high values for the younger age groups may have resulted from partial recruitment of only bigger-sized individuals of younger ages, due to mesh selectivity. Hence it is.assumed that the mono-molecular or von Bertalanffy (1939, 1949, 1957) growth equation, "which gives a curve for growth in length that fits well the growth rates of many species of fish" (Beverton, 1954, Beverton and Holt, 1957), adequately describes the growth of the male Anguilla nebulosa. The close agreement observed between the lengths at ages as estimated by the yon Bertalanffy growth equation and by other methods (Text-Fig. 5) confirms that this assumption is justified. Hence lengths at age 't + 1' were plotted against lengths at age 't' (Text-Fig. 4) and a least square line wes fitted to the resultant points, which gave the estimates of loo (the point of intersection of the least square line with the bisector drawn through the origin), and K ( = e-k).

lt+l = 1 ~ (1 - - e "k) q- IW "k.

268 V. RAYAPPA PANTULU AND VLIAI DEV SINGH

TABLE I

Mean back-calculated lengths o f immature and male Anguilla nebulosa at different ages

Age Number Age group of

(at capture) specimens Mean 1 2 3 4 5 6 7

. . . . . . . . . . . . . .

II . . . . . . . . . . . . . . . . . .

III 20 .. 55.22 139.86 250.14 . . . . . . . .

IV 18 .. 49.62 131.50 206-50 264.83 . . . . . .

V 14 .. 58-86 137.16 231-57 276.80 350.50 . . . .

VI 12 .. 43.86 117.60 177.40 259.40 304.80 363.00 ..

VII 12 .. 48.00 124.00 206.83 274.17 323.50 384.00 434.5

Total .. 76 Mean 51.11 130.02 214.48 268.80 326.26 373.50 434.5

The values of the parameters were estimated to be l~ = 682.89 • K = 0-14156.

The theoretical age at which the length of the fish was zero, ' t o ' was estimated by writing the growth equation as (von Bertalanffy, op. cit.)

log e (lo~ -- It) = (log elo~ - - kto) - - k t .

Hence plotting values of log e ( l~ -- It) against the corresponding age, gave estimates of to (I~everton, op. cit. ; Pantulu, 1961)which, in this case, was found to be to = 0.3214. Hence the yon Bertatanffy growth equation for immature and male A. nebulosa can be written as:

It = 682.89 (1 -- e -°'roSe ~t-0.3~1~).

Table II gives the estimates of lengths at different ages by various methods. The reasonably close agreement between these estimates, coupled with the facts that a proportional relationship exists between the growth of the fish and that of the otolith, and that the translucent bands are of annual nature, confirms the validity of the use of otoliths in the estimation of age and growth of A. nebulosa (Text-Fig. 5).

Otoiiths for Determination of Age & Growth of A. nebuiosa 269

700"

SO0"

4 O 0 - ,,i. ',.._., u,,I r- ~ ~ "

z

2 0 0 -

* 0 0 -

,oo 200 300 ~ o s~o 6(~o 760 LENGTH AT T I M E ~., M,M

TEXT-YIG. 4. Ford-Walford plot of length at time ' t ' against length at ' t 4- 1'.

DISCUSSION

q-he fact that the population of eels from the estuarine waters is repre- sented predominantly by males, confirms the observation made by Syrski (1874) and Tesch (1928) that "as a rule males are to be found in estuaries near the mouths of rivers, whereas females generally live in the upper reaches of rivers" Since it is well known that all eels under a certain size are not sexually defined, and that the sex-organ of the young eel is capable of deve- loping either as male or female according to the environment in which it is found (Mazza, 1913, Grassi, 1921; D'Ancona, 1924, Tesch, 1928), it is reasonable to presume that no sexual differences in growth would exist in immature or sexually undefined eels. From the few female specimens examined, it appears that females grow faster than males, an observation

B5

270 V. RAYAPPA PAN~rULU AND VIJAI DEV SINGFI

500 ' ~= 682 "89

• OTOL ITH $TUOI Ie>

o LENGTH FREQUENCIES

" i , , ' • " I I t 2 3 4 5 6 7 9 I1

AGE

TEx~-Fm. 5. Theoretical growth CUlVe of Anguilla nebulosa (immature and male) obtained by fitting the von Bertalanffy growth equation (continuous line). Dark circles represent estimates by otolith studies and open circles by length frequency analysis.

which is in general agreement with known facts about other species o f Anguilla (Tesch, op. cit. ; Cairns, op. cit.).

Pantulu (1956), who estimated the age of.4. bengalensis ( = nebulosa) by means of scales, found the mean length of eels with one scale-ring to be 183.56mm. According to the present study, eels ranging in length from 177.4 mm. to 250.14 mm. (mean 214.48 mm.) have been estimated to be three years old. Considering that scales first form in elvers acclimatised to fresh- waters for six to seven months after metamorphosis, and that the first ring is laid approximately a year after (Pantulu, 1956), and as the glass eels are found to be about 1½ years old (vide page 264), the lengths of eels with one scale- ring should correspond to those with three otolith rings. This agreement may probably be taken to indicate the validity of the interpretation of 'seewasserringe' (sea-water rings) made in this study. However, the agree- ment between scale and otolith studies extends only up to the fifth year, after which estimates made on the basis of scale studies are much higher than those of otolith studies. Pantulu (1956) pointed out that some caution was necessary in utilizing the scales for the estimation of age, which "mus t necessarily involve a certain degree of error". The reasons that led to the

Otoliths for Determination of Age & Growth of A. nebulosa 271

TABLE II

Comparison of mean lengths at various ages as estimated by different methods

Mean lengths in mm. as estimated by Modes of

Age length frequency Otolith Theoretical distribution studies growth equation

1 51.11 62.55 88.00 (4-18.0)

2 130-02 144.'57 t38.5 (139.90)

3 214.48 215.52 192.00 (4-26.34)

4 268.80 277.32 251.20 (4-24.41)

5 326-26 330'59 331-00(:k29"59)

6 373"50 377"23 397.00 (4-25.13)

7 434"5 419"84 468.~0 (4-25-13)

8 .. 452.69 520-00 (4-26-89)

9 .. 482.87 ..

10 .. 509"37

conclusion were, that rings were observed to form on the scales during practically all the months of the year; that no direct correlation was observed between the size of the scale and the number of rings on it and due to the wide variability in the number of rings on scales taken even from the same region on the body of the eel. The length frequency data presented in his paper (Pantulu, 1956) have been re-analysed by the use of the probability plot method (Harding, 1949 and Cassie, 1954) of separating the component normal curves in a polymodal frequency distribution. The modal values of different size groups and the respective standard deviations as estimated by this analysis are given in Table If. One additional mode at 138-5 ram. is revealed, and the existence of a mode at 397.00 mm. (corresponding to the one at 386 mm. which was ignored as being probably a false one) was confirmed by this analysis. The modal lengths of the length frequency distributions show a fair degree of agreement with estimates by the otolith method up to the sixth year, beyond which corresponding values

272 V, RAYAPPA PANTULU AND VIJAI DEV SrNorl

of modes are considerably higher, which may be due to paucity of samples and non-representation of intermediate size groups in them.

A comparison of the growth of different species of eels belonging to the genus Anguilla brings out certain interesting features (Text-Fig. 6).

4 0

a'O

/ e //j

AGE IN YEARS

T~xI-FIG. 6.

~ ,'o

Approximate 'growth rate' curves of different species of Anguilla.

Otoliths .for Determination of Age & Growth of A. nebulosa 273

A. nebulosa, the Indian eel, appears to be the fastest growing species of the genus. This is interesting when viewed in the light of " the statement often repeated in fisheries literature that in warmer waters the fish tend to be smaller than in cooler waters but, equally, that they grow faster in the former" (Beverton and Holt, 1959). In this instance while the growth of the Indian eel is faster, the size also is consistently bigger, at identical ages, than eels from cooler waters. As regards growth, a similarity in growth rate is generally noticeable between the European eels (Ehrenbaum and Murakawa, 1914; Tesch, 1928; Marcus, 1919) and New Zealand eels [Cairns, 1941 ; McFarlane (quoted by Cairns op. cit.) whereas the Winder- mere (Frost, 1945) and Indian eels appear to be more or less similar to each other and different from the rest.

SUMMARY

A critical examination of the periodic markings on otoliths has helped to establish that the markings could be used for the estimation of age and growth of A. nebulosa. The relationship between the otolith radius and total length of the fish has been determined, based on which a formula for back calculation of lengths at different ages is derived. A study of the otolith margins has shown that the formation of the translucent bands is annual in nature. A yon Bertalanffy growth equation has been fitted to the data and it is found that, in the size ranges studied, this growth equation adequately describes the pattern of growth of the eel. Earlier studies on the age and growth of the eel are discussed and a comparison with growth of other species of eels of the same genus has been attempted.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. B. S. Bhimachar for the many helpful suggestions given during the progress of the investigations and for his critical reading of the manuscript.

1. Ancona, U . D .

2. von Bertalanffy, L.

3o 4.

REFERENCES

.. "La croissance chez les animaux mediterranees," Rapp. Pr. Verb. Comm. lnt. ExpL Medit., 1924, 10.

.. "A quantitative theory of organic growlh" (Inquiries on growth laws II), Hum. Biol., Baltimore, U.S.A., 1938, 10 (2), 181-213.

.. "Problems of organic growth," Nature, 1949, 163, 156-58. . . "Quantitative laws in metabolism and grox~th," Quart. Rev.

fliol.~ 1957, 32~ 217-31,

274

5.

7.

V. RAYAPPA PANTULU AND VIJAI DEV SINGH

•. " N o t e s on the use of theoretical models in tl-.c study e f the dynamics of exploited fish populations," U.S. Fi.~hery Lab. BeauJott. N•C. Miac. Contr., 1954, 2, 181 miraeo.

~ a n d Holt, S.J . .. " O n the dynamics of exploited fish populations," .Fishery lnvestigationa Series, Lend., 1957, 20(2).

.. "Longevi ty and mortality iatcs of fish in nature," 1959, 5, Ciba Foundation Colloquia on Ageing, J. and A. Churchill Ltd., London, 1959, 5.

.. "Life-his toly of the t~o species of the 1New Zealand Fresh- water Eel ," 2V.Z.d. Sci. Tech., 1941, 23(2)B, 53 N-72 B; 1941, 4B, 132-48B; 1942, 56, 172-78B.

. . "Some uses of probability paper in the analysis of size fre- quency distributions," Aust. d. Mar• Freshw. Re~., 1954, 5 (3), 515-22.

" A g e and growth of eels," Zeitenawr. F. Fi6herie.s, 1914, 14. " T h e age and growth of eels (Anguilla anguilla) from the

Windermere catchment area ," J. Anita. Eco., 1945, 14 (1), 26-36; (2), 106-23.

.. " N u o v o rieerehe sulla storia naturale dell Anguilla," Mem. R. Comit. Talass. ltal., 1919, No. 67.

.. " T h e use of probability paper for the graphical analysis of polymodal frequency distributions," J. Mar. Biol Ass. U.K., 1949, 25, 141-53.

" T h e age and growth of some eels from a small Worcester. shire pond," J. R. Micr. Soc., 1922, 9-26.

Job. T. J. and Pantulu, V. R. " F i s h trapping in India," d. Asiat. Soc. Bengal (Sci.), 1953, 19, 175-96.

Marcus, K. .. " U b e r Alter undWachstum des Asles," Milt. Zool. Mus., 1919, 36.

Mazza, F. .. "Risul ta t i di ricerehe anatomoistologiehe sngli organi genitali delle anguilla dacqua dolee e dacqua salmastra," Boll. Soc. Zool• Ital. Ser. 3, 1913, 2.

.. " T h e growth rings on the otoliths of the oil sardine Sardi- nella longiceps (Cuv. & Val)," Curr. Sci., 1949, 18(1), 9-11.

•. "Studies on the biology of the Indian freshwater eel Anguilla bengalensis (Gray)," Prec. Nat. Inst. Sci. India, 1956, 22 (5), 259-80.

.. "Determinat ion of age and growth of Mystus guile (Ham. by the use of pectoral spines, with observation on its biology and fishery in the Hooghly estuary," Ibid., 1961, 27 (4), 198-235.

.. " O n the use of pectoral spines for the determination of ago and growth of Pangasius pangaMus (Hamilton Buch)" (In press),

Beverton, R• J. H.

8. Cairns, D.

9. Cassie, R. M.

10. Ehrenbaum and Murakawa I1. Frost, W . E . ..

12. Grassi, B.

13. Harding, J. P.

14. Hornyold ,A. O. . .

15.

16.

17.

18. Nair, R. V.

19. Pantulu, V. Rayappa

21.

20.

V. Rayappa Pantuiu and Viiai Bey Singh

Proc. Ind. Acad. Sci., B, Vol. LV, PI. XXVI

?Li: -L 7 . . . . . . . . . . : . . . . .

÷ \

F I C , S . 1 - 5

Otoliths for Determination of Age & Growth of A. nebulosa

22. Parrish, B. B. and Sharman, D. P.

23. Radhakrishnan, N.

24.

25. Rao, S. R.

26. Rollefsen, G.

27.

28. Smith, S.B.

29. Syrski, S.

30. Tesch, J. J.

275

"Some remarks on methods used in herring ' racial ' investi- gation with special reference to otolilh studies," Rapp. Cons. Explot. Mer., 1958, 143, 66-80.

• ."Occurrence of glowth rings cn the ctoliths of the Indian whiting, Sillago sihama (Forskhl)," Curt. Sci., 1954, 23 (6), 196-97.

.. " A contribution to the biology of Indian sand whiting Sitlagosihama (Forskfil)," Indian J. Fish., 1957, 4(2)~ 254-83.

.. " A study of the otoli ths of Psettodes erumei (BI. & Schu)," Prec. 22nd lnd. Sci. Cong. Calcutta, 1935, 3, 319 (abstracts).

.. "The otoliths of the cod ," Rep. Norweg. Fish. Invert.., 1933, 4(3).

•. " T h e spawning zone in cod otoliths and prognosis of stock," Ibid., 1935, 4(11).

. . " T h e relation between scale diameter and body length of Kamloops trout Salmo gairdneri kamloops," J. Fish. Rea. Bd. Canada, 1955, 12(5), 1956.

. . " O n the organs of reproduction and fecundation of fishes and especially of the eels," Rept. U.S.A. Fiah. Comm., 1876.

. . " O n sex and growth investigations on the freshwater eel in Dutch waters," d. Cons. Int. Expl. Mer., 1928, 3, 52-69.

EXPLANATION OF PLATE X X V I

FIG. 1. Otolith of a glass eel showing one sea-water ring. (Dotted lines represent the trans- lucent zones.)

Fie . 2. An unground otolith of a 300mm. long eel.

F ie . 3. A ground otolith of a 420mm. long eel showing seven translucent bands.

F1t~. 4. An enlargement of a portion of the otolith sl'.own in Fig. 3.

FIG. 5. An enlargement of a portion of otolith of a 330ram. long eel showing five translucent bands•