Aquaculture, 30 (1983) 369-374 369 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Short Communication

DETECTION OF THE FIRST LARVAL FEEDING IN CRASSOSTREA

GZGAS, USING THE EPIFLUORESCENCE MICROSCOPE

ALBERT LUCAS and CARLOS RANGEL

Laboratoire de Zoologie, Facultk des Sciences et Techniques, 29283 Best Cedex (France)

(Accepted 15 December 1981)

ABSTRACT

Lucas, A. and Rangel, C., 1983. Detection of the first larval feeding in Crassostrea gigas, using the epifluorescence microscope. Aquaculture, 30: 369-374.

The first larval feeding in Crassostrea gigas, detected by epifluorescence microscope, occurred 24 h after fertilization at 24°C and 30 h at 21°C. The amount of algal cells (Pauloua lutheri and Isochrysis galbano) necessary to have larvae with filled stomachs was estimated to be 800 cells per day at 24°C and 400 cells per day at 21’C.

INTRODUCTION

The first larval feeding of Crassostrea gigus (Thunberg), a common hatchery species, is studied in the present work. In those hatcheries where the tem- perature varies between 20°C and 2’7”C, algal food is given as soon as the D- larval stage appears (i.e. approximately 24 h following fertilization). How- ever, it is not known whether these unicellular algae are immediately in- gested. In other cases, cited by Ukeles (1975), unicellular algae are furnished immediately upon filtration.

In order to study the larval response to added food, the technique of epi- fluorescence microscopy was used (Babinchak and Ukeles, 1979), thus allowing the detection of unicellular algae in the stomachs of the larvae. A preliminary study showed that, at 25”C, over 80% of 24-h C. gigas larvae took up algae. It was decided to perform a more complete study in order to determine precisely the time interval before the larvae are capable of in- gesting phytoplankton.

MATERIALS AND METHODS

The broodstock were oysters cultivated in the Bay of Brest and condi- tioned at the Tinduff Aquaculture Farm (Bay of Brest). A single male was employed to fertilize three females, whose eggs were subsequently treated separately.

0044-8486/83/0000-00001$03.00 o 1983 Elsevier Scientific Publishing Company

370

The fertilized eggs of each female were divided into two equal batches, in order to create two series differing only in their water temperature: 21°C (A) and 24°C (B). The experiments were performed without light and aera- tion. The six experimental vessels contained 35 1 of filtered seawater with added equal parts of PauZoua (Monochrysis) lutheri (Droop) and Isochrysis galbana Parke (3.54 pm diameter). Each vessel received 291-10’ algal cells, but contained a different number of larvae, depending on the brood female of origin (Table I).

TABLE I

Characteristics of the three vessels of each series at the beginning of the experiments. In each vessel the algal concentration was 83,285 cells ml-l.

Female

1 2 3

No. 1 O5 larvae 39 93 75

No. lsrvae per ml 111 266 214

No. algal cells per larva 750 314 389

Samples were collected 18-42 h after fertilization. During this period, no algae were added and no filtration was performed.

Each sample of water and larvae was vacuum filtered on a Nuclepore poly- carbonate 12 pm filter, which was then placed on a microscope slide without cover slip, and observed. When the examination was immediate, the larvae were observed to be alive but incapable of locomotion; quite often, the in- gested algae displayed a rapid rotating movement in the larval stomach.

Observations were performed using a Balplan type Bausch and Lomb microscope, equipped with a 50 W HOB mercury lamp, and FITC exciter filter and an 0G530 barrier filter.

The following scale was adopted to characterize the fullness of the larval stomach: Stage 0: stomach empty, no fluorescence. Stage 1: l-9 algal cells present, when countable; otherwise the equivalent in

fluorescence surface. Stage 2: lo-20 algal cells present, when countable; otherwise the equivalent

in fluorescence surface (stomach half filled). Stage 3: more than 20 algal cells present or equivalent fluorescence surface

(stomach entirely or almost entirely filled).

RESULTS AND DISCUSSION

In the reference larval populations, which were studied between 11 and

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TABLE II

Percentage of normal D larvae in the various samplings. The remainder represents either non-developed eggs, embryos, trochophore larvae (especially in the early samplings) and dead or abnormal D larvae (relatively infrequent). Number of larvae observed per sampling: 100.

Sampling Series A (21°C) Series B (24°C)

(h) 1 2 3 Average 1 2 3 Average

18 15 18 18 17.0 92 88 90 90.0 21 63 71 66 66.7 96 94 98 96.0 24 89 95 90 91.3 98 91 98 96.7 27 85 91 88 90.0 96 98 97 97.0 30 97 96 97 96.7 100 92 94 95.3 42 99 97 100 98.6 99 95 96 96.6

TABLE III

Percentage of larvae at various stages of ingestion in the 21°C cultures. Number of larvae observed per sampling : 100.

Sampling Female Stage 0 Stage 1 Stage 2 Stage 3 (h)

3

18 2 3

Average

1

21 2 3

Average

1

24 2 3

Average

100 99

100 99.7

0 1 0 0.3

0 0

0 0 0 0 0 0

27

1 94 4 2 0 2 95 2 3 0 3 96 4 0 0

Average 95.0 3.3 1.7 0

1 54 28 87 10 78 13 73.0 17.0

16 2 3 0 8 1 9.0 1.0

13 77 4 72

15 69

30 2 3

Average

1 8 2 42 2 14 10

3 11 5

100 100 100 100

100 100 100 100

0

0 0% 0

0 0

0 0 0 0

0 0

0 0 0 0 0 0

0 0

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TABLE IV

Percentage of larvae at various stages of ingestion in the 24°C cultures. Number of larvae observed per sampling : 100.

Sampling (h)

Female Stage 0 Stage 1 Stage 2 Stage 3

1

18 2 3

Average

100 100 100 100

0

0

0

0

0

0

0

0

1

21 2 3

98 2 0 0 100 0 0 0 100 0 0 0

99.3 0.7 0 0 Average

1

24 2 3

Average

70 18 12 89 6 5 96 4 0 85.0 9.3 5.7

1

27 2 3

Average

17 14 18 51 55 13 17 15 42 18 19 21 38.0 15.0 18.0 29.0

1 26 10 23 41 2 33 27 27 13 3 30 19 33 18

Average 29.7 18.7 27.6 24.0

30

1 2 0 0 98

42 2 44 4 5 47 3 20 7 64 9

13 July 1981, the number of eggs developed in the six experimental batches varied from 94 to 97%. When the D larval stage was attained (18 h at 24°C and 24 h at 21°C) the percent of normal and active larvae was always at least 90% (Table II). The mean shell length varied from 70 pm (at 18 h) to 80 E.trn (at 42 h).

The results of the larval ingestion observed are shown in Tables III (series A, at 21°C) and IV (series B, at 24°C). The results are quite similar for the three populations, except for the 42 h sampling (in this case the means are not indicated).

At 42 h, a clear difference is observed in the larvae from female 1 and

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those from females 2 and 3 in series B (24°C). In fact, the larvae from female 1, at 24”C, contain twice as much food as the others: almost all of them have full stomachs (stage 3), whereas most of those from females 2 and 3 are incompletely filled and sometimes empty (44 and 20% empty stomachs, respectively).

Previous experiments showed that the presence of empty larvae corre- sponds to a lack of food. In this case, the 314 and 389 algal cells per larva is insufficient 42 h following fertilization (i.e. 24 h after beginning food up- take) ; a supplementary amount of food would have been necessary. How- ever, an initial ration of 750 algal cells appears to have been sufficient. This phenomenon was not observed at 21°C.

In conclusion, the D-stage was attained by all the individuals in this larval population of Crassostrea gigus after 18 h at 24°C and after 24 h at 21°C (Table II). An appreciable number of these larvae begin taking up food at 24 h at 24°C and 30 h at 21°C. This represents a delay of 6 h at 24°C and 8-9 h at 21°C.

These results should allow the shellfish farmer better to estimate the moment of first feeding. Thus, the first feeding at 24°C should take place approximately 24 h following fertilization. At 21”C, however, it may take place 30-36 h following fertilization. Depending on the hatchery timetable, the first feeding should thus be given either one or two days following fertili- zation at culture temperatures of approximately 2O”C, and one day follow- ing fertilization at culture temperature of about 25°C. In any case it is use- less to add phytoplankton to fertilized eggs, as this increases the chances of bacterial contamination of the medium.

The first feeding may consist of at least 400 cells per larva at 21°C when the second addition is performed 24 h later; at 24”C, twice this amount is re- quired .

The present study illustrates the influence of temperature on the feeding activity of larvae, already known to shellfish farmers and demonstrated directly by Lucas and Range1 (1981) or indirectly, using growth rate, by many authors, e.g. Bayne (1965).

ACKNOWLEDGEMENTS

The authors are grateful to Dr. R. Ukeles of Milford (U.S.A.) who fur- nished the algal stocks used, to M.A. Gerard of the Tinduff Shellfish Farm, who supplied to broodstock, and to M.P. Beninger who translated the ori- ginal French manuscript.

REFERENCES

Babinchak, J. and Ukeles, R., 1979. Epifluorescence microscopy, a technique for the study of feeding in Crassostrea uirginica veliger larvae. Mar. Biol., 51: 69-76.

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Bayne, B.L., 1966. Growth and the delay of metamorphosis of the larvae of Myths edulis (L.). OpheIia, 2 (1): l-47.

Lucas, A. and Rangel, C., 1981. Vitesses d’ingestion et de digestion du phytoplancton observges au microscope a Bphluorescence chez les larves de My tilus edulis (L.) (Bivalvia, Mohusca). Haliotis, 11: 171-180.

Ukeles, R., 1975. Views on bivalve larvae nutrition. Proc. 1st Int. Conf. Aquac. Nutr., 127-162.