Observations on ingestion and digestion of unicellular algae by Strombus gigas larvae (Mollusca, Gastropoda) using epifluorescence microscopy

Aquaculture. 92 ( 1991) 359-366 Elsevier Science Publishers B.V.. Amsterdam

359

Observdtions on ingestion and digestion of unicellular algae by Strombus gigas larvae (Mollusca, Gastropoda) using epifluorescence microscopy Dalila Aldana Aranda”, Albert Lucasb, Thierry Brule”, Maria Andradea, Eduardo Garcia”, Nathalie Maginot” and Marcel Le Pennecb “CinvestavIPN., A.P. 73 Cordemex, C.P. 97310Merida, Yucatan, Mexico hLaboratoire de Biologie Marine, Faculte des Sciences et Techniques, 29287 Brest Cede-x.France (Accepted 26 June I 990 )

ABSTRACT Aldana Aranda, D., Lucas, A., Bruit, T., Andrade, M., Garcia, E., Maginot, N. and Le Pennec, M., 199 I. Observations on ingestion and digestion of unicellular algae by Strombus gigas larvae (Mollusca, Gastropoda) using epifluorescence microscopy. Aquaculture, 92: 359-366. Epifluorescence microscopy was used to observe the ingestion and digestion of unicellular algae in gastropod larvae. Larvae started feeding 8 h after hatching. Three kinds of diet were tested during the period of this study: Isochrysis aff. galbana, Tetraselmis chuii and Isochrysis-Tetraselmis mixture. All the experiments were conducted at 29°C. It was shown that ingestion was faster with Tetraselmis than with Isochrysis. Within 10 min 43Ohof the larvae had ingested Tetrasefmis and no larvae had ingested any Isochrysis. Within 30 min, 60°h of the larvae had ingested Tetraselmis while only 25OYa had ingested Isochrysis. Using a scale based on the quality of fluorescence, it was possible to determine the time interval to digest a diet, which is shorter with Tetraselmis than with Zsochrysis.After 8 h the digestion index reached 45% for larvae fed with Tetraselmis while it was only 28% for larvae fed with Isochrysis. Using a quantitative scale based on measurements of the fluorescent surface in each larva, it was demonstrated that the amount of diet ingested is higher with Tetraselmis than with Isochrysis. These results indicate that Tetraselmis chuii forms an adequate diet for young veligers of Strombus gigas.

INTRODUCTION

Larval nutrition in gastropods, unlike bivalves, has been little studied. Only the following references seem to be available in the literature for Strombidae: Siddall ( 198 1) , Ballantine and Appeldoorn ( 198 3 ) , Davis and Hesse ( 198 3 ) , Pillsbury ( 1985)) and Aldana Aranda et al. ( 1989). *To whom correspondence

0044-8486/91/$03.50

should be addressed.

0 199 1 -

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B.V.

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D. ALDANA ARANDA ET AL.

Various methods have been used to evaluate the ingestion and digestion rates of diets in bivalve larvae. Clearance rate of food particles in the medium is one of the indirect methods (Walne, 1959; Le Roux, 1975; Malouf and Breese, 1977; Riisgard et al., 1980). Another indirect method is the enzymatic technique proposed by Samain and Boucher ( 1974). Among the direct methods of measuring diet ingestion, two have been widely used for larval feeding. The first utilises radioisotope labelling of algae (Walne, 1965; Ukeles and Sweeney, 1969) and the second technique is direct observation of the inside of the digestive tract where natural fluorescence of chlorophyll can be detected through epifluorescence microscopy (Babinchak and Ukeles, 1979; Lucas and Rangel, 198 1,1983; Le Pennec and Range1 Davalos, 1985 ). In this work, techniques used on bivalve larvae were applied to Strombus larvae. Due to its economic importance, the cultivation of Strombus gigus in the Caribbean Sea has received considerable attention. In this context, the purpose of the present work is to analyse larval nutrition of this species, which may be helpful for improving hatchery techniques. MATERIALS

AND METHODS

The fertilised eggs used in this study were collected, just when a female laid its eggs, by scuba diving at 10 m depth. The sample was taken in June 1989 at Banco Chinchorro in the Yucatan Peninsula, Mexico ( 19 ON and 87 oW) . The eggs were transported to the laboratory where epibionts and sand particles were cleaned off. The eggs were placed over a loo-pm mesh and kept immersed in a tank of sea water which was filtered using a 0.2~pm filter. During the experiments, the temperature was maintained at 29°C and the sea water was aerated. After hatching, veliger larvae were collected using a 45-pm mesh. Larvae were counted using live samples taken with a 0.1 -ml Eppendorf pipette from a well mixed suspension of larvae. The 8-h-old veligers were distributed into I-1 beakers at a density of 500 larvae/l; 20 beakers were prepared and during the course of the experiment 6000 larvae were observed. Algae were grown in F/2 medium (Guillard and Ryther, 1962) and cultured in 2-l Erlenmeyers containing 1 1 of culture medium. The mean algal density was determined from three samples, using a Neubauer cell. Algae in their exponential phase of growth were used as food. The selected species were Isochrysis aff. galbana (Tahiti) and Tetraselmis chuii. As the observations of digestion time needed a discontinuous feeding regime, fed larvae were transferred into a beaker without food after 2.5 h. In the beaker with food, algae were initially at a concentration ( 1.37 x 1O4cells/ ml) allowing normal ingestion for 2.5 h of feeding for each algal species. Soon after the beginning of the experiment, samples of 100 larvae were taken at the

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following times: 10 min, 30 min and 1, 2, 3, 4, 5, 6, 7 and 8 h. Each experiment was duplicated and results from the two series were averaged. The larvae were fixed in sea water with 1% formalin for microscopic observation. The microscopic observations were made either immediately or within a few days by storing samples in the cool to avoid spoilage. The microscope used (Baush and Lomb type Balplan) was equipped with an HBO mercury lamp of 100 W, an FITC exciter filter and an OG530 barrier filter. The four stages of nutrition defined by Babinchak and Ukeles ( 1979) and by Lucas and Range1 ( 198 1) were used in this study. The characteristics of these stages are: Stage 1: Whole algae visible in the stomach, fluorescing red. Stage 2: Lysed algae appearing as discrete bright red fluorescing particles without cellular structure. Some whole algae may be visible. Stage 3: No red particles in cells. Stomach fluoresces uniformly pale red, pink or orange, indicating nearly complete digestion of the algae. Stage 4: No fluorescent signs of algal feeding or digestion. The interpretation is that these larvae have never fed or that digestion has already taken place. To obtain quantitative estimates of the algae ingested, a scale proportional to the fluorescent surface, expressed in pm*, was established. The fluorescent surface was calculated with the help of a micro-ocular graticule of 350 pm per side, divided into 25 squares of 70 ,um per side. Each square was in turn divided into 25 units of 14 pm per side. This quantitative scale is given in Table 1. Two indices were utilised to compare the feeding behaviour of larvae under different experimental conditions. The indices defined by Salaiin ( 1987 ) were adapted for the present study as follows: Ingestion index, II = number of larvae at stage 1/number of larvae observed Digestion index, DI=number of larvae at stages 2+3/number of larvae observed. TABLE I Quantitative scale established to measure feeding using a graticule with 70 pm x 70 pm squares. The + indicates that one more elementary unit of 14 pm per side is fluoresent Stages

A

B c D

Area of fluorescence Number of squares

Area tfim’ )

o-o.75 (0.75)+-1.50 ( 1.50) +-2.25 (2.25)+-3.00

o- 3675 3871- 7350 7546-11025 11221-14700

Proportion of stomach tilling (O/o) o- 25 26- 50 51 -75 76-100

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RESULTS

Change of nutrition stages with time The results obtained for the three diets show how the four stages of nutrition (expressed in percentages) vary with time (Fig. 1). The larvae fed with Tetraselmis and Tetraselmis-Isochrysis mixture showed an ingestion rate of 30% to 40% during the first 10 min. During the same period, no algal ingestion of Isochrysis occurred. Within 30 min, 60% of the larvae had ingested Tetraselmis while only 25% had ingested Isochrysis. With the three diets used, less than 10% of the larvae ingested no food at all during the experiment.

b

Fig. 1. Variations in the ratio of the four nutritional stages defined for queen conch veliger larvae, when reared at 29°C and fed with Isochrysis aff. galbana (a), Tetraselmis chuii (b) and Isochrysis-Tetrasehnis mixture (c). n= 200 individuals per sample.

INGESTION AND DIGESTION OF UNICELLULAR

ALGAE BY STROMBL’S

GIGAS

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Nutrition indices Fig. 2 shows changes in the ingestion index for larvae fed with Tetraselmis and with Isochrysis. Ingestion was more rapid when the larvae were fed with Tetraselmis than with Isochrysis. In the same manner, digestion was also faster for Tetraselmis than for Isochrysis (Fig. 3 ).

Fig. 2. Change of the ingestion index (II) with time of queen conch veliger larvae reared at 29°C and fed with Isochrysis aff. galbana (*) or Tetraselmis chuii ( l ). 200 individuals observed for each value.

Fig. 3. Change of the digestion index (DI) with time of queen conch veliger larvae reared at 29°C and fed with Zsochrysis aff. galbana (*) or Tetraselmis chuii ( l). 200 individuals observed for each value.

D. ALDANA ARANDA ET AL.

364 TABLE 2

Quantitative observations of feeding in Strombus gigus larvae fed three diets at 29°C. Stages A-D are explained in Table I. The data express the percentage of larvae belonging to different stages at different times (200 observations for each time) Food

Time (h) 0 0.17 0.5 1 2

Tetraselmis

1 Isochr.vsis

Tetraselmis+

Isochrysis

Stages A

B

C

D

100 57 40 8 0

43 47 12 8

13 14 18

66 78

0

0

II

89

10 34 30

58

0 0.17 0.5 1 2 3

100 79 50 33 0

21 40 33 12

0 0.17 0.5

100 70 42

30 51

7

35 0 0

33 15 0

11 20 16

1 2 3

100

15 65 84

Quantity offood ingested A greater quantity of food was ingested when the diet was Tetraselmis compared to Isochrysis (Table 2 ). For example, in the third hour, feeding stage D was reached by 89% of larvae fed with Tetraselmis, but only by 58% of the larvae fed with Isochrysis. DISCUSSION

The different nutritional index stages described by Babinchak and Ukeles ( 1979 ) and Lucas and Range1 ( 198 1) are based on the close relationship that exists between the type of fluorescence and the stage of digestion. The value of this index has been confirmed by histological observations on the structure and ultrastructure of algae that have been ingested and digested (Range1 Davalos, 1983). To date, these observations have been made only on bivalve larvae. Veliger larvae of queen conch are able to feed upon the algae 8 h after hatching. This rapid feeding activity has already been observed in bivalve larvae

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(Lucas and Rangel, 1983; Le Pennec and Range1 Davalos, 1985; Salaiin, 1987). Epifluorescent microscopy does not differentiate the chloroplast of Tetraselmis and Zsochrysis inside the stomach of the larvae; consequently it was impossible, in this study, to know if there had been a selection of Tetraselmis in the algal mixture or not. Measurement of the area of fluorescence has been adopted in this study. This method represents a direct quantitative measurement of food ingested, in so far as this food is concentrated in a retracted stomach similar to a sphere. From these data, an area or surface measurement can be converted to a packed cell volume of algae corresponding to a proportion of filling in the stomach (Table 1). To date, many authors have used Tetraselmis chuii or Tetraselmis suecica to feed Strombus gigas or Strombus costatus (Siddall, 198 1; Ballantine and Appeldoom, 1983; Davis and Hesse, 1983; Davis et al., 1985; Pillsbury, 1985; Aldana Aranda and Torrentera, 1987; Aldana Aranda et al., 1989 ) . The present study confirms that this choice of diet is really a good one as, with Tetraselmis, the larvae ingest and digest more algae at a faster rate compared to Zsochrysis. ACKNOWLEDGEMENTS

This study was supported by grants No. PCCNCNA-05 1406 of CONACYT and CONACYT-CNRS 1989 (Science and Technology Council of Mexico and France), the Science and Technology Council of the French Embassy (CST) and the Foreign Office of France (DCST). We acknowledge SEPESCA of Mexico (Delegation Yucatan, Quintana Roo and Campeche ), SEDUE, Government of Yucatan, and Fishermen of “Banco Chinchorro” for their help. The authors are thankful to Drs. M.S. Shafee and A.R. Beaumont who helped them to translate the French version into English.

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