Wheatgerm flour in diets for Manila clam,

Ruditapes philippinarum, spat

M. Albentosa a,*, A. Perez-Camacho a,1,M.J. Fernandez-Reiriz b,2, U. Labarta b,2

aInstituto Espanol de Oceanografıa, Centro Oceanografico de La Coruna, Muelle de Animas, s/n,

Apdo. 130, 15001 La Coruna, SpainbConsejo Superior de Investigaciones Cientıficas, Instituto de Investigaciones Marinas,

Eduardo Cabello, 6, 36208 Vigo, Spain

Received 10 December 2001; received in revised form 1 March 2002; accepted 1 March 2002

Abstract

Various diets for the spat of Ruditapes philippinarum were assessed, consisting wholly or

partially of wheatgerm flour—diet A1: 100% of the daily food ration (4 mg AFDW (100 mg

LW)� 1) consisted of the microalga Isochrysis galbana, clone T-ISO; diet M2: 50% of the daily

ration consisted of microalgae and the other 50% of flour; diet M3: 25% microalgae and 75% flour;

and diet G: 100% flour. Two control diets consisting solely of microalgae were also assayed: diets

A2 and A3, consisting of 50% and 25% of the daily ration, respectively. The composition of the

wheatgerm used was: 18.2% of the total organic matter consisted of protein, 71.2% of carbohydrate

and 10.6% of lipids, while the microalgae consisted of 27.8% protein, 38.3% carbohydrates and

33.8% lipids. The diets prepared with these two components are characterised by an increase in their

carbohydrate content and a decrease in their protein and lipid content as the proportion of wheatgerm

increases. The highest growth rates were obtained for the spat fed diets A1 (final dry weight,

7.12F 1.15 mg individual � 1) and M2 (6.72F 0.30 mg individual � 1),with no significant difference

being observed between the two diets. The spat fed diet M3 reached a final dry weight of 4.12F 0.49

mg individual � 1, a figure which is greater than that obtained with the control diets A3 (1.44F 0.13

mg individual � 1) and A2 (2.98F 0.76 mg individual� 1). The growth of the spat fed exclusively

wheatgerm, diet G (1.55F 0.08 mg individual � 1), was similar to that obtained with control diet A3.

The proportion of organic matter in the spat at the end of the experimental period is related to the

0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0044 -8486 (02 )00121 -7

* Corresponding author. Tel.: +34-81-205362; fax: +34-81-229077.

E-mail address: marina.albentosa@co.ieo.es (M. Albentosa).1 Tel.: + 34-81-205362; fax: + 34-81-229077.2 Tel.: + 34-86-231930; fax: + 34-86-292762.

www.elsevier.com/locate/aqua-online

Aquaculture 212 (2002) 335–345

nutritional value of the diet, so that the spat fed the diets with the greatest nutritional value (the

highest growth rates) showed higher proportions of organic matter.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Wheatgerm flours; Clams; Spat; Ruditapes philippinarum; Growth diets

1. Introduction

The cultivation period for clam spat in hatcheries, i.e. from metamorphosis until a

length of 5 mm is reached, is characterised by a high demand for food, namely microalgae

that are grown in the hatchery itself. The replacement of all, or part, of a live food with a

low-cost inert alternative would lower the production costs of hatchery-produced spat,

thereby enabling a larger amount of spat to be produced. One of the potential feeds for use

with bivalves are flours derived from cereals, which have been used in the broodstock

conditioning (Haven, 1965; Gillespie et al., 1966) or in the production of artificial diets for

adults (Castell and Trider, 1974; Langdon and Siegfried, 1984).

Our research team has spent several years studying the nutrition of certain species of

bivalves during their growth stage with the aim of finding a total or partial replacement for

live food in their diet. Given the relevance of the carbohydrates in the nutrition of bivalves,

we decided to study the possibility of using carbohydrate-rich cereal flours in diets for this

species. Among the cereal flours studied, successful replacement of 50% of the live food

by corn meal (Perez Camacho et al., 1998) and wheatgerm (Albentosa et al., 1999) has

been shown for diets of the clam Ruditapes decussatus.

In the present study, wheatgerm flour was evaluated as a partial substitute for

microalgae in the diet of the spat of another species of clam, the Manila clam Ruditapes

philippinarum, a commercially interesting species whose cultivation has increased con-

siderably in recent years. Substitution was assayed at 50% and 75% of the daily ration,

with the microalgae Isochrysis galbana, clone T-ISO, being used as the live component of

the diet. The results of the growth of the spat fed the different experimental diets are

discussed in relation to the biochemical composition of the diet and its effect on the

biochemical composition of the spat at the end of the experimental period.

2. Material and methods

2.1. Experimental conditions

The spat used came from commercial hatcheries of R. philippinarum, and was

acclimatised in our laboratories over a minimum period of 10 days, during which time

it was fed on the microalga I. galbana, clone T-ISO and kept at a temperature of 20F 2

jC. After the acclimatisation period, spat of 1.26F 0.17 mm individual � 1 in length and

0.52F 0.02 mg live weight (LW) individual � 1 (0.26F 0.00 mg dry weight (DW)

individual � 1 and 0.034F 0.001 mg organic weight (AFDW, ash-free dry weight)

individual � 1) were selected. The experimental set-up was the same as that used by

M. Albentosa et al. / Aquaculture 212 (2002) 335–345336

Albentosa et al. (1999), enabling the different diets to be fed at intervals throughout the

day (six doses per day) in order to avoid food sedimentation. The initial volume in the

culture vessels was 3 l, increasing to 4 l at the end of the feeding period (24 h). Increases in

biomass due to growth during the experimental period were compensated by increases in

the initial volume of the culture vessels and by reducing the biomass in the systems. Each

diet was assayed with three replicates, and a fourth culture without spat was added in order

to quantify sedimentation rates. The initial number of specimens in each culture was 400,

giving an initial biomass of approximately 200 mg LW. The culture period was 29 days.

2.2. Diets

Six diets were assayed (Table 1), three composed exclusively of microalgae (diets A1,

A2 and A3); two mixed diets composed of microalgae and wheatgerm flour in different

proportions (M2 and M3) and one diet consisting solely of wheatgerm (G). Diets A1, A2

and A3 consisted of varying rations of the microalga I. galbana, clone T-ISO: 4 (the

optimal daily ration for the spat of R. philippinarum according to results obtained in our

laboratory, unpublished data), 2 and 1 mg AFDW (100 mg LW)� 1, respectively. The

mixed diet M2 consisted of 2 mg of microalgae and 2 mg of wheatgerm flour, while diet

M3 included 1 mg of microalgae and 3 mg of flour. Diet G consisted of 4 mg of flour.

The method used for cultivating the microalgae used in the diet and the handling

process for the wheatgerm were the same as those described in Albentosa et al. (1999).

Samples collected during the experimental period of these microalgae and flour were

taken for biochemical analysis.

2.3. Evaluation parameters of diets

Spat measurements were performed as follows: spat biomass in each experimental vessel

was weighed weekly after drying on absorbent paper for 10 min to remove surface water.

After the secondweek, a spat sample from each experimental culture was removed in order to

Table 1

Characteristics and biochemical composition of the experimental diets used in the culturing of the spat of R.

philippinarum

Diet Components Daily ration Protein Carbohydrate Lipid

mg AFDW mg %AFDW mg %AFDW mg %AFDW

A1 100% I. galbana 4 1.11 27.8 1.53 38.3 1.35 33.8

A2 100% I. galbana 2 0.56 27.8 0.77 38.3 0.68 33.8

M2 50% I. galbana+

50% wheatgerm

4 0.92 23.0 2.19 54.8 0.89 22.2

A3 100% I. galbana 1 0.28 27.8 0.38 38.3 0.34 33.8

M3 25% I. galbana+

75% wheatgerm

4 0.82 20.6 2.52 63.0 0.66 16.4

G 100% wheatgerm 4 0.72 18.2 2.85 71.2 0.42 10.6

The rations fed to the different experimental cultures are expressed in mg AFDW per 100 mg spat live weight per

day. The biochemical composition of the diets is expressed in rations supplied of each component (mg (100 mg

LW)� 1) and as a percentage of total organic matter.

M. Albentosa et al. / Aquaculture 212 (2002) 335–345 337

reduce biomass and used to determine dry and organic weights. Dry and organic (AFDW)

weights of the spat were determined at 100 and 450 jC, respectively. At the end of the

experiment, spat length was measured using a graduated binocular microscope.

2.4. Analytical methods

Protein was determined by the method of Lowry et al. (1951), after alkaline hydrolysis

with 0.5 N NaOH for 24 h at 30 jC. Total carbohydrates were quantified as glucose by

means of the phenol–sulphuric acid method (Strickland and Parsons, 1968). Lipids were

extracted by a modification of the Bligh and Dyer (1959) method (Fernandez-Reiriz et al.,

1989) as follows: lipid material was extracted by means of chloroform–methanol (1:2);

after centrifugation, the sediment was extracted again with chloroform–methanol (2:1). To

purify the extract, both supernatants were washed with a mixture of chloroform, methanol

and water (8:4:3), as described by Folch et al. (1957). Total lipids were gravimetrically

determined through evaporation of the solvent in the purified extract on aluminium sheets

at 60–80 jC.

2.5. Statistical methods

The results were analysed using the statistical package Statgraphics. Comparison of the

different parameters of evaluation between diets was carried out by an ANOVA with a

significance level of P < 0.05. Percent composition data and efficiencies were transformed

by angular transformation (arcsin Mpercentage) prior to analyses to ensure normality. The

homogeneity of variances was checked by means of the Bartlett test. Differences between

each of the diets tested, in the case of multiple comparisons, were analysed using the

Student–Newman–Keuls. In cases where data transformations were not sufficient to

produce the homogeneity of the variances, a rank analysis of the variance was carried out

with the Kruskal–Wallis test (Snedecor and Cochran, 1971; Zar, 1974).

3. Results

3.1. Growth

At the end of the experiment, the spat that reached the highest weight were those fed

exclusively microalgae at the highest ration (diet A1, 7.12F 1.15 mg DW individual � 1)

and those fed the mixed microalgae and flour diet using equal proportions of the two feeds

(diet M2, 6.72F 0.30 mg individual� 1), with no significant differences being observed

between the two diets (P > 0.05, ANOVA, SNK test). Significantly less growth was

recorded for the other mixed diet, 25% microalgae and 75% flour (diet M3, 4.12F 0.49

mg individual � 1), but this value was nevertheless significantly higher than the value

observed for the other two diets consisting solely of microalgae in rations of 2 mg AFDW

(diet A2, 2.98F 0.76 mg individual � 1) or 1 mg AFDW (diet A3, 1.44F 0.13 mg

individual � 1). The clams fed exclusively wheatgerm grew much less than those fed the

mixed diets (diet G, 1.55F 0.08 mg individual � 1), although this growth was similar to

M. Albentosa et al. / Aquaculture 212 (2002) 335–345338

that obtained with the microalgal diet of 1 mg AFDW (diet A3). Table 2 shows the results

of growth in weight and length of the spat fed the various diets. The differences in organic

weight were the same as those found for dry weight; however, the proportion of organic

matter in the spat differed according to the diet on which it had been fed. Spat fed diets A1

or M2 showed the highest content in organic matter, almost 15% of their dry weight (Table

2), while the proportion for spat fed diet M3 was 12%. In the case of the spat fed the other

diets, the two microalgal diets A2 and A3 and the flour-only diet G, the percentage of

organic matter present in the spat decreased to 10%.

3.2. Biochemical composition

Table 1 shows the biochemical composition of the different diets. In the three diets

consisting solely of microalgae, A1, A2 and A3, protein accounted for some 28% of total

organic matter, lipid for 34% and carbohydrates for 38%. The difference among these

three diets, A1, A2 and A3, lies in the absolute amount of each of these components that

were fed to the spat: 4, 2 and 1 mg of total organic matter per 100 mg live weight of the

spat, respectively. Diet G, which consisted solely of wheatgerm, was characterised by its

high carbohydrate content, 71.2%, 8.2% protein, and 10.6% lipid. The composition of the

mixed diets M2 and M3 therefore was between these two extremes, according to the

proportion of each of the two components, microalgae and wheatgerm, used in each diet.

The main biochemical components of the spat of R. philippinarum (Table 3) are

proteins, which account for 55–66% of total organic matter, followed by lipids and

carbohydrates, 20–27% and 15–23%, respectively. Significant differences were observed

in the biochemical composition of the spat according to the diet on which they were fed.

There was a negative relationship between the protein content of the spat, expressed as a

Table 2

Corporal parameters of the spat of R. philippinarum at the start of the experiment and after 29 days of being fed

on diets consisting wholly or partially of the microalga I. galbana, clone T-ISO and wheatgerm flour: diets A1,

A2 and A3, consisting of 100%, 50% and 25%, respectively, of the daily microalgal food ration (4 mg (100 mg

LW)� 1); diet M2, consisting of 50% microalgae and 50% flour; diet M3, consisting of 25% microalgae and 75%

flour; and diet G, consisting of 100% wheatgerm flour

Diet Components (mg Live weight Dry weight Ash-free dry weight Length

(100 mg

LW)� 1)

(mg

individual� 1)

(mg

individual� 1)mg individual� 1 %

(mm

individual� 1)

Initial 0.52F 0.02 0.26F 0.01 0.034F 0.001 12.82F 0.11 1.26F 0.17

A1 4 I. galbana 13.15F 2.24a 7.12F 1.15a 1.114F 0.253a 15.52F 1.15a 4.21F 0.30a

A2 2 I. galbana 5.75F 1.58c 2.98F 0.76c 0.313F 0.100c 10.41F 0.77b,c 2.99F 0.30b

M2 2 I. galbana+

2 wheatgerm

12.29F 0.45a 6.72F 0.30a 0.951F 0.014a 14.18F 0.67a 4.04F 0.20a

A3 1 I. galbana 2.71F 0.24d 1.44F 0.13d 0.158F 0.018d 10.96F 0.59b,c 2.36F 0.05c

M3 1 I. galbana+

3 wheatgerm

7.56F 0.83b 4.12F 0.49b 0.489F 0.040b 11.95F 0.92b 3.31F 0.40b

G 4 wheatgerm 2.61F 0.13d 1.55F 0.08d 0.150F 0.009d 9.65F 0.34c 2.28F 0.05c

The results of the multiple range test carried out on the corporal parameters are indicated by the superscript

indices a – d. When two or more results have the same superscript index, this means that there is no significant

difference between them.

M. Albentosa et al. / Aquaculture 212 (2002) 335–345 339

Table 3

Biochemical composition of the spat of R. philippinarum at the start of the experiment and after 29 days of being fed on diets consisting wholly or partially of the

microalga I. galbana, clone T-ISO and wheatgerm flour: diets A1, A2 and A3, consisting of 100%, 50% and 25%, respectively, of the daily microalgal food ration; diet

M2, consisting of 50% microalgae and 50% flour; diet M3, consisting of 25% microalgae and 75% flour; and diet G, consisting of 100% wheatgerm flour

Diet Organic weight Proteins Carbohydrates Lipids

(mg individual� 1)mg individual� 1 % mg individual� 1 % mg individual� 1 %

Initial 0.034 0.022 64.7 0.005 14.5 0.007 20.8

A1 1.114F 0.253 0.609F 0.111 55.1F 2.8c 0.261F 0.083 23.1F 2.4a 0.244F 0.059 21.9F 0.7b,c

A2 0.313F 0.100 0.188F 0.070 59.4F 3.7b,c 0.045F 0.012 14.7F 1.1c 0.080F 0.019 25.9F 2.6a,b

M2 0.951F 0.014 0.523F 0.035 55.0F 4.2c 0.168F 0.008 17.6F 1.1b 0.260F 0.053 27.3F 5.2a

A3 0.158F 0.018 0.104F 0.012 66.3F 2.0a 0.022F 0.003 14.2F 0.7c 0.031F 0.006 19.5F 2.5c

M3 0.489F 0.040 0.308F 0.017 63.0F 2.9a,b 0.084F 0.011 17.1F 0.8b 0.098F 0.017 19.9F 2.1c

G 0.150F 0.009 0.087F 0.008 58.0F 1.8b,c 0.033F 0.002 21.8F 0.3a 0.030F 0.001 20.2F 2.0c

The results are expressed in mg individual� 1 of each biochemical component and in percentages of the total organic matter considered. The results of the multiple range

test carried out on the corporal parameters are indicated by the superscript indices a – c. When two or more results have the same superscript index, this means that no

significant difference exists between them.

M.Albentosa

etal./Aquacultu

re212(2002)335–345

340

percentage of total organic matter, and its growth, expressed in terms of its organic weight

at the end of the experiment (r =� 0.6229, n = 18, P < 0.05), while a positive correlation

was observed between the carbohydrate content of the spat and its growth (r = 0.4816,

n = 18, P < 0.05).

The amount of each biochemical component of the diet influenced the growth of the

spat, with a high degree of correlation being observed between the quantity of lipids

supplied by the diet and growth of the spat (r = 0.9120, n = 18, P < 0.05). The correlation

between protein content of the diet and growth of the spat was also significant, but to a

lesser extent (r = 0.8235, n = 18, P < 0.05).

4. Discussion

Our results show that the growth achieved by spat of R. philippinarum fed the

microalga I. galbana corresponding to 50% or 25% of the maximum growth daily food

ration was noticeably enhanced when the algal diet was supplemented with wheatgerm

flour. Growth of the spat of R. philippinarum fed 2 mg of microalgae day � 1 more than

doubled when 2 mg of flour were added to the diet. The same effect was observed when

diet A3, consisting of 1 mg of microalgae, was supplemented with 3 mg of flour (diet M3)

(Table 2). Furthermore, growth for the mixed diet M2, consisting of 2 mg of microalgae

and 2 mg of flour, was comparable in terms of dry weight and organic matter with that

obtained with algal diet A1 (4 mg microalgae). In other words, the use of wheatgerm flour

allows the amount of microalgae used to culture R. philippinarum to be reduced by 50%

without affecting growth.

The three microalgal rations assayed in this experiment had a noticeable influence on

the growth of the spat, to the extent that growth rates (in organic matter) on rations of 2

and 1 mg were only 30% and 15%, respectively, of that obtained on a ration of 4 mg

AFDW (100 mg LW)� 1. This ration, which produced maximum growth, is similar to that

described by other authors for spat of the same species (Laing and Millican, 1991, 1992).

The percentages of organic matter in the spat were different depending on its growth

rates, and therefore to the nutritional value of each diet, an effect previously described by

our research team for the spat of R. decussatus (Albentosa et al., 1999). As shown in Table 2,

the percentage of organic matter was significantly higher in spat fed the maximum ration of

microalgae or on mixed diet M2, while the lowest percentage corresponds to the spat fed

exclusively wheatgerm. Consequently, the differences in growth in terms of dry weight

increase when compared to those for organic weight (Fig. 1). If relative growth in terms of

dry weight and length is compared to that for the control diet (Fig. 1), we can see that in

those cultures that obtained the highest growth rates (diets M2, and to a lesser extent, M3),

relative growth is similar for both of the above parameters, while for the cultures with the

lowest growth rates (diets G and A3), relative growth in comparison to the control diet is

almost three times greater in length than in dry weight. If we take the percentage of organic

matter as an expression of shell growth, it appears that shell production is the first priority in

the destination of the nutrients absorbed. This fact has been described in the bibliography in

cases of extreme nutritional stress (Gabbott and Bayne, 1973; Riley, 1976; Lane, 1986;

Laing, 1993) or during larval development (Labarta et al., 1999).

M. Albentosa et al. / Aquaculture 212 (2002) 335–345 341

The replacement of 2 mg of microalgae by 2 mg of wheatgerm (diet M2) led to a

reduction in the protein and lipid content of the diet in comparison with the microalgae-

only diet, and a consequent increase in the carbohydrate content. This change in the

biochemical composition of the control diet had no effect on growth of the spat, indicating

that some of the protein in the live food can be replaced by larger amounts of carbohydrate

found in the wheatgerm particles. It was observed that the amount of protein and lipid

available to the spat had a significant influence on its growth, while carbohydrates were

not a determining factor for growth given that the majority of the diets assayed contained

wheatgerm, which consists mainly of carbohydrates. Minimum protein requirements have

been one of the most frequently studied aspects of bivalve nutrition, although there are no

conclusive data regarding what the minimum requirement should be. In general, it is

considered that bivalves can make efficient use of carbohydrate-rich diets provided that

their protein requirements are met (Castell and Trider, 1974; Nell and Wisely, 1983;

Enright et al., 1986; Kreeger and Langdon, 1993), while the type of lipid is more important

than their quantity (Trider and Castell, 1980; Langdon and Waldock, 1981; Enright et al.,

1986; Chu and Greaves, 1991). In addition, nutritional requirements have been found to be

species-dependent (Albentosa et al., 1993, 1996).

The results obtained from the present study regarding the possibility of replacing

microalgae with wheatgerm in the diet of the spat of R. philippinarum are generally similar

Fig. 1. Growth in terms of individual dry weight, or ash-free dry weight (AFDW) or length of R. philippinarum

spat after 29 days of being fed diets consisting wholly or partially of the microalga I. galbana, clone T-ISO and

wheatgerm flour: A2 and A3, consisting of 50% and 25%, respectively, of the daily microalgal food ration; diet

M2, consisting of 50% microalgae and 50% flour; diet M3, consisting of 25% microalgae and 75% flour; and diet

G, consisting of 100% wheatgerm flour. Growth for each of the corporal parameters measured is expressed as a

percentage of the growth obtained with the control diet (diet A1, consisting of 100% of the daily microalgal food).

M. Albentosa et al. / Aquaculture 212 (2002) 335–345342

to those obtained for the same flour (Albentosa et al., 1999) or cornflour (Perez Camacho

et al., 1998) for R. decussatus spat. Nevertheless, we observed that growth of the spat fed

the 75% replacement diet, in relation to the control diet, and expressed in terms of organic

matter, was greater in R. decussatus than in R. philippinarum. In addition, the spat of R.

decussatus has a higher carbohydrate content and a lower lipid content than the spat of R.

philippinarum. Both these results could point to a difference in the nutritional require-

ments of the two species. On the one hand, the low protein content of the diet consisting of

75% wheatgerm (20% in both studies) may be insufficient to sustain growth of flesh and

shell in the spat of R. philippinarum. This would lead to the protein in the diet being

diverted to build the protein matrix of the shell, and there would therefore be a dietary

protein deficiency for the synthesis of body structures, a phenomenon that was not seen in

R. decussatus. On the other hand, the low lipid content in this replacement diet

(approximately 15%) may also be a limiting factor for flesh growth in R. philippinarum,

a species with a higher lipid content in its fleshy parts, but not for R. decussatus. All this

notwithstanding, the use of wheatgerm as a feed for the spat of R. philippinarum allows, as

was the case for R. decussatus, 50% of the maximum daily microalgal ration to be replaced

without affecting growth.

In recent years, there have been numerous attempts to find an alternative feed that could

act as a total or partial replacement for live food in the culturing of bivalve molluscs.

Among these, some of the most important studies have been those using dried cultivated

microalgae (Albentosa et al., 1997; Laing et al., 1990; Laing and Gil Verdugo, 1991; Laing

and Millican, 1991, 1992) or modified yeasts (Albentosa et al., 1989; Coutteau et al.,

1991, 1994; Coutteau, 1992). The use of a low-cost alternative feed such as the one used in

the present study: US$5 (kg DW)� 1, which is considerably less than the cost of

microalgae cultivated in the hatcheries (US$50–400 (kg DW) � 1) according to Coutteau

and Sorgeloos (1992) or that of heterotrophic microalgae (US$160 (kg DW) � 1 according

to Laing and Millican (1992)) would allow an increase in spat production in a hatchery

with no extra cost, or would cut production costs for existing volumes of production by at

least 50%. Taking spat production per cost unit in a hatchery to be 8.1 g when live food is

used, then this figure could rise to 10.7 g if 50% of the live food were to be replaced by

wheatgerm, or to 10.4 g if 75% of the live food were to be replaced.

Acknowledgements

We are grateful to C. Fernandez Pena for technical assistance in the culturing of

microalgae and spat, and to L. Nieto and B. Gonzalez for their help with the biochemical

analyses. This study was financed by CICYT-IEO-CSIC, project MAR99-0240-CO2.

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