The biochemical transformation of food by the mussel Mytilus galloprovincialis Lamarck at different temperatures

Camp. Biochem. Physiol.

Vol. 112A.Nos. 314, pp. 441-453, 1995 Copyright 0 1995Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95$9.50+ .OO

Pergamon 0300.9629(95)02012-9

The biochemical transformation mussel Mytilus galloprovincialis different temperatures

of food by the Lamarck at

A. V. Frolov and S. L. Pankov Mariculture Laboratory, Russian Federal Research Institute of Fisheries and Oceanography (VNIRO), Moscow, Russia The influence of temperature on the biochemical composition of the faeces of MytiZus has been examined in the range of temperatures from 10” to 25°C. Percentage content of ash and carbohydrates decreased with an increase in the temperature from 40.2% f 2.8% to 19.6% + 1.1% and from 25.9% f 2.3% to 19.9% + 1.9%, respectively. The content of lipids and proteins increased with an increase in the temperature from 5.8% f 0.4% to 14.9% + 1.9% and from 23.4% f 4.3% to 46.7% f 3.3%) respectively. Among neutral lipid classes, the most essential alterations were connected with triacylglycerols, sterol and wax esters (percentage content of these compounds increased from 0.2% & 0.1% to 7.2% SE 2.0% and from 3.4% f 0.5% to 9.0% f 1.2%, respectively) and with hydrocarbons (their content decreased from 63.5% f 6.9% to 32.2% -t 3.7% with an increase in the temperature). An extremely significant influence of temperature was shown on the percentage content of total phospholipids and polar lipids (their content increased from 0.2% f 0.5% to 4.2% + 0.2% and from 7.1% + 0.9% to 33.6% f 3.6%). Amino acid composition of proteins that presented in the faeces was rather constant, and only an insignificant increase in the correlation of essential/ nonessential acids took place at low temperatures. The fatty acid compositions of investigated lipid classes were different and altered in different ways with an increase in temperature, but the increase in the ratio of w3/w6 acids with increase in the temperature was the characteristic feature of all lipid classes. The most abrupt alterations in the biochemical composition took place in the interval of temperatures from 20” to 15°C. These alterations are connected with high energy substances and point to the process of cold adaptation of M. galfoprovincialis as connected with these compounds and in this interval of temperatures.

galloprovincialis

Key words: Mussel; Faeces; Amino acids; Carbohydrates. Comp. Biochem.

Temperature;

Fatty acids; Lipid class composition;

Proteins;

Physiol. 112A, 441-453,1995.

Introduction There are different approaches to the estimation of the food requirements of molluscs and Correspondence

ro: A. V. Frolov, Mariculture Laboratory. Russian Federal Research Institute of Fisheries and Oceanography (VNIRO). 17 A. V. Krasnoselskaya. Moscow 107140 Russia. Tel: 7-[095]-264-90-54; Fax: 7-[095]-264-91-87. Received 1 March 1994; revised 9 May 1995; accepted 12 May 1995.

their physiological condition. We may investigate the selective consumption and food preference to observe such indexes as the growth rate, mortality, fecundity and biochemical composition of tissues. We can also estimate the degree and specificity of these requirements, judging by the biochemical composition of their fecal pellets (Odum, 1953). This latter approach is not elaborated in the physi-

441

442

A. V. Frolov and S. L. Pankov

ology of molluscs. However, sometimes just with the aid of this method, we can get rather

fast, indispensable information about nutrition. We should make the reservation that this is only really applicable to molluscs that form rather well-shaped fecal pellets, as in the case of Mytilus galloprovincialis, and is inapplicable when fecals are liquid as with oysters (Ostrea edulis). The main shortcoming

of this method is in the fact that we cannot estimate the dissolved remains and liquid products of metabolism. It is well known that the flow of fecal material is an extremely important vector of organic metabolism of the oceans. Changes in the chemical transformation of fecals occurring during the time of sedimentation influence nearly all chemical indexes of the water column and define the type of chemical constituents of the bottom sediments. All of these aspects have been rather intensively studied (Gagosian et al., 1983; Angel, 1984; Fowlerand Knauer, 1986; Roy and Poulet, 1990). It has been shown that the deposition (its chemical and qualitative indexes) of organic matter alters considerably with the season and with the geographical situation (Prahl et al., 1980; Wakeham et al., 1980; Gagosian et al., 1983). The direction and degree of possible changes in the chemical composition of fecals depend on both the abiotic (temperature regime and pressure) and biotic factors (type of microbiological community and availability of consumers) and the initial chemical composition of the fecal pellets. This initial composition is a result of the physiological adaptation that occurs in response to these factors. Temperature is one of the most important factors influencing physiological adaptation, and we would like to focus our attention on this factor (Hochachka and Somero, 1973). In our work we studied the influence of temperature on the chemical composition of fecal pellets of M. galloprovincialis. We would like to answer, as clearly as possible, such questions as: To what degree it is possible to determine food requirements of mussels judging by the chemical composition of faeces, and what is the effect of temperature, without the influence of physiological conditions and biotic and abiotic factors.

Materials and Methods Mollusks (the middle size is 60 mm, 12 individuals) were collected on a natural oyster bank in May from a depth of 4 to 6 m at 10 to 11°C and placed in one plastic aquarium with aeration (vol. = 100 L). The temperature

was raised 5°C every 15 days. It was 10°C for the first 15 days, 15°C during the second 15 days, 20°C the third, and 25°C during the fourth 15-day period (before, samples of mollusts were adapted to the temperature for 10 days). Food was added into the aquariums in portions every 4 hours. The total algae biomass was about 0.5 g dry weight per day per aquarium. We used a mix of microalgae Phaeodactylum tricornutum (Bacillariophyceae), Platimonas viridis (Prasinophyceae), Dunaliella tertiolecta (Chlorophyceae), Nefiochloris salina (Xanthophyceae), 2 : 2 : 1: 1 weight proportions. All of the species were grown under standard conditions in 10-L glass vessels on Walne media (stationary growth phase, t = lS”C, illumination lo-13 Klux). Faeces were collected daily, centrifuged and placed in a vial with a methanolchloroform mixture (1: 2). We controlled the absence of pseudofaeces by microscope. Samples with lipids were fixed (without air) by chloroform (10 mg/ml) and stored at -33°C (2.5 months). Samples for protein amino acid analysis were fixed by ethanol (1 dry weight part of the sample to 20 parts ethanol). All of the methods used were applied for determination of biochemical compounds of algeae and faeces. Identijkation position

of the general biochemical

com-

Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Lipid was determined by weight method (extracting 3 times according to Bligh and Dyer, 1959). Carbohydrate was determined by the phenol-sulfuric acid method with glucose as a standard (Josefsson et al., 1972). Extraction

and fraction

of lipids

Lipids were extracted according to Bligh and Dyer (1959) and then separated into neutral and polar lipids by thin-layer chromatography on 20- x 20-cm plates coated with Kiselgel60 (Merck) in hexane-ether (9: 1, vol/ vol). Polar and neutral lipids were eluded with a chloroform-methanol mixture (1: 1, v/v) and chloroform, respectively. Neutral lipids were separated with the following mixture : hexane : ether: acetic acid (85 : 15 : 1, v/v/v) on lo- x 20-cm plates with Kiselgel60 (Merck). We used CAMAG Linomat III for plotting spots 3 mm long on the plates. Polar lipids were separated on 6- x 6-cm plates coated with KCK silica gel (3-5 mi-

Transformation

of

crons) with gypsum (10% from silica gel weight). The technique was described by Svetashev and Vaskovsky (1972). First direction for algae-chloroform : acetone : methanol : formic acid : HZ0 (100 : 40 : 20 : 20 : 8, v/v/ v/v/v), the second direction-acetone : benzene: formic acid : H,O (200: 30 : 3 : 10, v/v/v/ v) (Belenkii er al., 1984). Zdentzjication of polar lipids

Nonspecific detection of polar lipids was achieved by 10 N sulfuric acid in methanol (t = 180°C for 5 min). We scraped the silica gel zone containing the phospholipids into Pyrex test tubes. We carried out the determination of total phosphorus according to Vaskovsky et al. (1975) with sodium molybdate (Na,MoO, 2H,O) with the aid of a Shimadzu spectrophotometer (model UV 260) in a cuvette 10 x 10 mm, A = 820 nM. We used a cuvette 0.5 x 0.3 mm, A = 820 nM, and a spectrophotometer (Hitachi 220) for microprocedures. Glycolipids, cerebrosides and sulfolipids were determined after spraying in two stages: first, by 0.2% solution of antron reactive (CISH,,O,) in benzene, and then, by 5% sulfuric acid in H20, and heating in an oven (t = lOO”C, time = lo-15 min). Sulfolipids were violet, glycolipids were dark blue and phospholipids, light brown. Sulfolipids, cerebrosides and glycolipids were determined by the weight method (microbalance: CAHN C-31). Previously these compounds were developed in iodine streams and extracted by methanol: chloroform (1: 1, v/v). Total extract had been evaporated and then placed on the thin aluminum plates. After this plates were dried to the constant weight. Identification

of neutral lipids

Neutral lipids were detected by spraying with 5% ethanolic phosphomolybdic acid (Kritchevsky and Kirk, 1952) and heating the plates in an oven to 100” to 120°C for 5-10 min. The quantitative analysis of neutral lipid composition was made with the aid of a Shimadzu CS-9000 densitometer in transmission measurements (A = 640 nM). As a standard, we used neutral standards of Iatroscan firm (cholesterol palmitate, cholesterol lmonopalmitoilglycerol, 1,2-dipalmitoilglycerol, tripalmitoilglycerol). Adjustment of the lipid class composition was made by means of the weight method. Fatty acid analysis

Methyl esters of fatty acids were obtained in two stages: in the first stage, lipids were hydrolyzed with 12% KOH in aqueous metha-

food by the mussel

443

no1 (at 80°C for 2 h), then after acidification of the soap, the acids were extracted with hexane 3 times; the second stage consisted of esterification with 2.5% HCL in methanol (at 80” for 2 h). The fatty acid composition was determined with the aid of a Shimadzu GC-16 gasliquid chromatograph equipped with a flame ionization detector (T = 300°C) and a fused silica column 50 m long with i.d. = 0.32 mm, packed with free fatty acid phase (FFAP). The temperature of the column was programmed from 200” to 240°C (I”/min). Fatty acid identification was confirmed by gas chromatography-mass spectrometry using a Finigan-MAT instrument. Amino acid analysis

To estimate the amino acid composition of protein, we hydrolyzed the samples in 6 N HCL at 110°C for 24 h. Amino acids were analyzed by means of a Hitachi amino acid analyzer (model 835). We used four citrate buffers (pH range 3.2 to 4.9). Column 2.6 x 150, analysis cycle time 50 min, buffer flow rate 0.225 ml/min, column temperature 53°C ninhydrin flow rate 0.3 ml/min. All results are represented as I 2=X~t, i.e., ta {S,}

J

I: (f-xi)2 n(n - 1)

standard deviation

where A = 0.05, t = the value of t criterion, n = 5 for protein, lipid, and carbohydrate, n = 3 for neutral lipid class composition, ash and phospholipid class composition, n = 2 for amino acid composition.

Results Znjkence of temperature on the general biochemical composition, neutral and polar lipid class composition of faeces

Percentage content of ash and carbohydrate decrease with increase in the temperature from 40.2% ? 2.8% to 19.6% ? 1.1% and from 25.9% + 2.3% to 19.9% t 1.9%, respectively, whereas the lipid and protein contents increase from 5.8% ? 0.4% to 14.9% + 1.9% and from 23.4% rt 4.3% to 46.7% r 3.3’0, respectively. The most abrupt alterations take place in the interval from 15” to 20°C (Table 1) Percentage contents of mono- and diacylglycerols, free fatty acids and hydrocarbons decrease with an increase in the temperature from 3.2% ~fr 0.8% to 1.4% + 1.3%, from 5.2% 2 0.4% to 3.5% 2 0.4%, from 4.7% + 0.2%to 1.4% + 0.3%andfrom63.5% & 6.9% to 32.2% t 3.7%, respectively. Percentage

A. V. Frolov and S. L. Pankov

444

Table 1. General (dry weight %) biochemical composition (n = 5). neutral (weight %, total lipids) lipid class composition (n = 3) and polar (weight %, total polar lipids) lipid class composition (n = 3, ~1= 0.05) of faeces of M. galloprovincialis x = 8 + 1, {SJ

15”

10”

Comnonent

20”

Init. mix.

25”

General Ash Lipid Protein Carbohydrates

40.2 5.8 23.4 25.9

of: 2.8 2 0.4 ? 4.3 ? 2.3

36.1 1.9 26.5 26.2

2 2 ? ?

1.7 0.4 2.8 1.7

25.8 12.4 36.8 23.3

? ? ? 2

0.9 0.3 3.1 1.8

19.6 14.9 46.7 19.9

2 2 2 ”

1.1 1.9 3.3 1.9

14.2 18.1 48.0 18.7

2 2 2 ”

1.0 2.0 6.4 4.1

Neutrals Monoacylglycerols Diacylglycerols Triacylglycerols Free fatty acids Free sterols Sterol and wax esters Hydrocarbons

3.2 5.2 3.4 4.7 5.9 0.2 63.5

* * * ? + 2 f

2.0 4.0 6.9 3.6 9.9 0.5 59.1

rt r ? 2 t ? a

0.4 0.5 1.1 0.4 1.2 0.2 7.0

3.7 4.0 8.0 4.0 10.3 0.2 45.0

+ ? 2 ” * * 2

0.9 0.7 0.9 1.0 0.9 0.2 3.8

1.4 3.5 9.0 1.4 6.3 7.2 32.2

* * 2 s 2 ? 2

1.3 0.4 1.2 0.3 1.1 2.0 3.7

2.8 3.2 12.2 2.4 4.6 11.9 24.4

2 ” ” 2 t 2 2

1.5 1.0 1.5 0.1 1.5 2.0 6.6

f i 2 ?

1.10 3.20 0.60 0.25

1.20 1.10 23.50 14.19 7.50

* + ” 2 *

0.34 0.18 4.60 2.90 0.05

Polar Sulfolipids Cerebrosides-1 Cerebrosides-2 Monogalactosyldiglycerols Total phospholipids Total solar liDids (weight %, total lipids)

0.8 0.4 0.5 0.2 1.0 0.1 6.9

0.21 2 0.50

trace 0.62 2 0.55

7.11 2 0.97

11.21 -t 0.69

21.53 2 1.94

trace -

on the amino acid

Giving a characteristic to the amino acid composition, we can note that in general this is rather a stable parameter and does not alter

-

0.14 2 0.40 0.15 2 0.97 trace 3.91 2 0.90

content of triacylglycerols and sterol and wax esters increase from 3.4% * 0.5% to 9.0% * 1.2% and from 0.2% + 0.1% to 7.2% & 2.0%, respectively. Percentage content of free sterols is maximum at 20°C (10.3% + 0.9%) and decreases both with an increase (6.3% & 1.1% at 25°C) and with a decrease (5.9% ? 1.O% at 1oOC)in the temperature. If we compare the initial polar lipid class composition of algae before consumption and after passing through the digestive tract, we can see that such components as sulfolipids and monogalactosyldiglycerols are utilized rather completely. As to cerebrosides-1, these compounds disappear at temperatures below 20°C. We subdivided cerebrosides according to two spots: the first spot, named “cerebrosides-l”, appeared in the upper right corner, and the second one, named “cerebrosides-2” in the middle of the plate. Cerebrosides-2 are present, but in trace amounts at these temperatures. The influence of temperature is most conspicuous in the percentage content of total polar lipids. Percentage content of these compounds decreases with decrease in the temperature from 4.24% r 0.25% to 0.21% + 0.50% and from 33.55% + 3.56% to 7.11% +- 0.97%, respectively. The most abrupt alterations take place in the interval from 20” to 15°C. Influence of temperature composition of faeces

-

4.40 15.50 0.80 4.24

33.55 4 3.56

38.49 ? 4.73

significantly with temperature. From the statistical point of view, there are no differences between the initial and final percentage contents of total essential and nonessential acids, and, still, we can trace the increase and decrease in the content of separate acids with an increase in the temperature (Table 2). Among nonessential acids, it concerns first of all the proline and glutamic acid, and among essential acids, methionine and leucine, respectively. lnfruence of temperature on the fatty composition of total lipids of faeces

acid

The most interesting peculiarity of the influence of the temperature consisted of an increase of percentage content of total w3 acids and the ratio of the w3/w6 acids during a simultaneous decrease in the content of total w6 acids (Table 3). Percentage content of total polyunsaturated acids was rather constant. The most essential alterations were connected with 20: 5~3, 22~6~3 and 22: 3~3, that is, with the acids of the w3 row. Percentage content of total saturated acids decreased (from 51.09% to 42.71%), with increase in the temperature mainly at the expense of 16 : 0 acids (from 27.36% to 21.53%). Influence of temperature on the fatty acid composition of polar lipids of faeces In general, the main alterations in this fraction of lipids were connected with 16: 0 and 18: 0 acids (Table 4). Percentage content of polyunsaturated acids was more stable. However, this stability is a result of two opposite

Transformation Table

2. Amino

of food by the mussel

of faeces of M. galloprov_incia~s at different n = 2, a = 0.05, X = X f 1, {S,}

acid composition

445 temperatures

(weight

%, total acids)

10”

15”

20”

25”

Init. mix.

Threonine Valine Methionine

6.25 k 0.19 6.76 k 0.99 2.32 k 0.06

6.17 * 1.65 6.28 2 0.38 2.50 f 1.73

6.16 Ir 0.57 6.21 k 0.13 2.21 f 1.14

6.09 k 0.76 6.10 k 0.76 1.31 f 5.21

5.49 f 0.38 5.89 + 0.38 2.41 ? 0.38

Isoleucine Leucine Phenylalanine

5.05 k 0.44 9.70 -c 1.72 6.01 2 0.76

5.21 ? 2.03 9.45 2 1.53 6.47 f 0.25

5.06 ” 0.71 9.52 k 0.76 6.00 f 1.27

4.80 k 0.76 9.00 2 1.97 5.82 2 0.94

4.51 * 0.38 9.46 + 0.25 5.88 2 0.51

Lysine Histidine Arginine

5.11 * 0.12 0.82 f 0.51 5.28 t 0.12

5.20 ” 0.25 1.12 -c 0.70 5.12 +- 0.70

5.32 + 0.31 0.92 2 0.25 5.31 f 0.19

5.50 k 0.76 1.02 f 0.32 5.30 ” 0.70

6.62 ” 1.01 0.85 * 0.06 6.50 f 0.13

Amino

acid

Total essential

47.30

Asnartic Se&e Glutamic

10.22 k 1.01 5.80 2 0.31 11.96 ‘- 0.25

10.50 ?z 0.38 5.90 2 0.94 12.06 ” 0.98

10.53 2 0.38 5.79 t 0.13 12.37 2 0.38

10.46 2 0.51 5.80 f 0.38 12.70 k 0.41

10.36 f 0.90 5.11 f 0.95 13.49 2 0.13

6.87 t 1.27 7.99 2 0.06 1.97 k 1.27

6.44 k 0.06 6.87 2 1.53 2.00 k 1.90

6.50 -e 0.89 6.95 2 0.13 1.90 2 0.13

6.70 2 1.01 7.18 k 1.53 1.91 f 0.88

5.92 f 0.25 7.26 2 0.13 1.82 2 1.27

4.26 2 0.38 3.45 2 0.14 52.60 2 4.69

4.48 2 0.13 4.00 2 0.88 52.33 2 6.80

4.32 k 0.32 4.12 k 0.25 52.51 2 2.58

4.82 f 0.67 4.23 -t- 0.11 53.77 * 5.50

3.64 2 1.14 4.62 ? 0.40 52.31 2 5.17

0.90

0.91

0.89

acid acid

Glycine Alanine Cystine Tyrosine Proline Total nonessential Ratio

es/non

es.

* 4.91

47.54

k 9.22

processes: an increase in the content of such acids as 20:5w3, 22~6~3, and 18:2w6, and a decrease of 22: 5~3, 20: 3~6, 20: 4~3. The main variation of the fatty acid composition of faeces from the fatty acid composition at algae mix consists in the content of the 18: 1 acids. Fatty acid composition of algae mix is characterized by a larger content of 18 : 1w9 and 18: lw7 as compared to that of faeces. Influence of temperature on the fatty acid composition of the total fraction of triacylglycerols, diacylglycerols, free fatty acids and free sterols of faeces

Contrary to the other investigated fractions, the fatty acid composition of these lipids was very stable, and this concerns, without exception, all of the acids and fatty acid classes (Table 5). Injiuence of temperature on the fatty acid composition of wax and sterol esters offaeces

All of the essential alterations in this fraction of neutral lipids were connected with the following: among saturated acids - 16:O (increase), 24 : 0 (decrease); among monounsaturated acids - 16: 1~7, 18:lw7, 2O:lw9 (increase), 18 : 1w12 (decrease) and among polyunsaturated acids - 20 : 5~3, 22 : 5~3 (increase), and 18: 3~3, 18:2w6 (decrease) (Table 6). Besides that, we would like to note in this case a very abrupt increase in the ratio w3/w6 acids (from 0.91 to 2.40) with increase

46.70

2 5.33

45.59

f

0.85

12.18

47.63

* 3.48

0.91

in the temperature from 10” to 25°C. The total percentage content of monounsaturated acids increases with a decrease in the content of polyunsaturated acids. Another distinctive feature of this fraction is a very large difference between the initial and final fatty acid compositions.

Discussion Physiological compensation is a common adaptation to the temperature of the environment at any level of the biochemical organization of animal life. First of all, these processes are connected with the energy budget and its metabolism (Newell, 1979; Bayne and Newell, 1983). Each thermal alteration in the environment is fast and quantitatively transferred to the aquatic organism, especially with respect to ectotherm animals. If we compare the initial general biochemical composition of algal mix and the composition at different temperatures, we note that there is a clear relation between the temperature and percentage contents of different compounds. We can distinguish right away several particularities that point out that the absorption efficiency of mussels is inversely related to temperature (percentage content of ash is the main index of this relation). A similar increase in assimilation efficiency was obtained by Ivleva “( 1970)” for Leander adspersus and by Widdows and Bayne (1971) for M. edulis.

A. V. Frolov

446 Table

3. Fatty vincialis

and S. L. Pankov

acid composition of total lipids of faeces of M. galloproat different temperatures (weight %, total acids) 10”

15”

20”

25”

Init. mix.

12:o 14:o 15:o i-15:0 ai-15:O

0.12 5.88 1.27 5.17 0.46

0.15 5.07 1.44 2.34 1.44

0.22 4.51 1.25 3.48 1.17

0.16 4.60 1.17 3.21 1.99

0.14 4.80 0.36 0.05 0.05

16:0 17:o i-17:0 18:O 19:o

27.36 1.73 0.20 6.26 0.25

26.57 1.00 0.14 6.53 0.34

22.60 1.00 0.34 5.98 0.20

21.53 1.18 0.31 6.16 0.18

17.82 0.31 0.10 1.36 0.05

20:o 21:o 22:o 23:0 24:0

0.36 0.53 0.40 0.12 0.98

0.45 0.13 0.22 0.08 0.75

1.01 0.60 0.48 0.08 0.56

0.39 0.81 0.54 0.08 0.40

0.12 1.73 0.10 0.05 0.57

Fattv

acids

14: 16: 16: 16: 16:

lw7 1~12 lw9 lw7 1~5

0.10 1.12 0.41 8.40 0.14

0.20 1.00 0.66 10.82 0.15

0.23 0.94 0.59 12.48 0.19

0.36 0.76 0.70 9.60 0.21

0.06 0.44 0.20 11.93 0.20

16: 17: 17: 18: 18:

lw3 lw9 lw3 1~12 lw9

0.82 0.78 0.11 3.31 7.00

0.92 1.19 0.06 6.23 8.27

1.44 il.89 0.05 2.56 7.65

0.67 0.99 0.12 2.83 6.10

1.17 0.17 0.19 4.09 22.60

18: 18: 20: 20: 22:

lw7 1~5 lwll lw7 lw7

3.80 0.16 1.51 2.02 0.02

3.86 0.17 0.20 1.17 0.06

6.11 0.69 0.30 0.05

6.87 0.29 1.05 0.87 0.14

3.60 0.81 0.60 0.40 0.02

16:4w6 16:3w6 16:3w3 16:2w6 17 : 2~6

0.13 1.00 0.09 0.82 0.99

0.27 0.36 0.66 2.29 0.69

0.27 2.70 0.86 1.47 0.58

0.13 1.06 0.14 0.07 1.08

0.20 4.96 0.40 2.65 0.27

18:3w6 18:3w3 18:2w6 19:5w3 19:2w6

1.72 1.28 2.76 0.18 0.40

1.92 0.99 2.81 0.20 0.37

0.64 1.13 2.20 0.10 0.14

0.94 1.77 2.80 0.12 0.46

0.80 3.48 2.80 0.07 0.14

20: 5w3 20 : 4~6 20 : 4w3 20 : 3w3 20: 3~6

3.07 0.10 0.68 0.33 0.88

4.50 0.16 0.53 0.28 0.21

4.27 0.18 0.18 0.24 0.24

6.20 0.33 0.10 0.33 1.44

7.87 0.23 0.09 0.11 0.07

20 : 2~6 22:6w3 22:5w3 22 : 4~6 22 : 3w3

0.16 0.68 0.19 0.40 1.46

0.84 0.69 0.38 0.40 0.16

0.73 0.77 0.31 0.50 0.16

0.24 1.46 0.28 0.19 0.73

0.12 1.08 0.08 0.14

51.09 29.70 17.32 7.85 9.36 0.84

46.65 34.96 18.71 8.93 10.32 0.87

43.48 34.17 17.67 9.46 9.65 0.98

42.71 31.56 19.87 13.24 8.74 1.51

27.61 46.42 25.56 14.49 12.24 1.18

Total Total Total Total Total Ratio

saturated monounsaturated polyunsaturated w3 acids w6 acids w3lw6 acids

Transformation

447

of food by the mussel

Table 4. Fatty acid composition of polar lipids of faeces of M. gailoprovinciafis at different temperatures (weight %, total acids) lo”

15”

20”

25

12:o 14:o 15:o i-15:0 ai-15:o

0.17 6.18 1.30 0.71 0.36

0.14 4.10 0.70 0.90 0.49

0.08 3.05 0.86 1.33 0.26

0.27 5.32 1 .oo 2.36 0.49

6.18 0.23 0.06 0.02

16:O 17:o i-17:0 18:O 19:o

29.74 1.55 0.13 4.22 0.24

26.39 1.00 0.23 3.33 0.13

21.92 0.86 0.17 2.94 0.13

19.46 0.79 0.25 0.05 0.17

17.63 0.10 0.13 0.41 -

20:o 21:o 2210 24:0

0.27 0.09 0.24 0.52

0.02 0.01 0.20 0.45

0.17 0.16 0.36

0.22 0.05 0.18 0.38

0.03 -

14: 16: 16: 16: 16:

lw7 1~12 lw9 lw7 1~5

1.49 0.27 11.58 0.21

0.15 1.49 0.15 11.18 0.99

0.17 1.55 0.25 16.24 1.02

0.20 1.21 0.25 18.80 0.32

0.04 4.59 0.58 12.02 0.21

16: lw3 17 : lw9 17: lw7 17: lw3 18: lw12 IS: lw9

0.47 0.75 1.75 4.74

0.30 0.66 0.07 1.15 4.34

0.82 0.08 0.18 3.87 5.15

0.99 0.08 0.03 2.91 4.75

18: lw7 18: lw5 20: lwll 20: lw9 20: lw7 22 : lw7

3.37 0.07 0.76 0.94 0.05

5.17 0.05 0.87 0.55 0.04

6.78 0.07 0.95 0.53 0.07

6.51 0.07

16:4w6 16:3w6 16: 3~3 17:2w6

0.07 0.32 0.60 0.90

0.07 1.13 0.40 0.94

0.06 1.56 0.30 0.67

0.13 0.72 0.37 0.73

0.08

18: 3w3 18 : 3~6 18:2w6 19:5w3 19:2w6

0.67 1.20 1.39 0.10 0.13

0.81 1.22 1.24 0.71 0.13

0.93 1.54 2.13 0.59 0.07

0.99 1.17 2.44 0.02 0.11

3.16 1.82 0.51 0.08

20: 5w3 20 : 4w3 20 : 3w3 20 : 3~6

5.19 0.89 2.36

6.26 1.16 0.70 2.13

7.68 0.40 0.33 0.35

8.63 0.11 0.35 0.88

7.35 0.10 0.20 0.50

: 2~6 : 6~3 : SW3 : 3~6 : 5w3

0.25 0.37 0.08 0.96

0.16 1.06 0.11 0.19

0.15 1.12 0.20 0.49

0.25 1.01 0.17 0.19 0.35

0.10 1.39 0.34 -

45.72 26.45 16.68 10.06 6.62 __ 1.52

38.09 27.16 18.42 11.40 7.02 1.62

32.29 37.73 18.57 12.22 6.53 1.87

30.99 37.40 17.85 12.03 5.89 2.04

25.13 51.90 24.33 12.54 11.75 1.07

Fatty

20 22 22 22 22 Total Total Total Total Total Ratro

acids

saturated monounsaturated polyunsaturated w3 acids w6 acids w3/w6 acids

1.20

-

68 0.42 0.08

Init. mix.

-

0.04 0.30

1.41 0.09 0.01 4.20 12.10 16.30 0.04 0.21 0.05 0.05 0.90 7.80 -

448

A. V. Frolov

and S. L. Pankov

Table 5. Fatty acid composition of total fraction of triacylglycerols, monoacylglycerols, diacylglycerols, free fatty acids and free sterols of faeces of M. galloprovincialis at different temperatures (weight %, total acids) 10”

15”

20”

25”

12:o 14:o 15:o i-15:0 ai-15:O

0.31 4.36 1.29 0.33 0.45

0.32 4.53 1.45 0.59 0.30

0.40 4.18 1.44 0.90 0.89

0.45 4.68 1.54 1.23 0.52

0.20 5.47 0.81 0.11 0.21

16:O 17:o i-17:0 18:O 19:o

21.52 0.85 0.42 4.83 0.20

22.34 0.90 0.45 5.16 0.18

22.67 099 0.60 5.89 0.25

22.30 1.11 0.52 6.97 0.27

18.92 0.52 0.32 3.60 0.12

20:o 2l:O 22:o 23:0 24:0

0.43 0.08 0.50 0.15 3.21

0.40 0.10 0.60 0.25 3.20

0.45 0.11 0.62 0.10 2.17

0.49 0.10 0.70 0.14 1.93

0.45 0.05 0.31 0.08 0.42

0.20 0.86 0.28 10.56 0.84

0.22 0.80 0.20 10.11 0.20

0.20 0.77 0.32 10.11 0.30

0.18 0.76 0.35 9.03 0.23

0.13 0.53 20.50 0.37

0.37 1.00 3.10 9.79 5.49

0.74 1.12 3.00 9.43 4.95

0.77 1.04 2.32 8.86 4.75

0.30 0.49 8.28 9.76 1.39

0.17 0.71 0.23 0.25

0.13 0.82 0.24 0.26

0.33 0.14 0.44 0.50 0.11 0.06

Fatty

14: 16: 16: 16: 16:

acids

lw7 lwl lw9 lw7 1~5

16: lw3 17 : lw9 18: 1~12 18: lw9 18: lw7

1.02 3.19 9.66 4.50

Init. mix.

18: 1~5 18: lw3 20: lwll 20: lw9 20 : lw7 22: lw7

0.11 0.76 2.27 -

0.12 0.70 0.20 0.19

16 : 4~6 16:3w3 16:2w3 17:2w6 18: 3~3

0.29 0.82 0.13 0.73 1.95

0.29 0.80 0.12 0.80 2.00

0.24 0.92 0.11 0.90 2.21

0.16 1.30 0.14 1.06 2.01

0.14 2.74 1.01 0.71 2.46

18:2w6 19:5w3 20 : 5w3 20 : 4w3 20: 3w3

3.53 0.13 2.74 0.36 0.32

3.40 0.15 0.40 0.20 0.30

3.41 0.21 0.54 0.23 0.27

3.52 0.26 2.47 0.13 0.26

4.18 0.63 2.21 0.11 0.30

20 : 3~6 20 : 2~6 22:6w3 22 : 5w3 22 : 4~6 22: 3w3

1.33 0.46 0.61 0.18 0.10 0.10

1.40 0.50 0.78 0.15 0.09 0.11

1.87 0.53 0.89 0.14 0.08 0.16

1.96 0.47 0.99 0.16 0.17 0.15

0.11 0.20 0.66 0.17 0.10 -

38.93 32.25 13.90 7.33 6.57 1.12

40.77 32.49 14.68 8.52 6.48 1.31

38.66 32.30 12.71 6.42 7.03 0.91

42.95 29.51 16.19 8.71 7.48 1.16

31.59 42.89 16.17 9.72 6.45 1.51

Total Total Total Total Total Ratto

saturated monounsaturated polyunsaturated w3 acids w6 acids w3lw6 acids

Transformation Table 6. Fatty

acid composition

galloprovincialis at different

449

of food by the mussel of wax and sterol esters of faeces of temperatures (weight %, total acids)

Init. mix.

10”

15”

20”

25

12:o 14:o 15:o i-15:0 a&15:0

0.36 5.65 1.10 0.47 0.50

0.34 5.30 1.11 0.56 0.19

0.40 5.35 1.23 0.77 0.78

0.39 5.10 1.09 1.09 0.45

0.07 2.01 0.41 0.10 0.18

16:O 17:o i-17:0 18:O

21.83 0.74 0.30 4.06

20.17 0.98 0.43 4.01

24.32 0.75 0.31 4.00

27.13 0.71 0.29 3.51

12.22 0.28 0.12 2.90

19:o 20:o 22:o 24:0

0.21 0.42 0.50 6.83

0.21 0.44 0.51 5.74

0.17 0.37 0.40 4.34

0.15 0.26 0.30 2.48

0.50 0.30 0.13 0.99

Fatty

acids

14: 16: 16: 16: 16: 16:

lw7 1~12 lw9 lw7 1~5 1~3

0.26 0.71 0.11 9.17 0.13 0.30

0.18 0.67 0.04 12.34 0.22 0.30

0.43 0.75 0.05 18.39 0.20 0.27

0.31 0.51 0.08 20.11 0.28 0.22

0.10 0.41 0.08 5.94 0.12 0.30

17: 17: 18: 18: 18: 18:

lw9 lw5 1~12 lw9 1~7 Iw5

0.94 0.15 7.20 7.04 4.12 0.13

0.90 0.17 8.48 6.57 5.10 0.10

1.11 0.09 5.20 6.13 6.00 0.11

1.12 0.04 4.00 6.85 6.36 0.08

0.44 2.11 27.62 6.90 0.12

18: 20: 20: 20: 22: 22:

lw3 lwll lw9 lw7 lw7 lw5

0.06 0.66 -

0.07 0.30 0.92 0.34 0.13 0.15

1.12 0.54 0.11 0.20

0.12

0.37 0.06 0.09

0.05 0.50 0.89 0.13 0.07 0.15

E3 0:21 -

16:4w6 16:3w6 16: 3~3 16:2w6 17:2w6

0.41 0.19 0.24 0.17 0.50

0.35 0.18 0.18 0.15 0.66

0.28 0.12 0.15 0.14 0.50

0.30 0.09 0.10 0.12 0.43

0.24 0.09 1.20 0.06 0.14

18: 3~3 18:2w6 19 : 5w3 19 : 2~6

3.90 5.39 0.10 0.11

2.18 5.40 0.04 0.12

1.11 2.13 0.03 0.10

0.20 1.69 0.07 0.17

2.21 14.44 0.20 0.53

20 : SW3 20 : 4w3 20: 3w3 20: 3~6 20 : 2~6

1.52 0.35 0.25 0.14 0.27

2.10 0.32 0.17 0.12 0.19

3.14 0.40 0.18 0.33 0.14

5.65 0.36 0.16 0.15 0.16

2.33 0.40 0.03 0.07 0.25

0.30 0.04 0.08 0.11 0.33

0.16 0.01 0.03 0.15 0.20

0.10 0.24 0.19 0.10 -

0.07 1.25 0.21 0.14 -

0.51 0.14 0.26 0.17 0.14

42.97 31.50 14.40 6.87 7.59 0.91

40.59 36.86 18.36 5.31 7.40 0.71

43.19 40.64 5.45 5.52 3.93 1.40

42.95 41.93 8.00 8.00 3.32 2.40

20.21 45.29 23.47 7.37 16.22 0.45

22 22 22 22 22 Total Total Total Total Total Ratio

: 6~3 : 5w3 : 4~6 : 3w3 : 3~6 saturated monounsaturated polyunsaturated w3 acids w6 acids w3/w6 acids

M.

450

A. V. Frolov and S. L. Pankov

As follows from Table 1, the adaptation to low temperature takes place at the expense of lipids and proteins. In this connection, we would like to note that the amino acid composition of faeces at different temperatures was rather constant; however, at low temperatures some increase in the correlation of essential/ nonessential amino acids was observed. There was a marked seasonal shift from reliance on carbohydrate, as the main energy reserve in summer, to greater reliance on proteins. Analogical results were also noted by Gabbot and Bayne (1973) for M. edulis and by Moerland and Side11 (1981) for fish Fundulus heteroclitus. A decrease in the percentage content of lipids in faeces characterizes, first of all, an increased demand for these compounds. Why does it take place? This is rather a complex and many-sided question, and we cannot answer this question unambiguously. On the other hand, this might be connected with the expenditures of energy on filtration at a high viscosity of the water (but metabolic cost of these alterations is small) (Jorgensen et al., 1986). On the other hand, it might be connected with the fact that the filtration rate decreases with a decrease in the temperature (Jorgensen et al., 1990) and increases the residence time of food in the digestive tract, which leads to an increase in the time period of enzyme influence on the food, which increases the assimilation efficiency (Wilbur and Hilbish, 1989). And last, at low temperatures the intensity of many important reactions related to the “main lines” of catabolism increases during the cold acclimation. It especially concerns the reactions that occur in the production of “high energy currency” (ATP, NADP, H and so on) (Hochachka and Somero, 1973). Additionally, we would like to mention that in unfavorable conditions lipids are used up preferably as compared to other substances (Kattner, 1989; Kattner and Krause, 1989). Most of our attention was devoted to the investigation of the interrelation between the temperature and faecal lipids, because these compounds are connected with all of the processes in the organisms-from the structure and energy to catalysis and regulation. Alterations in the individual fractions of these compounds will characterize different aspects of the temperature influence and food requirements. The dynamics of alteration of the percentage content of the total lipids is an “external manifestation” of complex and many scale processes of its utilization. In the neutral lipid class composition, there are the following main tendencies: an increase in the content of

hydrocarbons and free fatty acids and a decrease of triacylglycerol and sterol and wax esters. Hydrocarbons are not utilized in the digestive tract of mussels because an increase in their content at low temperatures is a result of the preferential use of other lipid fractions. Alterations among the main “fuel elements” of neutral lipids (mono-, di- and triacylglycer01s and free fatty acids) rather clearly show the processes of triacylglycerol degradation. What is more, the fatty acid composition of the total fraction of these elements was rather independent of the temperature. The most essential changes were connected with monoenoic acids: 16: lw7 and 18: 1~12, which are, as a rule, located in the l-position in triacylglycerol molecules and subjected to the greatest influence of the digestive enzymes. Triacylglycerols are hydrolyzed to free fatty acids and mono- and diacylglycerols and, in this form, are transported by plasma and, to a lesser extent, by hemocytes (Holland, 1978; Sheridan, 1988). However, in an experiment with radiolabelled lipids, Huca et al. (1984) have concluded that in mollusc Deplodon deplodontus, triacylglycerol can be directly absorbed from the digestive tube and incorporated in the hemolymph. The labelled triacylglycerols were found only in the hemocytes. Triacylglycerols are the most accessible fraction of lipids for digestive enzymes, that is, their utilization occurs without the complications associated with the destruction of the membrane structure (as would occur with phospholipids). Sterol and wax esters in the other fraction of neutral lipids were subjected to the temperature influence to the greatest extent. Similar results were obtained by Prahl et al., 1985. In an experiment with sprat, gobies and scad, they have shown that all of the dietary wax esters consumed by fish were completely hydrolyzed during their passage through the digestive tract. They also noted that the major differences between the composition of the released fatty acids of the faeces and those of the food consisted of a higher proportion of C,,:, acid and polyunsaturated acids in faeces. Unlike neutral lipids, the percentage content of polar lipids in faeces decreased more abruptly with a decrease in the temperature. In this case, the most essential alterations occurred in sulfolipids. These compounds are chemically unstable and are quickly destroyed under the influence of digestive enzymes. We also observed significant alterations in the percentage content of glycolipids. These compounds are localized in microalgae in the membrane of chloroplasts, that is, in the structures that are also subjected to the chemical

Transformation

of food by the mussel

influence, provided the cellular membrane has been damaged. It should also be remembered that these alterations not only characterize the activity of digestive enzymes, but also serve as indicators of physiological requirements for different substances. The preferential use of polar lipids and especially of phospholipids is probably connected with the processes of reconstruction of the membranes. This reconstruction affects both the molecular species and headgroup (Hazel and Landrey, 1988; Pruitt, 1988) and proportions of individual substances (Houslay and Stanley, 1982; Farkas et al., 1984) and leads to the rapid homeoviscous adaptation to the temperature (Carey and Hazel, 1989). With a decrease in the temperature, these alterations are followed by an increase in the activity of different enzymes of the lipid metabolism such as phosphotransferases (Hazel, 1990) and phospholipase AZ (Neas and Hazel, 1985). Besides, there are definite requirements not only for separate classes of organic molecules, but for inorganic moleculesphosphorus, for example, This is the principal compound of all phospholipids (Chapelle, 1986), which is actively incorporated in membrane structures with a decrease in the temperature. The dynamic nature of the membrane structures is an extremely important feature of the membranes in the determination of such processes as enzyme kinetics, transport of low molecular weight substances, protein synthesis by membrane-bound ribosomes (Cossins, 1977). The physical state of the membrane bilayers depends on their phospholipid composition (Martin et al., 1976). The most important mechanism of the adaptation to temperature is the alteration of the fatty acid composition of phospholipids. This can take place via acyl-chain unsaturation, redistribution of existing acyl chains or via an alteration in the length of the fatty acid (Ramesha and Thompson, 1984; Hazel et al., 1987). However, one should not forget that the fatty acid composition of phospholipids also depends on the fatty acid composition of food. There is ample evidence to show that the composition of the food affects the composition of membrane lipids: for fish (Farkas et al., 1980), for rotifers (Frolov et al., 1991a,b; Frolov and Pankov, 1992a), and for oysters (Frolov and Pankov, 1992b). Since we deal with the “external products of metabolism,” i.e., with faeces, we can only estimate the preferential use of individual substances that refer to the fatty acid composition. First of all, the processes of adaptation are connected with preferential accumulation

451

of the polyunsaturated fatty acids (PUFA) and especially the long chain PUFA in phospholipids. This phenomenon was shown for many organisms: fish Cyprinus carpio, Hypophatalmithryx molitrix, Aristichtis nobilis, Ctenophyrungon idella, Esox Lucius (Farkas et al., 1980; Farkas and Roy, 1989), leech Hirudo medicinalis (Spinedi et al., 1987), crustacea Balanus balanoides (Tooke et al., 1985), Carcinus maenas (Chapelle, 1978), Chorismus antarcticus (Clarke, 1977) crayfish P. clarkii

(Farkas

and Nevenzel,

1981), and mollusc (Piretti et al., 1988). Among the PUFA, the most important role belongs to the docosahexaenoic acid (22 : 6w3), and many investigators attribute the processes of cold adaptation to this acid. Planktonic mysid Neomysis integer (Morris, 1971), fish Tilapia nilotica (Satoh et al., 1984) crustacea Cyclopus vicinus and Daphnia magna (Farkas et al., 1984) may be taken as examples. Similar tendencies have been traced in our experiment. We observed a decrease in polyunsaturated acids, preferentially in the w3 row, with a decrease in the temperature. The most essential alterations in this case were associated with the eicosapentaenoic acid (20 : 5~3). Its percentage content decreased from 8.63% to 5.19% with a decrease in the temperature from 25” to 10°C. As for the docosahexaenoic acid, percentage content of this acid also decreased, but as soon as the initial percentage content was low (1.39%), the absolute value of the decrease of its content was low too. The same tendencies for these acids were observed in all other lipid fractions, which emphasizes their necessity. Scapharca

inaequivalvis

Conclusion From the results of our work, we can determine several main propositions concerning the influence of temperature on the biochemical composition of faeces of M. galloprovincialis. At low temperatures, the efficiency of food assimilation is higher than at high temperatures, and the ash content is the main index of this phenomenon. Adaptation to low temperatures mainly takes place at the expense of the preferential use of lipids and proteins. Among lipids, triacylglycerols, sterol and wax esters in the neutral fraction and phospholipids, sulfolipids and cerebrosides in the polar lipids are utilized in the first place. At low temperatures, there is also a preferential accumulation of fatty acids of the w3 row, mainly of the eicosapentaenoic (22 : 5~3) and docosahexaenoic (22 : 6~3) acids.

4.52

A. V. Frolov and S. L. Pankov

So we see that the method of determining the food requirements of M. galloprovincialis from the biochemical composition of fecal pellets can be successfully used in experiments of this type. However, one should not use this method exclusively, because there are many physiological conditions and external factors that influence the digestion and assimilation of food.

compounds through the oceanic water column. Progr. Oceanogr. 16, 147-194. Frolov A. V., Pankov S. L., Geradze K. N. and Pankova S. A. (1991a) Influence of salinity on the biochemical composition of the rotifer Brachionus plicarilis (Muller): aspects of adaptation. Camp. Biochem. Physiol. 99 A, 541-550. Frolov A. V., Pankov S. L., Geradze K. N., Pankova S. A. and Spectorova L. V. (1991b) Influence of the biochemical composition of food on the biochemical composition of the rotifer Brachionus plicatilis. Aqua-

Acknowledgements-We

Frolov A. V. and Pankov S. L. (1992a) The effect of starvation on the biochemical composition of the rotifer Brachionus plicatilis (Muller). .I. Mar. Biol. Ass. U.K.

culture 91, 181-202.

thank G. A. Vaitman for the chromatographic analysis of amino acids and T. I. Piminova for the chromatographic fatty acid analysis.

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