Biochemical studies on marine zooplankton—IV. investigations on Meganyctiphanes norvegica (M. Sars)

Deep-Sea Research, 1969, Vol. 16, pp. 141 to 156. PergamonPress. Printed in Great Britain.

Biochemical studies on marine zooplankton--IV. Investigations on Meganyctiphanes norvegica (M. Sars) J. E. G. RAYMONT*, R. T. SRINIVASAGAM* and J. K. B. RAYMONT* (Received 9 October 1968)

Abstract--The biochemical composition of deep frozen samples of the euphausid, Meganyctiphanes norvegica, collected over the 6-month period November-May, has been investigated. The mean values for the whole period showed substantial amounts of protein (57~o dry weight) and of lipid (18~ dry weight), but carbohydrate content was insignificant (2~o). Chitin and ash amounted to 18yo. Protein varied from a mean monthly minimum in February (50~) to a maximum in April (62~); lipid from a minimum in April (10~) to maximum in November (29~). The fluctuations suggest a reciprocal relationship between protein and lipid content. The very large fluctuations in total lipid suggested by some workers for certain zooplankton species have not been substantiated. Some preliminary data on the biochemical composition of Thysanoessa inermis are included. INTRODUCTION

THE NEED for biochemical studies on marine zooplankton is now widely accepted in view of the relatively few studies which have been made and of their importance in any understanding of the metabolism of zooplankton. Previous publications by RAYMONT, AUSTIN and LINFORD (1964, 1966, 1968) have emphasized the importance of investigating single species of zooplankton, since, as FISHER (1962) and many other authorities have recognized, exceedingly few studies have concentrated on one species, and the biochemical composition of mixed zooplankton can vary greatly, in part owing to the changing composition of the plankton population. We have further emphasized the necessity of investigating all the major biochemical fractions on one species, if possible virtually simultaneously on the same plankton haul, since analyses of only one major constituent (e.g. protein, total lipid) may present a somewhat misleading picture. On the other hand, a complete proximate analysis permits observation of any reciprocal relationship between the constituents. To some extent also the accuracy of the analyses may be better checked if all biochemical fractions are determined simultaneously. Knowledge of the proximate biochemical composition of a variety of zooplankton species is a prerequisite for detailed investigations of the constituent lipids, amino acids and other nitrogenous components, carbohydrates, etc. In turn, the knowledge of the variations in these fractions may lead to some understanding of the biochemical pathways in zooplankton. Our earlier work has mainly concerned itself with a mysid (Neomysis integer) which is recognized as a neritic and only semi-planktonic species, though some preliminary investigations have been made on certain oceanic decapods (RAYMONT, AUSTIN and LrNFORD, 1967). There is a clear need, however, to investigate the *Department of Oceanography, University of Southampton, Southampton, England,

141

142

J.E.G.

RAYMON'r, R. T. SmNIVASAOAM and J. K. B. RAYMONT

biochemical composition of more typical marine zooplankton, especially species from higher latitudes. It has been suggested that these may show differences in biochemical composition from zooplankton from low latitudes, in particular in lipid content (LITTLEPAGZ, 1964; SHEARO, 1953). The authors are, therefore, greatly indebted to Dr. K. Wiborg of the Fisheries Institute, Bergen, Norway, for his kindness in offering to collect samples of certain euphausids from Norwegian waters and ~,~ send them to Southampton for analysis. This paper deals essentially with results on Meganyctiphanes norvegica (M. Sars) but includes some notes on another euphausid, Thysanoessa inermis (Kroyer).

MATERIAL

AND

METHODS

Meganyctiphanes norvegica were collected from four localities in southern Norway .... Gulafjord, Byfjord (Bergen), Hardangerfjord (Mundheim) and Bomlafjord. Since the latitude of these areas differed only from 59 ° 45'N to 60 ° 58'N, no distinction has been made in the results between material from the four areas. The euphausids were sorted rapidly and deep frozen; then sent by air to Southampton where they were stored in a deep-freeze prior to analysis. The collections, approximately monthly, covered the period from November 1966 to May 1967. The material consisted mostly of relatively large specimens divided into two size groups, except during January when they were much smaller and belonged almost entirely to O-group. Some tests were made on the best method of maintaining the material as several months elapsed before all analyses could be completed. All the January, February, March and April analyses quoted were obtained on material kept sealed in tubes until shortly before analysis. On the other hand, some of the material, from November and May respectively, was partly sorted into petri dishes and maintained in the deep-freeze until analysis was carried out. This proved to be a less suitable way of maintaining the material; even in the deep freeze the specimens tended to dry and the determination of dry weight of such material was unreliable. Some of the experiment~; carried out on the November and May material were, therefore, disregarded. Ho~ ,,ever, some material was analysed sufficiently rapidly for satisfactory data to bc obtained, though the results for these two months are probably somewhat less reliable than those for the remaining periods. Even for material maintained in sealed tubes, dry weight determinations showed some degree of variability, the differences ranging from 21 to 26.5 ~ dry weight of wet biomass. This may reflect actual changes in percentage dry matter, but in part is probably due to a slow drying of the material. This was emphasized by some of the November and May samples which were analysed much later and gave dry weight determinations as high as 28.5 ~. It therefore became essential, when carrying out analyses of the major biochemical fractions, to determine at the same time a dry weight ratio in order to use the correct conversion factor. Although the Factor of × 5 suggested by FIsI-ma (1962) for conversion of wet weight to dry weight can give approximately accurate values, the simultaneous determination of dry weight with biochemical analyses is preferable. Determinations of protein were made by the biuret method; for total lipid the gravimetric estimation of FOLCH, LEES and SLOANE STANLEY (1956) was adopted; estimates of carbohydrate were made according to the method of DUBOIS, GILLS, HAMILTON, REBERS and SMITH (1956), with slight modification of each method a,

Biochemical studies o n marine z o o p l a n k t o n - - I V . Investigations o n Meganyctiphanes norvegica 143

Table 1. The values for the five major biochemical constituents in M e g a n y c t i p h a n e s n o r v e g i c a from November to May. F o r protein a n d carbohydrate each value quoted represents the m e a n of several aliquots, the n u m b e r being stated in brackets below (see text). Overall means for each of the five fractions are also quoted. All values are percentage dry weight.

November

Protein

January

February

March

April

May

Overall means

54.2 : 51'1 57-8 : 58-1 49"3 : 47.9 62-1 : 59-9 64.0 : 65"5 65'7 : 60-3 "1 (4) (6) (5) (5) (3) (5) (5) (5) (5) (5) (5) (11) 50.5: (5)

59.2 (5)

58.7 51.5 (5) (5)

53.5 57.5 (5) (5)

59.7 64.8 (5) (5)

60.4 5 8 . 5 : 5 7 - 5 (5) (5) (5)

56.0

50.3 (5)

44-3 50.6 (5) (5)

52-9 62.8 (5) (5)

57.8 (5)

(5)

56.5

58.7 (5) Lipid

32.0 : 28.7 1 5 " 4 : 1 4 " 4 : 3 2 ' 1 : 2 5 . 3 10.0:11.3 7'6:8-3 25-2:14"2"1 29.0 : 27.6 12-7 : 13"8 26"2 : 20-5 17-8 : 5-8 7.6 9.9 16'8 16'9 13"7 : 17"2 23'2 : 31-5 11'2 7-4 13"5 14"6 : 15"6 6'8 18.6 12"4 : 18'5 12.9 9"0 15-1 : 15'0 16"2

Carbohydrate2"3 : 1.8 (6) (6)

1.9 : 2.0 (6) (6)

2.6 : 3.0 (*) (*)

1.7 : 2.3 (6) (6)

1.9 : 1.9 (6) (6)

2.7 : 2.1 (5) (6)

1.8 : 2.0 (6) (6)

2,0 : 1-8 (*) (*)

1.8 : 2.4 (6) (6)

1.6 : 2"0 (6) (6)

2.2 (6)

1"8 (6)

1.9 : 2"0 (3) (6)

1"8 : 1'6 (6) (6)

1"7 : 1.8 (6) (6)

2.0 : 1.8 (6) (5)

18'4

2.0

1-6 (6)

1.9 : 1.9 (6) (6) Ash

Chitin

12.4 13-6 13.9 12.0 11.6 12.5 8.6

13.3 12.3 12.0 11.5 12-5 13.6

(4%)]"

13.0 : 14.3 17.5:15.8 12.4 : 14.4 14.2 : 12-8 13.1

6"3 4-8 6.4 5"7

4.9 6"6 4-3 4.6

9.4 10-3 11-I 14.4 10.9

9.2 14.7 : 14.8 13.7 : 14.9 14-2 10.8 15.7 : 14.9 14.9 : 16.5 14-4 11.3 16.0 : 14.9 14.7 11.4 16.3 14-2 12.6 14.7

4.0:3.0 3-9 : 4.8

3-3:4-2 2.4 : 4-1

4.1 : 4.1 3.7 : 4.6

13.6

J

4.0

] I Total

4.2 94-7

*Four data for carbohydrate in February represent special analyses carried out on whole animals. ~No chitin analysis for November. Value m e a n of other months.

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RAYMONT R. T. SRINIVASAGAMand J'. K. B. RAYMON~F

described by RAYMONT,AUSTIN and LINFORD(1964), except that in the lipid analysis washing with the chloroform-methanol-KC1 mixture was reduced to a single treatment only. Some preparation of the material previous to the biochemical analysis was necessary however. For lipid determinations usually only one animal was used, but in some months, notably January, two to three animals were "pooled " to give the necessary weight of material. For protein and carbohydrate determinations, however, a standard procedure was adopted which involved homogeneization of" Meganyctiphanes. Either 300-500 mg wet weight, equivalent to 1-3 or, in January, up to 6 animals, were homogenized in distilled water to give a final volume of l0 ml, or more rarely 1000 mg wet weight were made up to 20 ml with distilled water, using a Warning blender and a glass Potter-Elvehjem homogenizer. Normally five aliquots, each of 1 ml, were then removed from the homogenate and each tested separately for protein. The average protein value obtained on each occasion is recorded in tht: results, together with the number of aliquots employed (Table 1). For carbohydrate analyses, a suitable quantity of the homogenate was removed and diluted four time~ with distilled water; after thoroughly stirring the mixture usually six aliquots of I ml each were removed and tested for carbohydrate. Data quoted in the results for carbohydrate are means of the separate determinations on each occasion, and the number of aliquots is also recorded (Table 1). Ash contents were determined on animals which had first been used for dry weight determinations and thus dried to constant weight at 70°C. The animals were then ashed to constant weight in a muffle furnace at 500°C, weighings being made after cooling in a desiccator. Chitin was determined according generally to the method of RAYMONT, AUSTIN and LINFORD (1964) but the prepared exoskeletons were not washed in dilute HCI and in some analyses the material was treated in KOH for a longer period. Relatively few determinations were made on chitin, and no values are available for the month of November, owing to shortage of material. Insufficient material was available for regular analyses of non-protein nitrogen, apart from chitin, but a few analyses were carried out recently on a separate sample of MeganyctiphanestakeninJanuary 1967in Norwegian waters. The mcthodfollowcd was that described by AUSTIN (1965) in which homogenized material is treated witl~ 20 ~o trichloroacetic acid and the precipitated protein fraction is separated by centrifuging from the supernatant. Both fractions are then analysed separately for N content using the usual Kjeldahl method. Recovery from both fractions when added together compared extremely well with N analyses of whole animals. However, preliminary results suggested a relatively very high NPN fraction amounting to nearly one-third of the total nitrogen. This i~ almost certainly a misleading result and is probably related to some partial breakdown of protein, since this euphausid material was subjected to prolonged cold storage. No further NPN analyses were therefore undertaken; any future investigation of this fraction should preferably be on fresh material.

RESULTS

The whole of the results for the major biochemical fractions over the 6-month

Biochemical studies on marine zooplankton--IV. Investigations on Meganyetiphanes norvegica 145 period are shown in Table 1. These data exclude a few early tests which were carried out before procedures were standardized, and some of the determinations carried out on the excessively dried material of November and May respectively (of. page 143). Table 1 includes, in addition to the mean values for protein and carbohydrate for each set of analyses the grand overall means for each of the major five fractions. The total of these overall means amounts to 94.7 % (Table 1). This general level of recovery may be regarded as reasonably satisfactory in view of some additional nonprotein nitrogen which was not regularly estimated. Variation in the mean monthly amounts of the five major fractions have also been examined (Table 2).

Table 2. Meganyctiphanes--biochemical .fractions.

Summary of monthly means,

(Percentage dry weight)

November

January

Protein Lipid Carbohydrate Ash Chitin

51'9 29"3 2"1 12.3 (4)*

58-0 15.0 1"9 14.2 5"5

Total

99.6

94.6

February

March

April

May

49.5 26.5 2'1 11-0 3"9

57-1 11.2 1.9 15.0 3-5

62"0 10"2 1.8 15'3 4"1

60"5 18'3 2'3 14'1 4"0

93.0

88.7

93'4

99.2

*Calculated value from other months. No measurements made. (a)

Protein

The monthly average values for protein in Meganyctiphanes norvegiea for the period November to May varied from a minimum of ~ 50% dry body weight (February) to a maximum of 62% (April) (cf. Table 2 and Fig. 1). The April and May protein contents were rather similar and suggest that a gradual building up of body tissue occurred over this time from the relatively low late winter value. The late autumn (November) protein content was almost equal to that of February; however, a rise to 58 % occurred in January (Table 2). Apart from this higher January content the data would support a rise in protein from late autumn to spring, and this might well reflect improved feeding conditions with the general augmentation of the spring plankton occurring in Norwegian waters. The difference in the January sample may reflect a difference in the age composition of the material; only in this month were most of the Meganyetiphanes of the O-group. It seems probable that the changes in the protein content from February to May cannot be attributable to fauRy analyses, since although the total recovery of all the fractions showed the largest variations over these months (see Table 2) the gradual build-up of protein was reasonably consistent. The limits of variation in any month for protein are not excessive (cf. Fig. 1) but over the whole period the protein varied from a maximum of ,~ 66% (April and May) to a minimum of ~ 44% (February) (cf. Table 1). A scatter diagram plotting sample wet weight of the euphausid (which varied from about 350 to 1000 mg) against percentage protein does not reveal any obvious change in the amount of pro-

146

J.E.G.

RAYMONT,R. T. SRIN1VASAGAMand J. K. B. RAYMONT

70-

Protein

50 40 -

t~

Lipid

t/

..___I / '

m

-

l

Carbohydrate_

2,4

Z

I-6 16 -

1

A,h

,

~

Chitin

12 m

- e

~

e

J N 1966

I D

,,

I, J 1967

,

,I,, F

T , M

I A

I ,,

M

MONTH Fig. 1. Meganyctiphanesnomegica. The monthly means for the major biochemical constituents, protein, lipid, carbohydrate, ash and chitin shown as percentage of the dry weight, together with the limits of variation in each month. tein with size of animal. However, the range in animal size was far greater than these data indicate since, especially in January, several animals (up to six) were " p o o l e d " to obtain a sample weight sufficient for analysis. Individual Meganyctiphanes were thus averaging in January only 50-60 rag; some indeed weighed for other analyses were only about 40 mg wet weight since they were mostly O-group specimens. The relationship between protein and body size in O-group animals is still uncertain. however, since analyses of individual specimens were not carried out.

(b) Lipid Our analyses strongly suggest that this fraction is the most variable. The monthly means show a mimmum (10/o dry body weight) for the April samples, whereas in November almost × 3 (29 ~ dry body weight) of total lipid was present (Table 2) •

"

O/

Biochemical studies on marine zooplankton--IV. [nvestigations on Meganyctiphanesnorvegica 147 Although lipid fell in January, it rose again to a value almost as high in February as in November; fairly low levels however persisted from March to May inclusive (Table 2 and Fig. 1). The individual variations in total lipid are also greater than for protein; as little as 6 ~ and 7 ~ in samples in March and April as against a maximum of 32 ~ in November and February (Table 1). In any one month variation between individuals was often considerable, e.g. in March a range of 5.8 ~ to 17.8 ~ and in April from 6-8~ to 18.6~ (Table 1). Nevertheless as Fig. 1 indicates, there is a real fall in average total lipid from the high levels of November and February to the low values of March and April. It was possible that percentage lipid varied with size of individual. A scatter diagram plotting sample size (as wet weight) varying from less than 50 to more than 800 mg, against weight of lipid appeared to show no very clear trend (cf. Fig. 2). The data, however, need to be grouped according to month of sampling and it is also necessary to bear in mind that some samples included more than one animal. In X 30

X X X

20 A I1. _J

X Nov.1966 o Jan.1967 • Feb.

IO

zx Mar. • Apr.

• Moy 0

I

I 200

I

I 400

I

! 600

I

I 800

I

WET W E I G H T (Mg) Fig. 2. The relationship betweenweight of total lipid and.wet weightof sample in Meganyctiphanes. The data for each month are shown separately. March and April, for instance, only single animals were used for all lipid analyses and the weights of individuals varied approximately between 300 mg and 450 mg in March, and between 350 mg and 850 mg in April, respectively. In these two months when lipid was generally low there would appear to be some tendency to an increase in percentage body lipid with individual size (cf. Fig. 2). According to FISHER (1962) percentage lipid content increases with body size in Meganyctiphanes. This relationship does not appear so obvious in our November and February samples

148

J . E . G . RAYMONT,R. T. SRn~aVASAOAMand J. K. B. RAYMONT

when the average level of lipid was maximal and when mostly single animals weighing some 300-400 mg were used for analysis. However, it is perhaps significant that one sample consisting of three very small animals (total weight 75 mg) analysed in February and excluded from the results on account of their small size, gave a combined lipid content only shghtly higher than half the average for that month. More significant are the results from January. The lipid data form a fairly homogeneous group but all are much lower than in both the previous and the following months of November and February respectively (cf. Fig. 2). As contrasted with the November and February samples, however, almost all the January Meganyctiphanes were much smaller; the sample weights used were only about 150--220 mg but even these were composed of two or three " p o o l e d " animals. The small size ot Meganyctiphanes in January consisting almost entirely of O-group animals would thus appear to be associated with low lipid. One month (May) has not so far been discussed. The individual size was generally small (average ,-~ 50 mg), but the range and the number of analyses was also very limited. It is not possible, therefore, to comment critically on the relationship between size and lipid content for this month. (c)

Carbohydrate

The amount of total carbohydrate was exceedingly low, averaging about 2 ~ /0 dry body weight throughout the period. The differences between the monthly means are relatively small; maximum 2.3% in May; minimum 1.8% in April (Table 2). Although the fluctuations are small, the pattern of changes follows that shown by the lipid component (Fig. 1). The range of values in any one month also appears to have little significance; March shows the largest range which was only from t.6% to 2.4~o for homogenized material (cf. Table 1). A particular test of carbohydrate content was made in February. Apart from the normal tests on homogenized Meganyctiphanes, each test usually involving six experiments, some four animals of very small size (7-16 mg wet weight) were specially selected and each separately determined for total carbohydrate without homogenization. The results are reasonably comparable though shghtly higher carbohydrate (maximum 3 %) was once recorded (Table 1). (d) Ash and chitin Since chitin was not determined by direct chemical analysis but by difference in weight between the dried KOH prepared exoskeletons before and after ashing, these two fractions ash and chitin are reported on together. Chitin, however, was much less in weight than total ash averaging just over 4 ~o dry weight, whereas ash averaged 13.6~o (Table 1). The variations in the monthly means were from 3.5~ (in March) to 5.5~o (in January) for chitin; from 11~o (February) to 15.3~o (April) for ash (Table 2). It is likely that the chitin values are somewhat less reliable than the ash contents, owing to the relatively small amounts of material involved and the small number of determinations made. The variations in ash and chitin do not follow one another precisely though both are relatively high in January, April and May (cf. Fig. 1). Together ash and chitin amounted to ,~ 15 ~o of the dry body weight in February as against a maximum of almost 20 ~o dry body weight in the preceding month (Table 2).

Biochemical studies on marine zooplanktonI--V. Investigations on Meganyctiphanesnorvegica 149

(e) Total of biochemical fractions Apart from any non-protein nitrogen which was not analysed regularly (cf. page 145), the total mean amounts of protein, lipid and carbohydrate, together with ash and chitin, for any one month should theoretically approach 100~o dry body weight. A summing of these fractions for each month might, therefore, form a useful check on the accuracy of our analyses. Table 2 suggests that the recovery of the various biochemical fractions was reasonably satisfactory, especially bearing in mind that the material was collected in Norway, deep frozen, transported and then stored in a deepfreeze for some months in Southampton before analyses were completed. The storage of plankton for subsequent biochemical analysis is an exceedingly difficult problem. Recent preliminary work in this Department (FUDGE, unpublished M.Sc. dissertation, Southampton Univ.) comparing a variety of preservation methods, suggested that freeze-drying gave the most consistent results comparable with analyses of fresh material, but that deep freezing was also reasonably satisfactory, provided the containers were kept closed. The present results on the variations in the biochemical fractions of Meganyctiphanes may, it is believed, be accepted with some confidence since the totals of the fractions exceeded 90 ~ and even approached 100 ~, except for March (89 ~o) (Table 2). The addition of any non-protein-nitrogen which has been suggested by CORNER, COWEY and MARSHALL (1965) as approximating to I0~o for other crustacean zooplankton would lend further confidence to these results. DISCUSSION

Although some monthly variations in protein content are apparent for Meganyctiphanes the average over the six month period is 57 ~ dry body weight (Table 1). This value is lower than that found for three different mysid species. Neomysis integer from the Southampton area has an average of 71 ~o protein and the value is surprisingly constant over the year (RAYMONT, AUSTIN and LINFORD, 1964, 1966). Leptomysis lingvura, a Mediterranean species, was found to have an almost identical value (70~o) for protein (RAYMONT and LINFORD, 1966) while SEGU1N (unpublished results), working on Praunusflexuosus from Southampton, also obtained ~-~ 72 ~ dry body weight as protein. Admittedly these mysids cannot be regarded as typically marine zooplankton species; however, results such as those of NAKAI (1955) for several species of marine copepods showed a wide range of protein from 35 ~ to 83 ~ dry weight. Nakai also found for the euphausid, Euphausia pacifica, a protein value of 7 9 ~ dry body weight. ORR (1934b) found a range for Calanus of 30-77 ~ ; COWEYand CORNER(1963) suggest a content approaching 50 ~o protein for the same species. KREy (1950) quotes 71-77~ protein for "copepods." VINOGRADOVA(1964) gives 40-60 ~ protein for general plankton, with 43 ~ for Calanus and higher values (more than 50 ~) for two mysid species. In an earlier study on Euphausia superba (VINOGRADOVA,1960) the protein content was relatively constant, ranging from 55 ~ to 61 ~ dry weight both seasonally and in young and adults. This value is remarkably close to our range for Meganyctiphanes and our values are well within the range for zooplankton generally. The mean value of 57 ~ protein for Meganyctiphanes also accords well with the preliminary results obtained from this laboratory for three species of oceanic decapods (Acanthephyra, Gennadas, Sergestes) collected from the Gulf of Aden (RAYMONT,

150

J . E . G . RAYMONT,R. T. SRINIVASAGAMand J. K. B. RAYMONI'

AUSTIN and LINFORD, 1967). These decapods gave values of protein of 60~, 62 ~, and 58 ~ respectively. The lipid content of Meganyctiphanes, while varying considerably (cf. page 148); shows an average of ~ 18~o; the highest monthly mean was just under 30),~ dry body weight and the minimum ~ 10~o. This range for Meganyctiphanescorresponds fairly well with values given by LITTLEPAGE(1964) for the somewhat neritic Antarctic euphausid, Euphausia crystallorophias. He quotes over a period of nine months :t variation in total lipid of 9.4-35.5 ~o dry weight; the drop in content appears to recur fairly steadily as winter progresses and the rise is associated by Littlepage with the Antarctic spring flowering of phytoplankton. VINOGRADOVA(1960) gives values for the oceanic Antarctic euphausid Euphausia superba which suggest a seasonal fluctuation. For adults lipid varied from 11 ~ in autumn to 26 ~ dry weight in summer, with juveniles showing much lower values of only 2.5 ~o dry weight. (Some of this euphausid material was formalin preserved and soxhlet extraction was used in analysis). Vinogradova points out that some data on euphausids from northern waters (Sea of Japan; Okhotsk Sea) also indicate higher lipid in summer than in winter. FISHER (1962) gives values for E. superba which are generally lower than Vinogradova's (3 ~o-21 ~) but extend over two months only. The range of variation of the two Antarctic euphausids is of the same order as our results for Meganyctiphanes from somewhat less high latitudes. Related zooplankton species have been considered to show variations in lipid content with latitude. LITTLEPAGE(1964) believes that Crustacea from high latitudes have higher body lipid than related genera from warmer waters and SHEARD(1953) considered that lipid storage increases in euphausids with a fall in environmental temperature, and thus percentage lipid is higher at high latitudes. LINFORO (1963) found in a few preliminary analyses of frozen Meganyctiphanes from Millport a total lipid content of about 25 ~, a value which is well inside our range for Norwegian material. A more direct comparison comes from the analyses of Fisher on Meganyctiphanes from British waters (FISHER, 1962). He shows somewhat greater average seasonal variations in total lipid (6-30 ~ approximately dry weight) though it must be remembered that his figures were expressed as percentage wet weight and we have used his suggested conversion factor. A much greater variation appears, however, in Fisher's data when the individual values for the different months are considered. From his graphs the larger Meganyctiphanes may exceed 14~ lipid wet weight (i.e. more than 7 0 ~ dry weight). At the opposite extreme some specimens had < 5 ~o dry weight as lipid. Fisher points out that geographical area is significant; Mediterranean specimens were generally lower in lipid with a minimum of < 3 ~ dry weight. CONOVERand CORNER (1968) quote two values (17-9~o and 23-5~o dry weight) for Meganyctiphanes from the Gulf of Maine. For Thysanoessa raschii Fisher quotes average lipid values only; even so these vary from ~-~ 5 ~ to 44 ~o dry weight. A much greater range (--~ 5-70 ~ dry weight) is given for Euphausia krohnii from the North Atlantic and this range applies to one month only. NAKAI (1955) dealing with Euphausiapacifica also obtained some very low values, about 3 ~o dry weight. We have not so far found the very low values which Fisher records, nor the very high lipid content which he obtained on certain occasions. Our data are much more similar to those of Littlepage and Vinogradova. It is some-

Biochemical studies on marine zooplankton--IV. Investigations on Meganyctiphanes norvegica 151 what difficult to envisage the reduction in body protein which presumably occurs if lipid amounts to some 60 70 or more of the dry body weight. Meganyetiphanes is recognized as an active swimmer (cf. HARDY and PATON, 1947) and any reduction in protein might be expected to involve inter alia some body musculature. Apart from euphausids, high lipid contents have been proposed for other zooplankton, especially for copepods from high latitudes. CONOVER(1962, 1964) for example, for the very cold water Calanus hyperboreus has claimed variable lipid which at times may reach even about 60 70 dry body weight. The variation is believed to reflect storage and at times extensive utilization. More recently CONOV~Rand CORNER (1968) using soxhlet extraction, suggest variation in lipid from ,~ 1570 to > 5070 dry weight for ~_ ?_ C. hyperboreus; extensive utilization in late winter was followed by remarkable increase in lipid during April-May. Nitrogen also showed considerable change. For Calanus finmarchicus and Calanus helgolandicus from British waters ORR (1934b) reported from 10-47 70 lipid; LINFORD (1963) recorded only 157o dry body weight for Calanus helgolandicus from Plymouth waters, and FISHER (1962) a range of 8-45 70 dry weight for C. finmarchicus from various areas. C. finmarchicus from the Gulf of Maine showed variation in lipid from N 20 70 to nearly 50 7o dry weight (CONOVERand CORNER, 1968). Maximum values for copepods as regards lipid content are given as 4070 by KLEM (1932) and 5470 for Calanus plumchrus by NAKAI (1955). On the other hand, Nakai states that Acartia possesses only about 6 70 lipid. Other relatively high figures for total lipid from copepods include values by LITTLEPAGE (1964) for the Antarctic copepod Euchaeta antarctica (maximum 467o dry weight); by VINOGRADOVA(1964) of up to 50 70 dry weight for Calanus from the Black Sea area; and by HAQ (1967) who found relatively high values (25-37 70) for Metridia longa from the Gulf of Maine, though somewhat lower values (10-307o dry weight) are suggested by CONOVERand CORNER(1968) for the same species. ORR (1934a) gives a range for adult and stage V Euchaeta norvegica from Scottish waters of 18-3670; FISHER (1962) quotes 25-55 70 dry weight lipid for the same species and COl~OVER and CORNER'S (1968) data from the Gulf of Maine are similar. Preliminary analyses for the three decapods, Acanthephyra, Gennadas and Sergestes, from the Gulf of Aden gave lipid values of 12, 15 and 29 ~ respectively dry weight (RAYMONT, AUSTIN and LINFORD, 1967). For two species of Acanthephyra, FISHER (1962) obtained a variation of ~-~ 3-15 70 total lipid of dry body weight. The range in lipid between the three decapods is in fair agreement with the seasonal fluctuations now reported for Meganyctiphanes norvegica. There is a widely held view that lipid is a major food reserve for many zooplankton species. This theory stems largely from the relatively very high lipid content recorded by some investigators and also from the wide range of values in individual species. Some authorities have proposed that these lipid fluctuations may be relatively rapid. SUSHKINA (1961) has claimed that while young copepodites of Calanus (Cop. I-III) are generally low in fat, Cop. IV and V show great variability. Fat specimens do not show obvious vertical migration, but lean copepodites migrate, moving to the upper layers to feed. Adult ~ ~ may also show high lipid but this is affected by reproduction. PETIPA (1964), pursuing this idea, believes that the lipid globules in Calanus may form a very labile reserve. She claims that a very rapid accumulation of lipid may occur during feeding and that lipid may be equally rapidly utilized as an energy source, so that even a diurnal rhythm in lipid may be witnessed in Calanus helgolandicus.

152

J. F_,,. G-. RAYMONT, R. T, SRINIVASAGAMand J. K. B, RAYMOm-

Although only some 5 % dry body weight occurs as non-variable pipid, Petipa states that an additional 10--65 ~o may occur as a reserve. However, over a six month period the Meganyctiphanes which we analysed from Norway have not revealed mean lipid contents exceeding 30 ~o dry weight. It is, of course, possible that certain zooplankton from very high latitudes may show exceptionally high lipid values, but this presumably needs confirmation especially by a simultaneous analysis of all biochemical fractions. Certainly the total lipid content in Meganyctiphanes shows considerable variation (,-~ I0-30~o dry weight) over six months. This might support the view that it is widely used as a substrate. We would suggest, however, that although lipid may be utilized it may not necessarily be the only important food reserve. NAtal (1955) was one of the first investigators to suggest a reciprocal relationship between the protein and the lipid content of zooplankton. Our results for Meganyctiphanes strongly support his suggestion; Fig. 1 shows that the fluctuations in lipid are almost a mirror image of the changes in protein. This is perhaps better illustrated by calculating the protein, lipid and carbohydrate in each month as percentage of the total organic matter excluding chitin (cf. Table 3, Fig. 3). The reciprocal pattern between protein and lipid is obvious; thus in April when protein is maximal (83.9 %), lipid is minimal (13.8%). By contrast in November, when protein is at its lowest

Table 3. Meganyctiphanes--biochemical fractions monthly means as percentage of total organic matter excluding chitin.

Protein Lipid Carbohydrate

November

January

62-3 35.2 2.5

77-4 20.0 2-5

o .....

I

c75 u

February

63.4 33.9 2.7

--

March

April

May

81.3 16.0 2.7

83.9 13.8 2.4

74.6 22"6 2'8

"- I

II

'

I-'-I Protein nln Carbohydrate

0

N 196£

J

~~

-M'

X

M

1967

MONTH Fig. 3. The amounts of p r o t e ~ lipid and carbohydrate in Meganyctiphanes for each month

sampled, expressedas

percentages

of total organic matter, excluding chitin.

Biochemicalstudies on marine zooplankton---IV.Investigationson Meganyctiphanesnorvegica 153 (62.3 ~), lipid is highest (35.2 ~o). This reciprocal pattern between lipid and protein might indicate that a portion of the lipid and of the protein might be utilizable as substrates, both being capable after some preliminary breakdown of entering the same metabolic cycle. To this extent they may be regarded, therefore, as complementary. The idea of utilization of part of the lipid and protein receives some support from the generally very low levels of carbohydrate. Figure 3 and Table 3 show the relatively insignificant contribution of this constituent; the mean carbohydrate amounts to 2 of the total dry weight or, reckoned as percentage of the total organic matter, it varies only between 2.4 ~ and 2.8 ~ (Table 3). This very low contribution by carbohydrate is almost indentical with what we have already reported for Neomysis (RAyraoNa', AUSTINand LINFORD, 1964, 1966) and for Leptomysis (RAYMONTand L1NFORD, 1966). Earlier RAYMONa"and KRISHNASWAMY(1960) and RhYMONT and CONOVER (1961) had carried out some preliminary investigations on the carbohydrate content of Calanus spp. The data suggested very low carbohydrate, probably not much exceeding 1 ~ of the dry body weight. A few determinations were also made by RAYMONa" and CONOVER (1961) on the three euphausids, Meganyctiphanes, Thysanoessa and Nematoscelis, from American waters. The same very low amount of carbohydrate was reported; Nernatoscelis appeared to be slightly higher than either Meganyctiphanes or Thysanoessa but all hardly exceeded more than about 1 ~ of the dry body weight. The more recent analyses of the deep sea oceanic decapods (Ra'~raONT, AUSTIN and LINFORD, 1967) also gave values of only 2 ~ dry body weight for total carbohydrate. It thus appears that zooplankton from quite a variety of habitats (estuarine, inshore, oceanic) and from several different geographical localities (Great Britain, Gulf of Aden, Western North Atlantic, Norway, Mediterranean) all show the same low carbohydrate content. Very few comparisons can be made on the carbohydrate contents reported by other workers since few investigators have estimated carbohydrate directly. VINOGghDOVA(1960) has suggested widely varying carbohydrate contents ranging from 6 ~ to as high as 28 ~ for Euphausia superba. Similarly VI~OGghDOVA (1964) obtained relatively high carbohydrate ranging from 16~ to 34 ~o of the dry weight for general plankton taken from the Black Sea, and rather lower carbohydrate (9 ~o) for the species, Calanus helgolandicus. In all these determinations, however, it appears that carbohydrate was not analysed directly, but the amounts were obtained by subtraction of the sum of the percentage ash, protein and lipid from a theoretical 100K. In the absence of direct simultaneous carbohydrate analysis, these relatively high carbohydrate values might need re-examination. Some comparison may be made between zooplanktonic decapods, copepods, euphausids and mysids as regards the ash and chitin content, which presumably refers mainly to differences in the exoskeleton. Thus while Meganyctiphanes had an average content of ash and chitin of 18 ~ , the three decapods analysed earlier ranged from 20 ~ to 25 ~o ash plus chitin. As compared with this, Neomysis and Leptornysis had only 14~o and 16~o respectively (RAYMONTand LINrORD, 1966). NAr,~I (1955), dealing with copepods, quotes only ash contents--some 1-3~o---but ORR (1934a) gives chitin as 3-5 ~ and this agrees well with a value by BghNDT and RABEN(1919) of 4-5 ~o chitin for copepods. The total, ash plus chitin, for copepods thus apparently does not exceed 10 ~ dry weight. VINOGghDOVA'S(1964) figure for a variety of copepods and cladocerans tends to be even lower for ash and chitin; the total organic

154

J.E.G.

RAYMONT,R. T. SRtrCtVASAOAMand L K. B. RAYMONT

matter is stated to be 90-95 ~o dry weight and for Calanus reaches even 97 ~. Nakai quotes a rather higher ash content for Euphausiapacifica (7 ~o), and this would agree reasonably well with VINOGRADOVA'S(1960) figure for Euphausia superba (7-8 ~ dry weight), HARRISand RmE¥ (1965) and CuRt. (1962) however quote figures as high as 21 ~ ash for some zooplankton species. Meganyctiphanes would appear, therefore, as might be expected, to have a relatively greater weight of exoskeleton than the small copepods; the amount is rather similar to that of mysids but it is slightly below that of certain oceanic decapods. Our suggestions on the biochemical composition of zooplankton and of the relative importance of the various fractions receive some support from certain analyses made on a few samples of Thysanoessa taken at approximately the same localities and over the same period as Meganyctiphanes. Part of the material, particularly Thysanoessa raschii, was not in good condition and gave somewhat erratic results. However, samples of Thysanoessa inermis, taken in February and May respectively, were suitable for analysis and a few results are included for comparison. The same methods were used in analysing the various fractions as for Meganyctiphanes, protein and carbohydrate measurements being made from homogenized samples, and the results (Table 4) are expressed in terms of percentage dry weight. For February the mean dry weight in T. inermis was 23 ~o of the wet weight; for May it was somewhat lower--19.5 ~,

Table 4. Biochemical analyses of" Thysanoessa inermis. All fractions expressed as percentage dry weight. For protein and carbohydrate the figuresin brackets denote the number of aliquots analysed on each occasion (of. Meganyctiphanesresults).

February Protein Lipid

Chitin Total

May

Mean

52.6 : 51.9 : 52.3 (6) (6) (5)

52-3

51-0 : 56.8 (6) (6)

53.9

19.6:23.3:22.5:23.8

22.3

12-7 : 11.4

12.1

1.8

3.0 : 3.0 (6) (6)

3.0

Carbohydrate 1.9 : 1.8 : 1-8 (6) (6) (6) Ash

Mean

11.4 : 12.4 : 11.6 : 11.6 : 12.0 2-8 : 1.7

11.8 2.3 90.5

11.3 : 17.2 : 10.3 3.6

12'9 3.6 85.5

The few results permit only a few tentative comparisons with Meganyctiphanes. The mean protein contents for the two months for T. inermis (,,~ 52 ~o and 54~) are not very different from Meganyctiphanes (,.~ 50~o and 61 ~o respectively), though the rise in protein in May is not so obvious, Lipid content in Thysanoessa shows a marked drop from ,-~ 22 ~o in February to 12 ~o in May (Table 4). This is very similar to the decline seen in Meganyetiphanes from 26.5~o (February) to 18.3~ (May) (Table 2). Possibly both protein and lipid values for Thysanoessa recorded for May are somewhat erroneously low; the material had been deep frozen for a long period and the total percentage recovery was only 85.5 Yo (Table 4). However, the general trend in changes in protein and especially in lipid is remarkably similar in the two euphansids.

..... orvegica155 Biochemical studies on marine zooplankton--IV. Investigations on Meganyct~pnanes n Regarding the other fractions, carbohydrate in Thysanoessa was the same low value as in Meganyctiphanes and indeed as in other zooplankton which we have investigated (RAYMONT, AUSTIN and LINFORD, 1966, 1967). Ash is virtually the same for the two species in February; during May both species show somewhat higher ash content. The very few analyses of chitin on Thysanoessa would suggest a somewhat lower value than that recorded for Meganyctiphanes but the general similarity in biochemical composition in Thysanoessa and Meganyctiphanes is obvious. It is perhaps worth stressing that although we have very limited data for only two months for T. inermis, and although appreciable lipid changes occurred between February and May, we again failed to record the extremely great fluctuations in lipid content suggested by some earlier workers (cf. page 154). Our observations on Meganyctiphanes suggest, therefore, that this species is similar to other zooplankton which we have studied in that material is stored almost entirely as protein and lipid. These two fractions show considerable fluctuations whereas carbohydrates remain very low. (Our very limited analyses of Thysanoessa would support this view). Although we would accept the view that lipid is probably utilized as a food reserve, as Vinogradova and others have proposed for zooplankton species, there is perhaps some indication that protein may also be utilized as has been suggested by several workers for other zooplankton species (cf. COWEY and CORNER, 1963; CONOVER, 1964; CONOVER and CORNER, 1968; RAYMONT, AUSTIN and LINEORD, 1968). Acknowledgements We are deeply indebted to Dr. K. F. WmORG, Fisheries Institute, Bergen, for the supply of the material. Our thanks are due to Dr. J. AUSTINfor assistance and advice with the analyses. The investigation was supported by a grant from the Natural Environment Research Council. REFERENCES AUSTIN J. (1965) Aspects of nitrogen metabolism in Neomysis integer (Mysidacea). Ph.D. Thesis, University of Southampton. BRANOT K. and E. RAB~,~ (1919) Zur Kenntnis der chemischen Zusammensetzung des Planktons und einiger Bodenorganismen. Wiss Meeresunters. (Abt. Kiel), 19, 175-210. CONOWR R. J. (1962) Metabolism and growth in Calanus hyperboreus in relation to its lifecycle. Rapp. P-v. R~um. Cons. perm. int. Explor. Mer., 153, 190-196. CONOVERR. J. (1964) Food relations and nutrition of zooplankton. Proc. Syrup. Exptl. Mar. Ecol. Occas. Public., No. 2. Univ. Rhode Island, 81-91. CONOVER R. J. and E. D. S. CORNER (1968) Respiration and nitrogen excretion by some marine zooplankton in relation to their life cycles. J. mar. biol. Ass. U.K., 48, 49-75. CORNERE. D. S., C. B. COWEYand S. M. MARSHALL(1965) On the nutrition and metabolism of zooplankton. III. Nitrogen excretion by Calanus. J. mar. biol. Ass. U.K., 45, 429-442. CowEv C. B. and E. D. S. CORNER(1963) On the nutrition and metabolism of zooplankton II. The relationship between the marine copepod Calanus helgolandicus and particulate material in Plymouth sea water in terms of amino acid composition. J. mar. biol. Ass. U.K., 43, 495-511. CURL H. 0962) Standing crops of carbon, nitrogen and phosphorus and transfer between trophic levels, in continental shelf waters south of New York. Rapp. P-v. Rdun. Cons. perm. int. Explor. MET., 153, 183-189. DUBOIS M., K. A. GILLS, J. K. HAMILTON,P. A. REBERSand F. SMITH(1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem., 28, 350-356. FISHER L. R. (1962) The total lipid material in some species of marine zooplankton. Rapp. P.-v. R~un. Cons. perm. int. Explor. Mer., 153, 129-136. FOLCH J., M. LEESand G. H. SLOANESTANLEY(1956) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem., 226, 497-509. HAQ S. M. (1967) Nutritional physiology of Metridia lucens and M. longs from the Gulf of Maine. Limnol. Oceanogr., 12, 40-51.

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RAX'MONT,R. T. SRmlVASAOAMand J. K. B. RAYMONT

H A z y A. C. and W. N. PATEN(1947)Experiments on vertical migration of plankton animals. J. mar. biol. Ass. U.K., 26, 467-526. I4Amtm E. and G. A. RILEY (1956) Oceanography of Long Island Sound. Bull. Bingham oceanogr. Coll., 17, 31-65. KL~.M A. (1932) Contributions to the study of the oils of marine crustacea. I. The oils of Meganyctiphanes norvegica M. Sars and Calanus finmarchicus Gunn. Hvalrdd. Skr., 6, 24 pp. Km~Y J. (1950) Eine neue Methode zur quantitativen Bestimmung des Planktons. Kielcr Meeresforsch., 7, 58-75. LI~ORD E. (1963) The lipid content of Mysidacea and of some other marine zooplankton. Ph.D. Thesis. University of Southampton. Lrrr~pAGE J. L. (1964) Seasonal variation in lipid content of two antarctic marine Crustacea. Actual. Scient. Ind. 1312, 463-470. NAKAt Z. (1955) The chemical composition, volume, weight, and size of the important marine plankton. Spec. Publs., Tokai Fish. Res. Lab., 5, 12-24. eRR A. P. (1934a) The weight and chemical composition of Euchaeta norvegica Boeck. Prec. R. Soc. Edinb. B, 54, 51-55. Omt A. P. (1934b) On the biology of Calanusfinmarchicus Part IV. Seasonal changes in the weight and chemical composition in Loch Fyne. J. mar. biol. Ass. U.K., 19, 613-632: P~n,A T. S. (1964) Diurnal rhythm of the consumption and accumulation of fat in Calanus helgolandicus (Claus) in the Black Sea. Dokl. Akad. Nauk S.S.S.R., 156, 1440-1443. (N.L.L. Translation). RAYMoNT J. E. G., J. AUSTIN and E. Lt~ORD (1964). Biochemical studies on marine zooplankton I. The biochemical composition of Neomysis integer. J. Cons. perm. int. Explor. Mer, 28, 354-363. RAYMONr J. E. G., J. AUSTIN and E. LrNFORD (1966) Biochemical studies on marine zooplankton IH. Seasonal variation in the biochemical composition of Neomysis integer. In : Some Contemporary Stt~lies in Marine Science, H. BARNES,Ed., 597-605. Allen and Unwin, London. RAX'MO~rJ. E. G., J. AUSTINand E. LrNFORO(1967) The biochemical composition of certain oceanic zooplanktonic decapods. Deep-Sea Res., 14, 113-115. RAYMONT, J. E. G., J. AUSTINand E. LINFORD(1968) Biochemical studies on marine zooplankton. V. The composition of the major biochemical fractions in Neomysis integer (Leach). J. mar. biol. Ass. U.K. (in Press). RAYMO~rr J. E. G. and R. J. CONOWR (1961) Further investigations on the carbohydrate content of marine zooplankton. Limnol. Oceanogr., 6, 154-164. RAYMom" J. E' G. and S. KRmm,TASWAMY(1960) Carbohydrate in some marine planktonic animals. J. mar. biol. Ass. U.K., 39, 239-248. RAYMONTJ. E. G. and ]E. LINFORD(1966) A note on the biochemical composition of some Mediterranean zooplankton. Int. Rev. ges. Hydrobiol., 51, 485-488. SI-mARDK. (1953) Taxonomy, distribution and development of the Euphausiacea (Crustacea). B.A.N.Z. Ant. Res. Exp. 1929-31, Rep. Set. B, 8, 1-72. SUSHKINA A. P. (1961) Vertical distribution of Calanus finmarchicus (Gunn) and its fat content. Dokl. Acad. Sci. U.S.S.R., Oceanologia (Translated Am. Gcophys. Un.), 136-141, 82-84. V~OmOa~OVAZ. A. (1960) Study of the biochemical composition of Antarctic krill (Euphausia superba Dana). Doki. Akad. Nauk. S.S.S.R., 133, 680-682. ('N.L.L. Translation). V~OGRm~OVA Z. A. (1964) Some biochemical aspects of a comparative study of plankton from the Black Sea, the Sea of Azov and the Caspian Sea. Okeanologiya, 4 (2), 232-242. (N.L.L. Translation).

Note: Just before the proof for this paper was received, a review by E. D. S. CORNERand C. B. C o w r y (Biol. Rev., 1968, 43, 393-426) was published, dealing with some aspects of variability in the biochemical fractions in zooplankton.