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The adenosine triphosphate content of some marine bivalve molluscs
J. exp. mar. Biol. Ecol., 1977, Vol. 28, pp. 269-283 @ Elsevier/North-Holland Biomedical Press
THE ADENOSINE
TRIPHOSPHATE BIVALVE
CONTENT
OF SOME MARINE
MOLLUSCS
ALAN D. ANSELL The Dunstaffnage
Marine Research Laboratory,
Oban, Argyli,
Scotland
Abstract:
The total ATP-content of representatives of 23 bivalve families was found to vary from 1.26-0.26 % of the dry tissue weight, with the dry tissue containing about 40 % carbon. Representatives from certain families consistently had more than average and others less than average ATP values, and there were greater differences between species from different families than between closely related species. Variations in the oxygen uptake of whole animals accounted for little of the observed variation in ATP-content. The ATP-content of individual tissues from the same individual showed wide variation, as did that of the same tissue from different species. The highest values of ATP were found in muscle tissues. High maintained levels of ATP were associated with the ability of the species to use energy rapidly for short periods, for example, in escape responses.
INTRODUCTION
The measurement of adenosine triphosphate (ATP) has been suggested as a means of estimating the biomass of planktonic organisms (Holm-Hansen & Booth, 1966; Hamilton & Holm-Hansen, 1967; Holm-Hansen, 1969) and the total living organic biomass in sediments (Ernst, 1970; Lee et al., 1971a,b; Hodson, Holm-Hansen & Azam, 1976). Measurements from a variety of organisms have shown a fairly constant relationship between organic carbon or other measure of total organic biomass and ATP content. In common with other measures of biomass or metabolic activity there are, however, marked differences between individual species so that although for many ecological purposes the general relationship between ATP and other measures of biomass may be acceptable, the deviations of individual species from such are of importance. The intimate involvement of ATP in energy metabolism suggests that differences in the level of ATP between species may be related to differences in rates of energy metabolism and in turn to their motility of production potential. Estimates of ATP from individual species from a community may, therefore, provide a means of assessing their relative potential contribution to the organic production of that community. In phytoplankton species there is evidence that this may be the case: in non-silicified micro-algae, Berland et al. (1972) showed that there was a significant correlation between the ATP/plasma ratio and the division rate in culture, even though cellular volumes were quite different; ATP content was higher in diatoms, and the authors suggest that this is related to silicon shell synthesis. There are no similar data to test this hypothesis for invertebrates. This paper gives the results of a series of measurements of ATP in various bivalve 269
270
ALAN D. ANSELL
molluscs made with the aim of determining the range of between-species variation relative to taxonomic position, habitat, and metabolic rate.
MATERIALANDMETHODS COLLECTION LOCALITIES The bivalves were freshly collected from localities on the west coast of Scotland, near The Dunstaffnage Marine Research Laboratory. Determinations were all made within three days of their collection, the animals being kept alive in running sea water until analysed. A taxonomic list of the species, together with the locality from which each was collected, is given in Table la. During July, 1975, a limited number of determinations were made with bivalves from shallow sandy areas on the Mediterranean coast, near Marseille; a list of these is given in Table Ib. The species examined include representatives of 23 bivalve families, both depositfeeding and suspension-feeding as well as epifaunal and infaunal species. In several instances two or more species belonging to the same family have been examined (Table I). In addition, in some cases individuals of the same species from different localities were examined. THE ESTIMATION OF ATP Extraction Extraction of ATP from the tissues of bivalves by the use of boiling Tris-buffer (Helm-Hansen, 1969) proved unsatisfactory because the hot buffer coagulated the proteins, and even when combined with homogenization, the disintegration of the tissues was not adequate to give reproducible and complete extraction of ATP. All attempts to use boiling Tris-buffer resulted in low and non-reproducible results. Extraction of ATP from fresh material by means of a strongly acid medium is an alternative. Acetate buffer at pH 1.5 was tried but results from this were again unsatisfactory, because, even after neutralization with sodium hydroxide and dilution of the final extract there was an increase in light emission, indicating interference with the enzyme reaction by some constituent of the buffer. The method chosen for extraction was a modification of that used by Lee et al. (1971a) for the extraction of lake sediments. The bivalve tissues or, in the case of thinshelled species such as Thyasira gouldi, the entire animal, were ground with ice-cold 0.6 N sulphuric acid in a pre-cooled Potter-Elvejhem homogenizer. The homogenate was then diluted to 25 ml with 0.6 N sulphuric acid and centrifuged. Aliquots of the supernatant were neutralized with an equal volume of ice-cold 0.6 N sodium hydroxide and diluted at least 10 times with Tris-buffer (pH 7.75). Further dilution with Trisbuffer was used as necessary to bring the final ATP concentration within the range of the standards used for calibration. Following 0.6 N sulphuric acid extraction of sedi-
ATP IN BIVALVES
271
ments, Lee et al. (1971a) used a cation exchange resin to remove interfering substances which were also extracted from the sediment by the acid. With bivalve tissues this was not necessary: there was no significant difference in the ATP of extracts passed over a cation exchange resin and that of untreated extracts. For each species, analyses were carried out for several animals over a range of size. Each living animal was quickly blotted dry, weighed, measured, and then either opened to remove the tissues, which were immediately homogenized, or homogenized intact. All extractions were followed immediately by analysis of the extract for ATP; storage of the acid extracts, or of neutralized and diluted extracts, even in the frozen state, could lead to an apparent loss of ATP over periods of 12 h or more of up to one third and no satisfactory method of storing such extracts was found. Estimation
The diluted extracts were analysed using a J.R.B. ATP-photometer (J.R.B. Associates, La Jolla, California). The luminescence of 200 j~l of firefly-lantern (Sigma Chemical Co. Ltd, FLE-50) hydrated with Tris-buffer was first measured in the photometer. 500 ~1 of extract were then added and the resulting luminescence measured (the instrument records the integrated light emission over a 60-see period starting 1.5set after mixing of the enzyme sample). The difference between these two measurements, representing the luminescence induced by the ATP in the sample, was compared with standards prepared from pure ATP (Sigma Chemical Co. Ltd, A 3 127). Several tubes of a concentrated standard (0.1 pg ATP/ml) were stored deep frozen and these were used daily to provide calibration standards by serial dilution. The stored standards were found to be stable for a period of more than one year. Suitable precautions were taken to minimize errors due to changes in the firefly extract during the course of a series of determinations. All Tris-buffer used throughout was prepared in large batches, sterilized and stored in 250 ml lots.
The reproducibility of the ATP assay was estimated using replicate samples of an extract of Tellina ten&. Eight samples of 1 ml were taken from the homogenate of a single animal and diluted to 25 ml. All the subsequent steps of neutralization, dilution, and ATP assay were replicated. The mean and standard deviation of these samples were 239.5k6.4 pg ATP. At the 95 % confidence level, the range would, therefore, be expected to be f 2.2 of the mean. This compares with a value of f 10 % of the mean found by Holm-Hansen & Booth (1966) for determinations with bacteria and a diatom, but their value includes initial sampling errors. Recouery and precision
The recovery of ATP by this method in bivalves was determined by adding known amounts of ATP to the homogenates immediately after grinding with ice-cold sulphuric acid. 26 recovery determinations were made using tissues of Abra alba and
Bronn
Chlam_vs opercularis (Linnaeus) Chlamys septemradiata Miiller
Pectinidae
(Linnaeus)
Myrtea spinifera (Montagu) Phacoides (Lucinoma) borealis
Lucinidae
islatzdica (Linnaeus)
Cyprina
Thyasira gouldi (Philippi) Thyasiru flexuosa (Montagu)
Cyprinidae
Glossus humanus (Linnaeus)
Glossidae
Thyasiridae
Lima hians (Gmelin)
Limidae
Cblamys tigerina (Miiller) Pecten maximus (Linnaeus)
Mytilus edulis Linnaeus Modiolus phaseolinus (Philippi) Modiolus modiolus (Linnaeus)
Mytilidae
Linnaeus
Glycymeris
glycymeris
Nuculana minuta (Miiller)
& Marshall
Nucula turgida Leckenby
Nucula sulcata
Nucula tenuis (Montagu)
Species
Bay
Bay
Loch Etive, Station E. 6
Loch Linnhe
Loch Etive, Station E. 24 Loch Linnhe
Dunstaffnage
Loch Creran Firth of Lorne Loch Creran
Loch Spelve Clyde, Arran Deep Clyde, Loch Long Clyde, Upper Loch Fyne Loch Creran Loch Creran
Creagan ropes Firth of Lorne Firth of Lome
Dunstaffnage
Clyde, Arran Deep Clyde, Loch Long Clyde, Arran Deep
Clyde, S. of Little Cumbrae Clyde, Loch Long
L. Etive, Station E. 6. Clyde, S. of Little Cumbrae Clyde, Loch Long
L. Etive, Station E.24 Clyde, S. of Little Cumbrae Clyde, Loch Long
Locality
56
22
12 15 20
20 100 80 120 12 12
100 80 100
90 50
56 80 50
22 80 50
Depth (m)
(mean fS.D.) of bivalves from Scottish west coast waters: n, no. of animals analysed: taxonomy
Glycymeridae
Nuculanidae
Nuculidae
Family
ATP content
TABLEIa to Moore
*0.11 &0.15 50.24 ho.08
0.23 0.37 f0.02
0.61
0.34 kO.10 0.52 *O.ll
0.68 *to.10 0.73 *0.12
1.07 0.80 0.90 0.53
0.55 &to.06 0.57 *to.07
0.72 50.22 0.81 ztO.20 0.72 f0.17
0.64 f0.09 0.52 &0.07
0.42 f0.07 0.48 kO.08 0.50 f0.11
0.47 *0.06 0.57 ho.08 0.74 hO.31
ATP (% of dry tissue)
according
2
8
8 8
4 7
8 4 6 8
8 7
6 2 8
8 8
5 8 8
8 2 2
n
(1969).
5 i% p
E P
>
h) 3
Loch Spelve Loch Creran Loch Spelve
Abra nitida (Miiller)
cuspidata
(Montagu) Brown
Cochiodesma
Cuspidaria
Cuspidariidae
praetenu
Mya truncata Linnaeus
Myidae
Laternulidae
Corbrda gibbu (Olivi)
Corbulidae
Jeffreys
Ensis arcuatus
(Pennant)
Cul~ellus peliucidus
Solenidae
Cuitellidae
Loch Spelve Loch Spelve Loch Spelve Clyde, S. Little Cumbrae Clyde, S. Little Cumbrae Clyde, Loch Long Loch Creran
Abra alba (Wood)
Scrobicularidae
Bay Clyde, Arran Deep Clyde, Arran Deep
Ardmucknish
Loch Etive, Station E. 6. Clyde, Loch Long Dunstaffnage Bay
Loch Spelve Clyde, Hunterston
Ardmucknish Bay Clyde, Kames Bay Millport
Clyde Barassie Shore
TeNina fabula Gmelin Tellina tenuis da Costa
(da Costa)
Tellinidae
oittatus
Donax
Loch Creran
Donacidae
(da Costa)
Bay Bay Bay
,-..-
Spisula
Dunstaffnage Dunstaffnage Ardmucknish
Loch Etive Loch Etive Loch Creran shore
--_______-_
Locality
1a (continued)
Mactridae
subtruncata
Dosinia exoleta (Linnaeus) Venerupis rhomboides (Pennant) Venus striatula (da Costa)
Veneridae
Cardiidae
Astarle montugui (Dillwyn) Astarte ellipitica (Brown) Card&m edule (Linnaeus)
.-_I__
Species
Astartidae
Family
TABLE
10
100
100
10
56 80
20 30 20
20 20 20 80 80 50 30
Intertidal L.W.S.T. Intertidal L.W.S.T. 10 Intertidal
0.44 kO.15 0.47 f0.11
0.67 rt0.19
0.44 kO.09 0.55
1.23 ho.25 1.14 kO.23
0.80 kO.14 0.74 kO.27 1.26 io.15
0.90 hO.37 0.87 kO.14 0.65 fO.11 0.92&0.38 0.90 kO.37 0.83 f0.26 0.70 10.09
0.75 io.10 0.64 ho.17
0.78 kO.38
0.69 ho.07
0.52 10.13 0.82 kO.12 0.56 .$,0.15
0.26 f0.05 0.36 kO.07 0.77 kO.18
10 10
Intertidal ,> 0
ATP (% of dry tissue)
Depth (m)
3 4
5
20 1
8 4
8 9
8
8
4 8 8
3 8 8
~~-
n
N d
> $
Tellinidae
-..---Veneridae Mactridae Donacidae
Family
&AD.)
Tellina tenuis (da Costa)
Venus galiina Linnaeus Spisda subtr~ncutu (da Costa) Donax semistriatus Poli Donax trunculus Linnaeus
Species
ATP (mean
Xb
Camargue coast Camargue coast Camargue coast
----~_~ Prado, Marseille Prado, Marseille
Locality
--
content of bivalves from Mediterranean
TABLE
5 5 4 1 4
Depth (m)
waters.
0.61 ho.13
0.77*0.15 0.75+0.14 0.62+0.1 I
0.18+0.05
ATP (% of dry tissue)
13
14 14
12 7
n
% $ F:
2; P
ATP IN BIVALVES
215
Cultellus pellucidus. Individual
recoveries ranged from 87.7-132.1 %, with a mean and standard deviation for recovery of 104.6pg ATP/lOO pg added f 12.0 pg ATP. At the 95 7; confidence level, the range would, therefore, be expected to be +4.64 % of the mean. The mean recovery found did not differ significantly from 100 % (t = 1.598; 25 d.f.). DETERMINATION
OF BIVALVE
TISSUE
WEIGHT
AND COMPOSITION
Since rapid extraction is necessary for accurate ATP determination, it was not possible when using acid extraction to determine both dry tissue weight and ATP in the same animal. In order to express the ATP content as a percentage of tissue dry weight, separate determinations of tissue dry weight were, therefore, made with animals of the same size range, collected at the same time, and treated in the same way as those used for ATP determinations. Measurements of carbon and nitrogen were made on the dry tissue from these determinations, using a Perkin Elmer Mode1 240 Elemental Analyzer. OXYGkN
CONSUMPTION
For some species, the relationship between oxygen consumption and dry tissue weight was also determined using either a Gilson Respirometer or Winkler oxygen determinations in closed vessels (Ansell, 1973). For the determinations with the Gilson Respirometer each animal was placed in a 25 ml flask containing 10 ml of filtered sea water. 20 % potassium hydroxide solution was placed in the centre well. The flasks were allowed to equilibrate for 1 h. The rate of oxygen consumption was determined as the regression of respirometer-micrometer reading on time, with appropriate allowances for blank flask readings, over a period of 4 h, but ignoring those periods, which occurred infrequently at the start of measurement, when the change in micrometer readings with time was not linear. All the results are given as pgO,/h/animal. The she11length, total weight, and wet and dry tissue weights of each experimental animal were determined at the end of each set of observations. Regressions of oxygen consumption @g/h/animal) on dry tissue weight (mg), were fitted, after a logarithmic transformation. Analysis of covariance showed no significant differences in slope between these relationships and for comparison between species, therefore, the mean metabolic-weight specific rate of oxygen consumption (Qo,), i.e., R/ W”.75 was then used. RESULTS RANGE
OF ATP CONTENT
In none of the species examined was there any variation in the percentage of ATP with size, and the former gives a good estimate of biomass for individual species; there
276
ALAN D. ANSELL
were, however, significant differences in the percentage content of different species (Table Ia, b). For bivalves from Scottish west coast waters the percentage of ATP ranged from 1.26 in ~~tei~~~ ~el~~~i~~~ to 0.26 in Astarte ~o~tagui. For those species for which carbon and nitrogen determinations were also made the % carbon varied between 35 and 43 % of dry weight with a mean and standard deviation of 39.21+2.40 and so the differences in carbon content are probably not significant and ATP content of bivalves from Scottish waters may be taken as varying from 3.15 to 0.66 % of the carbon, the latter being x 40 % of the dry tissue. The ATP content of bivalves from A
Nuculidae
0
Nuculanidae
co
Mytilidae
0
0
Pectinidae
0
0
Limidae
0
0
Thyasiridae
0
Lucinidae
00
Astartidae
0
Catdiidae 0
00
Veneridae
0
Mactridae
0
0
Donacidae
0
Tellinidae
00
Scrobicularidae
0
0 0
Cultellidae Solenidae Corbulidae
0
Laternulidae
0
0.5
Percentage
Cuspidar
idae
t-0
ATP
Fig. 1. Values of ATP content of whole animals: A, individual values for bivalves from different families; B, the distribution of values for all species combined.
277
ATP IN BIVALVES
the Mediterranean ranged from 0.77 to 0.18 %, but the number of species examined was very small; the ‘A ATP content of carbon for Mediterranean species ranged from 2.05 to 0.46 %. ATP CONTENT AND TAXONOMIC RELATIONSHIPS
The distribution of values of ATP content for all the bivalves examined is summarized in Fig. 1. The values show a more or less normal distribution with a mean and standard deviation of 0.65kO.23. When the distribution of the values for each bivalve family is considered (Fig. 1A) it is clear that there are greater differences between families than within families; greater than average ATP values are particularly found in the Cultellidae, Solenidae, Scrobicularidae, and Pectinidae, and lower than average values in the Thyasiridae, Lucinidae, Astartidae, Corbulidae, and Cuspidaridae. Although there are some differences between values for different populations of the same species these are generally less than the differences between species, and may not be significant. ATP CONTENT AND OXYGEN CONSUMPTION
The weight-specific oxygen consumption, Qb,, for some of the species for which ATP determinations were made are given in Table II. All the measurements were TABLE II ATP (% dry wt) and weight specific oxygen consumption (Qb, x 103), measured at 10 “C&so. for bivalves from Scottish west coast waters. Species Nucula tenuis Nucula sulcata Nucula turgida Nuculana minuta Mytilus edulis Modiolus phaseolinus Chlamys septemradiata Lima hians Astarte montagui Astarte elliptica Cardium edule Spisula subtruncata Donax vittatus Tellina jkbula Tellina tenuis Abra alba Cultellus pellucidus Corbula gibba Cochlodesma praetenu
ATP
Qoz
0.46 kO.07 0.42 ztO.07 0.64 50.09 0.72 kO.22 0.55 10.06 0.57 *0.07 0.80 kO.15 0.73 *0.12 0.26 f0.05 0.36 10.07 0.77 *0.18 0.69 ho.07 0.78 f0.38 0.75 *0.10 0.64 kO.17 0.899f0.312 1.259+0.126 0.442 kO.040 0.669hO.234
0.32*0.19 0.47kO.25 0.1710.05 0.51&0.16 2.0250.33 0.71 ho.16 1.9110.51 1.39hO.66 0.56rtO.22 0.4610.20 1.6810.40 1.315 1.60+0.60 0.49hO.27 0.52&0.09 1.30&0.50 0.86hO.19 0.96kO.47 0.83 10.09
(pg OJanimal/h)/ATP 0.099 0.160 0.043 0.116 0.559 0.194 0.401 0.31 I 0.274 0.179 0.363 0.307 0.344 0.110 0.130 0.251 0.128 0.314 0.200
IJ.‘~
ALAN
278
D. ANSELL
made at 10 “C. The regression of % ATP content on weight specific oxygen after logarithmic transformation is, log ATP = 0.201 log Q& - 0.188; variations in oxygen consumption at 10 “C between species account for little of the observed variation in ATP content. The ratio oxygen consumption/ATP0.75 varies from 0.1 to 0.559 (Table II). ATP CONTENT
AND MORPHOLOGY
The similarity of the ATP content between closely related species, and the failure of differences in metabolic rate (as indicated by oxygen consumption) to be related to the observed differences in ATP content between species, suggested that part of the differences in ATP content between less closely related species may be the result of morphological differences. Different bivalve families show considerable differences in the relative proportions of different organ systems and to investigate how far such differences in morphology may cause differences in whole-animal ATP content, a series of comparisons of the ATP content of individual tissues or organs from different species were made. The tissues were removed rapidly by dissection of freshly-opened, living bivalves, and the extractions and ATP-determinations made immediately as for whole animals. A second sample of each tissue was removed, at the same time, for determination of the moisture content, so that the ATP values could be expressed in terms of tissue dry weight; the carbon and nitrogen content of the dried tissues were also determined. The results are summarized in Table III. The highest values of ATP are in tissues which are predominantly muscular, especially the foot, mantle (mainly comprising the thickened muscular mantle margins), the siphons, and adductor muscles. Low values were found for the gills and digestive glands. The mature ovary also gave relatively low values, which were consistently lower than those found for the testis. Where possible, the adductor muscles were split to give separate estimates of the ATP for the so-called fast and slow components. In a number of cases there was a significantly higher level of ATP in the fast component of the adductor than in the slow. The greatest variation in the ATP between species, as indicated by the standard deviations in Table III, was found in those tissues which are predominantly muscular. Thus, the gills, digestive gland, and male and female gonads, showed relatively small variations between species compared with the mantle, the foot, and the anterior and posterior fast adductor muscles. The wide variation in ATP content found between different tissues indicates that variations in the relative proportions of the different tissues in different species will lead to differences in the ATP content of whole animals. The differences in ATP content of individual tissues between species, however, also reflect the differences found between species in whole animal ATP content, so that, e.g., the ATP content of the fast adductor muscles is greater in those species with a higher overall ATP content than in these species with a low ATP content. High values of ATP for the
ATP IN BIVALVES
suoyd!s
I I I I I I I I I I
p?ssl(q !
,%I d
Io!Ialuy
Jomppe MOIS Jo!Jawy Joxwppe tsr?j Jo!Jaluy
JO!JalSOd 3oisnppv aq Jo!Ja$sod
279
I
I
I
I
0
2’
’ i ’ 1’
I
35 dd
I
280
ALAN D. ANSELL
whole animal, therefore, result not only from differences in proportions of different organs, but also, and perhaps mainly, from differences in the amount of ATP in the same organ between species. DISCUSSION
The values given above for the ATP of bivalves are high when compared with those for other marine organisms which have been examined. Holm-Hansen (1970) gives 0.35 % as a mean value for the ATP (as a percentage of body carbon) for unicellular algae, based on 30 species, and Berland et al. (1972) found values of 0.169-0.557 % of body carbon for seven species of marine algae in culture. Goerke & Ernst (1975) give values of 1.3-0.9 % for two nematodes from marine muds. Balch (1972) found a mean value of 0.78 % of total body carbon (or 1.89 % of non-lipid carbon) for the planktonic copepod Calanus jknarchicus. Thiel & Holm-Hansen (unpubl. data quoted by Hodson, Helm-Hansen & Azam, 1976) found that the carbon: ATP ratios for a variety of meiofaunal and macrofaunal animals (up to 3.0 mg dry weight) isolated from shallow and deep (1200 m) sediments, averaged close to the ratio of 250 found in microbial organisms. The present values for bivalves from the Scottish west coast give ratios of carbon: ATP varying from 31.7 to 15 1.5 with a mean of 57.9. Moreover, the distribution of deviations from the mean ratio is clearly not random (Fig. l), but correlated with taxonomic differences and showing a hierarchy in which closely-related species, e.g., from the same genus, generally show a greater resemblance than those species belonging to more remotely related taxonomic groups. Differences have also been found in the ATP content of individual organs (Table II) as would be expected, but the differences in ATP content of whole animals cannot be accounted for entirely in terms of differences in the relative proportions of the dfferent organs. Neither do the differences found show a clear correlation with differences in the oxygen consumption (Table II). The ATP values found here for whole-animals and for individual organs, are in good agreement with most of the limited number of values for the ATP of bivalve molluscs and mollusc tissues which have been published. The values for MytiZus edulis (whole animals) agree well with those given by Addink & Veenhof (1975) and Wijsman (1976a, b) but are higher than the values found by Zs.-Nagy & Ermini (1972) for M. gulloprovinciulis. Wijsman (1976a) gives values for the ATP of adductor muscle, digestive gland and mantle of M. edufis which are within the ranges found here for other bivalves, and which agree most closely with those of the related mytilid, Modiolus modiolus. Beis & Newsholme (1975) give similar values for the ATP of the posterior adductor muscle of Mytilus edulis, and their values for the “snap muscle” (fast adductor) of Pecten agree closely with those found here. Higher values of ATP in the bivalves examined seem to reflect the ability of the animal to use energy rapidly for short periods, i.e., in bursts of activity which would not normally affect the rates of oxygen consumption as measured. Bivalves may use
ATP IN BIVALVES
281
energy in this way for locomotion, especially during normal burrowing after disturbance, for recovery of position in the sediment following burial, and in escape responses such as swimming or leaping (Ansell, 1968). These habits and activities are difficult to quantify, but the results of the ATP determinations seem to indicate that there is a correlation between the scope for such activity and the ATP content. For example, among the epifaunal species examined, the free-living Chlamys opercularis, which like other pectinids is able to swim actively, has a greater ATP content than the nestbuilding species Lima hiuns, which can also swim, and both have a greater ATP content than the two byssally-attached species Mytilus edulis and Modiolusphaseolinus, whose movements are more limited. Among the infauna, the very active razor shell, Cultellus pellucidus has the highest ATP content; like other solenids, this species can burrow rapidly to escape predation and, under certain conditions, makes leaping movements. The rapid and relatively deep-burrowing Abra and Tellina species contain greater amounts of ATP than sluggish, shallow-burrowing species such as Venus striatula, Corbula gibba, and Cardium edule, while these in turn have a greater ATP content than the very slow and shallow-burrowing Astarte species. In general, there is a clear relationship between a higher ATP content and a potential for more active movement. This relationship is supported by the distribution of high values of the ATP content in individual organs. The highest values were found in the organs which are immediately involved in such bursts of activity, notably the fast element of the adductor muscle in the pectinids examined, and in the foot and mantle of the solenid, Ensis arcuatus.
The difference in ATP content between the ‘fast’ and ‘slow’ elements of the adductor muscle also supports the conclusion that high levels of ATP are maintained in those muscles which are involved in bursts of activity. The fast element which, in the pectinids and in some other groups consists of transversely banded, striated muscle, contracts quickly and is involved in producing rapid adductions of the shell valves, for example, during burrowing in infaunal species such as Ensis (Trueman, 1967), and during swimming in pectinids (Yonge, 1936). The slow element consists of nonstriated muscle fibres which are responsible for the long-maintained contractions which keep the valves closed, for example, during exposure. The division is clear in some species, but, in others, one or other type of muscle only may be present, or the two types of fibre may intermingle (Hoyle, 1964; Morrison & Odense, 1973). The present results show that in certain tissues, the ATP content is maintained at a high level, but they provide no information on the rates at which ATP may be consumed or regenerated. In muscle systems, maintained high levels of ATP should provide a capacity for faster response, but continued activity must depend on the total energy immediately available, or on the capacity for the glycolytic pathways of the muscle to provide energy. The regeneration of the ATP used during the rapid contractions involved in burrowing or escape responses of the more active bivalves probably takes place in the first instance at the expense of the phosphagen, arginine
282
ALAN D. ANSELL
phosphate, which acts as an immediately available energy store in molluscan muscles, while this store in turn is regenerated, at the expense of stored carbohydrates, by normal aerobic or in some species and under certain circumstances, by anaerobic metabolic pathways. For the fast adductor muscle of Pecten muximus, the arginine phosphate pool is known to be large (52.16 pmol/g of frozen muscle; Beis & Newsholme, 1975) compared with the posterior adductor muscle of Mytilus edulis (1.99 pmol/g). Pectinid muscles also contain high levels of total carbohydrate reserves (Ansell, 1974; Comely, 1974). The full significance of the maintained higher level of ATP in some bivalve muscle systems can be assessed only when more complete information is available on all the processes which provide energy for the normal operation of these systems. REFERENCES ADDINK, A. D. F. & P. R. VEENHOF,1975. Regulation of mitochondrial matrix enzymes in Mytilus edulis. L. In, Proc. 9th Europ. mar. biol. symp., edited by H. Barnes, The Aberdeen University Press, Aberdeen, pp. 109-l 19. ANSELL, A. D., 1968. Defensive adaptations to predation in the Mollusca. Proc. Symp. Mollusca, mar. biol. Ass. India, Pt. 2, pp. 487-512. ANSELL, A. D., 1973. Oxygen consumption by the bivalve Donax vittatus (da Costa). J. exp. mar. Biol. Ecol., Vol. 11, pp. 311-328. ANSELL,A. D., 1974. Seasonal changes in biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea Area. Mar. Biol., Vol. 25, pp. 85-99. BALCH, N., 1972. ATP content of Calanusjinmarchicus. Limnol. Oceanogr., Vol. 17, pp. 906-908. BEIS, I. & E. A. NEWSHOLME,1975. The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem, J.. Vol. 152, pp. 23-32. BERLAND,B. R., D. J. BONIN,P. L. LABORDE& S. Y. MAESTRINI,1972. Variations de quelque facteurs estimatifs de la biomasse, et en particulier de I’ATP, chez plusieur algues marines planktoniques. Mar. Biol., Vol. 13, pp. 338-345. COMELY, C. A., 1974. Seasonal variations in the flesh weights and biochemical contents of the scallop Pecten maximus (L.) in the Clyde Sea Area. J. Cons. perm. int. Explor. Mer, Vol. 35, pp. 281-295. ERNST, W., 1970. ATP als Indikator fur die Biomasse mariner Sedimente. Oecologia (Berl.) Bd 5, S. 56-60. GOERKE,H. & W. ERNST, 1975. ATP content of estuarine nematodes: contribution to the determination of meiofauna biomass by ATP measurements. In, Proc. 9th Europ. mar. biot. symp., edited by H. Barnes, The Aberdeen University Press, Aberdeen, pp. 683-691. HAMILTON,R. D. & 0. HOLM-HANSEN,1967. Adenosine triphosphate content of marine bacteria. Limnol. Oceanogr., Vol. 12, pp. 319-324. HODSON,R. E., I. HOLM-HANSEN& F. AZAM, 1976. Improved methodology for ATP determination in marine environments. Mar. Biol., Vol. 34, pp. 143-149. HOLM-HANSEN,O., 1969. Determination of microbial biomass in ocean profiles. Limnol. Oceanogr., Vol. 14, pp. 740-747. HOLM-HANSEN,O., 1970. ATP levels in algal cells as influenced by environmental conditions. PI. Cell Physiol., Tokyo, Vol. 11, pp. 689-700. HOLM-HANSEN,0. & C. R. BOOTH,1966. The measurement of adenosine triphosphate in the ocean and its ecological significance. Limnol. Oceanogr., Vol. 11, pp. 510-519. HOYLE, G., 1964. Muscle and neuromuscular physiology. In, Physiology of Mollusca, Vol. 1, edited by K. M. Wilbur & C. M. Yonge, Academic Press, New York, pp. 313-351. LEE, C. C., R. F. HARRIS,J. D. H. WILLIAMS,D. E. ARMSTRONG& K. J. SYERS,1971a. Adenosine triphosphate in lake sediments: I. Determination. Soil Sci. Sot. Am. Proc., Vol. 35, pp. 82-86.
ATP IN BIVALVES
283
LEE, C. C., R. F. HARRIS,J. D. H. WILLIAMS,J. K. SYERS& D. E. ARMSTRONG,1971b. Adenosine triphosphate in lake sediments: II. Origin and significance. Soil Sci. Sot. Am. Proc., Vol. 35,
pp. 86-91. MOORE,R. C., 1969 (Editor). Treatise on invertebratepaleontology. Pt. N. Vol. 1. Mollusca 6, Bivalvia. The Geological Society of America Inc. and the University of Kansas Press, Kansas, 489 pp. MORRISON,C. M. &P. H. ODENSE,1973. Gross structure of the adductor muscles of some pelecypods. J. Fish. Res. Bd Can., Vol. 30, pp. 1583-1585. TRUEMAN,E. R., 1967. The dynamics of burrowing in Ensis (Bivalvia). Proc. R. Sot. B, Vol. 166, pp. 4599476. WIJSMAN,T. C. M., 1976a. Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. camp. Physiol., Vol. 107, pp. 129-140. WIJSMAN,T. C. M., 1976b. ATP content and mortality in Mytilus edulis from different habitats in relation to anaerobiosis. Neth. J. Sea Res., Vol. 10, pp. 140-148. YONGE, C. M., 1936. The evolution of the swimming habit in the Lamellibranchia. Mem. Mm. r. Hist. nut. Belg., T. 3, (Melanges Paul Pelseneer), pp. 77-100. ZS.-NAGY, I., & M. ERMINI, 1972. ATP production of the tissues of the bivalve Mytilus galloprovincialis (Pelecypoda) under normal and anoxic conditions. Comp. Biochem. Physiol., Vol. 43B, pp. 593-600.
THE ADENOSINE
TRIPHOSPHATE BIVALVE
CONTENT
OF SOME MARINE
MOLLUSCS
ALAN D. ANSELL The Dunstaffnage
Marine Research Laboratory,
Oban, Argyli,
Scotland
Abstract:
The total ATP-content of representatives of 23 bivalve families was found to vary from 1.26-0.26 % of the dry tissue weight, with the dry tissue containing about 40 % carbon. Representatives from certain families consistently had more than average and others less than average ATP values, and there were greater differences between species from different families than between closely related species. Variations in the oxygen uptake of whole animals accounted for little of the observed variation in ATP-content. The ATP-content of individual tissues from the same individual showed wide variation, as did that of the same tissue from different species. The highest values of ATP were found in muscle tissues. High maintained levels of ATP were associated with the ability of the species to use energy rapidly for short periods, for example, in escape responses.
INTRODUCTION
The measurement of adenosine triphosphate (ATP) has been suggested as a means of estimating the biomass of planktonic organisms (Holm-Hansen & Booth, 1966; Hamilton & Holm-Hansen, 1967; Holm-Hansen, 1969) and the total living organic biomass in sediments (Ernst, 1970; Lee et al., 1971a,b; Hodson, Holm-Hansen & Azam, 1976). Measurements from a variety of organisms have shown a fairly constant relationship between organic carbon or other measure of total organic biomass and ATP content. In common with other measures of biomass or metabolic activity there are, however, marked differences between individual species so that although for many ecological purposes the general relationship between ATP and other measures of biomass may be acceptable, the deviations of individual species from such are of importance. The intimate involvement of ATP in energy metabolism suggests that differences in the level of ATP between species may be related to differences in rates of energy metabolism and in turn to their motility of production potential. Estimates of ATP from individual species from a community may, therefore, provide a means of assessing their relative potential contribution to the organic production of that community. In phytoplankton species there is evidence that this may be the case: in non-silicified micro-algae, Berland et al. (1972) showed that there was a significant correlation between the ATP/plasma ratio and the division rate in culture, even though cellular volumes were quite different; ATP content was higher in diatoms, and the authors suggest that this is related to silicon shell synthesis. There are no similar data to test this hypothesis for invertebrates. This paper gives the results of a series of measurements of ATP in various bivalve 269
270
ALAN D. ANSELL
molluscs made with the aim of determining the range of between-species variation relative to taxonomic position, habitat, and metabolic rate.
MATERIALANDMETHODS COLLECTION LOCALITIES The bivalves were freshly collected from localities on the west coast of Scotland, near The Dunstaffnage Marine Research Laboratory. Determinations were all made within three days of their collection, the animals being kept alive in running sea water until analysed. A taxonomic list of the species, together with the locality from which each was collected, is given in Table la. During July, 1975, a limited number of determinations were made with bivalves from shallow sandy areas on the Mediterranean coast, near Marseille; a list of these is given in Table Ib. The species examined include representatives of 23 bivalve families, both depositfeeding and suspension-feeding as well as epifaunal and infaunal species. In several instances two or more species belonging to the same family have been examined (Table I). In addition, in some cases individuals of the same species from different localities were examined. THE ESTIMATION OF ATP Extraction Extraction of ATP from the tissues of bivalves by the use of boiling Tris-buffer (Helm-Hansen, 1969) proved unsatisfactory because the hot buffer coagulated the proteins, and even when combined with homogenization, the disintegration of the tissues was not adequate to give reproducible and complete extraction of ATP. All attempts to use boiling Tris-buffer resulted in low and non-reproducible results. Extraction of ATP from fresh material by means of a strongly acid medium is an alternative. Acetate buffer at pH 1.5 was tried but results from this were again unsatisfactory, because, even after neutralization with sodium hydroxide and dilution of the final extract there was an increase in light emission, indicating interference with the enzyme reaction by some constituent of the buffer. The method chosen for extraction was a modification of that used by Lee et al. (1971a) for the extraction of lake sediments. The bivalve tissues or, in the case of thinshelled species such as Thyasira gouldi, the entire animal, were ground with ice-cold 0.6 N sulphuric acid in a pre-cooled Potter-Elvejhem homogenizer. The homogenate was then diluted to 25 ml with 0.6 N sulphuric acid and centrifuged. Aliquots of the supernatant were neutralized with an equal volume of ice-cold 0.6 N sodium hydroxide and diluted at least 10 times with Tris-buffer (pH 7.75). Further dilution with Trisbuffer was used as necessary to bring the final ATP concentration within the range of the standards used for calibration. Following 0.6 N sulphuric acid extraction of sedi-
ATP IN BIVALVES
271
ments, Lee et al. (1971a) used a cation exchange resin to remove interfering substances which were also extracted from the sediment by the acid. With bivalve tissues this was not necessary: there was no significant difference in the ATP of extracts passed over a cation exchange resin and that of untreated extracts. For each species, analyses were carried out for several animals over a range of size. Each living animal was quickly blotted dry, weighed, measured, and then either opened to remove the tissues, which were immediately homogenized, or homogenized intact. All extractions were followed immediately by analysis of the extract for ATP; storage of the acid extracts, or of neutralized and diluted extracts, even in the frozen state, could lead to an apparent loss of ATP over periods of 12 h or more of up to one third and no satisfactory method of storing such extracts was found. Estimation
The diluted extracts were analysed using a J.R.B. ATP-photometer (J.R.B. Associates, La Jolla, California). The luminescence of 200 j~l of firefly-lantern (Sigma Chemical Co. Ltd, FLE-50) hydrated with Tris-buffer was first measured in the photometer. 500 ~1 of extract were then added and the resulting luminescence measured (the instrument records the integrated light emission over a 60-see period starting 1.5set after mixing of the enzyme sample). The difference between these two measurements, representing the luminescence induced by the ATP in the sample, was compared with standards prepared from pure ATP (Sigma Chemical Co. Ltd, A 3 127). Several tubes of a concentrated standard (0.1 pg ATP/ml) were stored deep frozen and these were used daily to provide calibration standards by serial dilution. The stored standards were found to be stable for a period of more than one year. Suitable precautions were taken to minimize errors due to changes in the firefly extract during the course of a series of determinations. All Tris-buffer used throughout was prepared in large batches, sterilized and stored in 250 ml lots.
The reproducibility of the ATP assay was estimated using replicate samples of an extract of Tellina ten&. Eight samples of 1 ml were taken from the homogenate of a single animal and diluted to 25 ml. All the subsequent steps of neutralization, dilution, and ATP assay were replicated. The mean and standard deviation of these samples were 239.5k6.4 pg ATP. At the 95 % confidence level, the range would, therefore, be expected to be f 2.2 of the mean. This compares with a value of f 10 % of the mean found by Holm-Hansen & Booth (1966) for determinations with bacteria and a diatom, but their value includes initial sampling errors. Recouery and precision
The recovery of ATP by this method in bivalves was determined by adding known amounts of ATP to the homogenates immediately after grinding with ice-cold sulphuric acid. 26 recovery determinations were made using tissues of Abra alba and
Bronn
Chlam_vs opercularis (Linnaeus) Chlamys septemradiata Miiller
Pectinidae
(Linnaeus)
Myrtea spinifera (Montagu) Phacoides (Lucinoma) borealis
Lucinidae
islatzdica (Linnaeus)
Cyprina
Thyasira gouldi (Philippi) Thyasiru flexuosa (Montagu)
Cyprinidae
Glossus humanus (Linnaeus)
Glossidae
Thyasiridae
Lima hians (Gmelin)
Limidae
Cblamys tigerina (Miiller) Pecten maximus (Linnaeus)
Mytilus edulis Linnaeus Modiolus phaseolinus (Philippi) Modiolus modiolus (Linnaeus)
Mytilidae
Linnaeus
Glycymeris
glycymeris
Nuculana minuta (Miiller)
& Marshall
Nucula turgida Leckenby
Nucula sulcata
Nucula tenuis (Montagu)
Species
Bay
Bay
Loch Etive, Station E. 6
Loch Linnhe
Loch Etive, Station E. 24 Loch Linnhe
Dunstaffnage
Loch Creran Firth of Lorne Loch Creran
Loch Spelve Clyde, Arran Deep Clyde, Loch Long Clyde, Upper Loch Fyne Loch Creran Loch Creran
Creagan ropes Firth of Lorne Firth of Lome
Dunstaffnage
Clyde, Arran Deep Clyde, Loch Long Clyde, Arran Deep
Clyde, S. of Little Cumbrae Clyde, Loch Long
L. Etive, Station E. 6. Clyde, S. of Little Cumbrae Clyde, Loch Long
L. Etive, Station E.24 Clyde, S. of Little Cumbrae Clyde, Loch Long
Locality
56
22
12 15 20
20 100 80 120 12 12
100 80 100
90 50
56 80 50
22 80 50
Depth (m)
(mean fS.D.) of bivalves from Scottish west coast waters: n, no. of animals analysed: taxonomy
Glycymeridae
Nuculanidae
Nuculidae
Family
ATP content
TABLEIa to Moore
*0.11 &0.15 50.24 ho.08
0.23 0.37 f0.02
0.61
0.34 kO.10 0.52 *O.ll
0.68 *to.10 0.73 *0.12
1.07 0.80 0.90 0.53
0.55 &to.06 0.57 *to.07
0.72 50.22 0.81 ztO.20 0.72 f0.17
0.64 f0.09 0.52 &0.07
0.42 f0.07 0.48 kO.08 0.50 f0.11
0.47 *0.06 0.57 ho.08 0.74 hO.31
ATP (% of dry tissue)
according
2
8
8 8
4 7
8 4 6 8
8 7
6 2 8
8 8
5 8 8
8 2 2
n
(1969).
5 i% p
E P
>
h) 3
Loch Spelve Loch Creran Loch Spelve
Abra nitida (Miiller)
cuspidata
(Montagu) Brown
Cochiodesma
Cuspidaria
Cuspidariidae
praetenu
Mya truncata Linnaeus
Myidae
Laternulidae
Corbrda gibbu (Olivi)
Corbulidae
Jeffreys
Ensis arcuatus
(Pennant)
Cul~ellus peliucidus
Solenidae
Cuitellidae
Loch Spelve Loch Spelve Loch Spelve Clyde, S. Little Cumbrae Clyde, S. Little Cumbrae Clyde, Loch Long Loch Creran
Abra alba (Wood)
Scrobicularidae
Bay Clyde, Arran Deep Clyde, Arran Deep
Ardmucknish
Loch Etive, Station E. 6. Clyde, Loch Long Dunstaffnage Bay
Loch Spelve Clyde, Hunterston
Ardmucknish Bay Clyde, Kames Bay Millport
Clyde Barassie Shore
TeNina fabula Gmelin Tellina tenuis da Costa
(da Costa)
Tellinidae
oittatus
Donax
Loch Creran
Donacidae
(da Costa)
Bay Bay Bay
,-..-
Spisula
Dunstaffnage Dunstaffnage Ardmucknish
Loch Etive Loch Etive Loch Creran shore
--_______-_
Locality
1a (continued)
Mactridae
subtruncata
Dosinia exoleta (Linnaeus) Venerupis rhomboides (Pennant) Venus striatula (da Costa)
Veneridae
Cardiidae
Astarle montugui (Dillwyn) Astarte ellipitica (Brown) Card&m edule (Linnaeus)
.-_I__
Species
Astartidae
Family
TABLE
10
100
100
10
56 80
20 30 20
20 20 20 80 80 50 30
Intertidal L.W.S.T. Intertidal L.W.S.T. 10 Intertidal
0.44 kO.15 0.47 f0.11
0.67 rt0.19
0.44 kO.09 0.55
1.23 ho.25 1.14 kO.23
0.80 kO.14 0.74 kO.27 1.26 io.15
0.90 hO.37 0.87 kO.14 0.65 fO.11 0.92&0.38 0.90 kO.37 0.83 f0.26 0.70 10.09
0.75 io.10 0.64 ho.17
0.78 kO.38
0.69 ho.07
0.52 10.13 0.82 kO.12 0.56 .$,0.15
0.26 f0.05 0.36 kO.07 0.77 kO.18
10 10
Intertidal ,> 0
ATP (% of dry tissue)
Depth (m)
3 4
5
20 1
8 4
8 9
8
8
4 8 8
3 8 8
~~-
n
N d
> $
Tellinidae
-..---Veneridae Mactridae Donacidae
Family
&AD.)
Tellina tenuis (da Costa)
Venus galiina Linnaeus Spisda subtr~ncutu (da Costa) Donax semistriatus Poli Donax trunculus Linnaeus
Species
ATP (mean
Xb
Camargue coast Camargue coast Camargue coast
----~_~ Prado, Marseille Prado, Marseille
Locality
--
content of bivalves from Mediterranean
TABLE
5 5 4 1 4
Depth (m)
waters.
0.61 ho.13
0.77*0.15 0.75+0.14 0.62+0.1 I
0.18+0.05
ATP (% of dry tissue)
13
14 14
12 7
n
% $ F:
2; P
ATP IN BIVALVES
215
Cultellus pellucidus. Individual
recoveries ranged from 87.7-132.1 %, with a mean and standard deviation for recovery of 104.6pg ATP/lOO pg added f 12.0 pg ATP. At the 95 7; confidence level, the range would, therefore, be expected to be +4.64 % of the mean. The mean recovery found did not differ significantly from 100 % (t = 1.598; 25 d.f.). DETERMINATION
OF BIVALVE
TISSUE
WEIGHT
AND COMPOSITION
Since rapid extraction is necessary for accurate ATP determination, it was not possible when using acid extraction to determine both dry tissue weight and ATP in the same animal. In order to express the ATP content as a percentage of tissue dry weight, separate determinations of tissue dry weight were, therefore, made with animals of the same size range, collected at the same time, and treated in the same way as those used for ATP determinations. Measurements of carbon and nitrogen were made on the dry tissue from these determinations, using a Perkin Elmer Mode1 240 Elemental Analyzer. OXYGkN
CONSUMPTION
For some species, the relationship between oxygen consumption and dry tissue weight was also determined using either a Gilson Respirometer or Winkler oxygen determinations in closed vessels (Ansell, 1973). For the determinations with the Gilson Respirometer each animal was placed in a 25 ml flask containing 10 ml of filtered sea water. 20 % potassium hydroxide solution was placed in the centre well. The flasks were allowed to equilibrate for 1 h. The rate of oxygen consumption was determined as the regression of respirometer-micrometer reading on time, with appropriate allowances for blank flask readings, over a period of 4 h, but ignoring those periods, which occurred infrequently at the start of measurement, when the change in micrometer readings with time was not linear. All the results are given as pgO,/h/animal. The she11length, total weight, and wet and dry tissue weights of each experimental animal were determined at the end of each set of observations. Regressions of oxygen consumption @g/h/animal) on dry tissue weight (mg), were fitted, after a logarithmic transformation. Analysis of covariance showed no significant differences in slope between these relationships and for comparison between species, therefore, the mean metabolic-weight specific rate of oxygen consumption (Qo,), i.e., R/ W”.75 was then used. RESULTS RANGE
OF ATP CONTENT
In none of the species examined was there any variation in the percentage of ATP with size, and the former gives a good estimate of biomass for individual species; there
276
ALAN D. ANSELL
were, however, significant differences in the percentage content of different species (Table Ia, b). For bivalves from Scottish west coast waters the percentage of ATP ranged from 1.26 in ~~tei~~~ ~el~~~i~~~ to 0.26 in Astarte ~o~tagui. For those species for which carbon and nitrogen determinations were also made the % carbon varied between 35 and 43 % of dry weight with a mean and standard deviation of 39.21+2.40 and so the differences in carbon content are probably not significant and ATP content of bivalves from Scottish waters may be taken as varying from 3.15 to 0.66 % of the carbon, the latter being x 40 % of the dry tissue. The ATP content of bivalves from A
Nuculidae
0
Nuculanidae
co
Mytilidae
0
0
Pectinidae
0
0
Limidae
0
0
Thyasiridae
0
Lucinidae
00
Astartidae
0
Catdiidae 0
00
Veneridae
0
Mactridae
0
0
Donacidae
0
Tellinidae
00
Scrobicularidae
0
0 0
Cultellidae Solenidae Corbulidae
0
Laternulidae
0
0.5
Percentage
Cuspidar
idae
t-0
ATP
Fig. 1. Values of ATP content of whole animals: A, individual values for bivalves from different families; B, the distribution of values for all species combined.
277
ATP IN BIVALVES
the Mediterranean ranged from 0.77 to 0.18 %, but the number of species examined was very small; the ‘A ATP content of carbon for Mediterranean species ranged from 2.05 to 0.46 %. ATP CONTENT AND TAXONOMIC RELATIONSHIPS
The distribution of values of ATP content for all the bivalves examined is summarized in Fig. 1. The values show a more or less normal distribution with a mean and standard deviation of 0.65kO.23. When the distribution of the values for each bivalve family is considered (Fig. 1A) it is clear that there are greater differences between families than within families; greater than average ATP values are particularly found in the Cultellidae, Solenidae, Scrobicularidae, and Pectinidae, and lower than average values in the Thyasiridae, Lucinidae, Astartidae, Corbulidae, and Cuspidaridae. Although there are some differences between values for different populations of the same species these are generally less than the differences between species, and may not be significant. ATP CONTENT AND OXYGEN CONSUMPTION
The weight-specific oxygen consumption, Qb,, for some of the species for which ATP determinations were made are given in Table II. All the measurements were TABLE II ATP (% dry wt) and weight specific oxygen consumption (Qb, x 103), measured at 10 “C&so. for bivalves from Scottish west coast waters. Species Nucula tenuis Nucula sulcata Nucula turgida Nuculana minuta Mytilus edulis Modiolus phaseolinus Chlamys septemradiata Lima hians Astarte montagui Astarte elliptica Cardium edule Spisula subtruncata Donax vittatus Tellina jkbula Tellina tenuis Abra alba Cultellus pellucidus Corbula gibba Cochlodesma praetenu
ATP
Qoz
0.46 kO.07 0.42 ztO.07 0.64 50.09 0.72 kO.22 0.55 10.06 0.57 *0.07 0.80 kO.15 0.73 *0.12 0.26 f0.05 0.36 10.07 0.77 *0.18 0.69 ho.07 0.78 f0.38 0.75 *0.10 0.64 kO.17 0.899f0.312 1.259+0.126 0.442 kO.040 0.669hO.234
0.32*0.19 0.47kO.25 0.1710.05 0.51&0.16 2.0250.33 0.71 ho.16 1.9110.51 1.39hO.66 0.56rtO.22 0.4610.20 1.6810.40 1.315 1.60+0.60 0.49hO.27 0.52&0.09 1.30&0.50 0.86hO.19 0.96kO.47 0.83 10.09
(pg OJanimal/h)/ATP 0.099 0.160 0.043 0.116 0.559 0.194 0.401 0.31 I 0.274 0.179 0.363 0.307 0.344 0.110 0.130 0.251 0.128 0.314 0.200
IJ.‘~
ALAN
278
D. ANSELL
made at 10 “C. The regression of % ATP content on weight specific oxygen after logarithmic transformation is, log ATP = 0.201 log Q& - 0.188; variations in oxygen consumption at 10 “C between species account for little of the observed variation in ATP content. The ratio oxygen consumption/ATP0.75 varies from 0.1 to 0.559 (Table II). ATP CONTENT
AND MORPHOLOGY
The similarity of the ATP content between closely related species, and the failure of differences in metabolic rate (as indicated by oxygen consumption) to be related to the observed differences in ATP content between species, suggested that part of the differences in ATP content between less closely related species may be the result of morphological differences. Different bivalve families show considerable differences in the relative proportions of different organ systems and to investigate how far such differences in morphology may cause differences in whole-animal ATP content, a series of comparisons of the ATP content of individual tissues or organs from different species were made. The tissues were removed rapidly by dissection of freshly-opened, living bivalves, and the extractions and ATP-determinations made immediately as for whole animals. A second sample of each tissue was removed, at the same time, for determination of the moisture content, so that the ATP values could be expressed in terms of tissue dry weight; the carbon and nitrogen content of the dried tissues were also determined. The results are summarized in Table III. The highest values of ATP are in tissues which are predominantly muscular, especially the foot, mantle (mainly comprising the thickened muscular mantle margins), the siphons, and adductor muscles. Low values were found for the gills and digestive glands. The mature ovary also gave relatively low values, which were consistently lower than those found for the testis. Where possible, the adductor muscles were split to give separate estimates of the ATP for the so-called fast and slow components. In a number of cases there was a significantly higher level of ATP in the fast component of the adductor than in the slow. The greatest variation in the ATP between species, as indicated by the standard deviations in Table III, was found in those tissues which are predominantly muscular. Thus, the gills, digestive gland, and male and female gonads, showed relatively small variations between species compared with the mantle, the foot, and the anterior and posterior fast adductor muscles. The wide variation in ATP content found between different tissues indicates that variations in the relative proportions of the different tissues in different species will lead to differences in the ATP content of whole animals. The differences in ATP content of individual tissues between species, however, also reflect the differences found between species in whole animal ATP content, so that, e.g., the ATP content of the fast adductor muscles is greater in those species with a higher overall ATP content than in these species with a low ATP content. High values of ATP for the
ATP IN BIVALVES
suoyd!s
I I I I I I I I I I
p?ssl(q !
,%I d
Io!Ialuy
Jomppe MOIS Jo!Jawy Joxwppe tsr?j Jo!Jaluy
JO!JalSOd 3oisnppv aq Jo!Ja$sod
279
I
I
I
I
0
2’
’ i ’ 1’
I
35 dd
I
280
ALAN D. ANSELL
whole animal, therefore, result not only from differences in proportions of different organs, but also, and perhaps mainly, from differences in the amount of ATP in the same organ between species. DISCUSSION
The values given above for the ATP of bivalves are high when compared with those for other marine organisms which have been examined. Holm-Hansen (1970) gives 0.35 % as a mean value for the ATP (as a percentage of body carbon) for unicellular algae, based on 30 species, and Berland et al. (1972) found values of 0.169-0.557 % of body carbon for seven species of marine algae in culture. Goerke & Ernst (1975) give values of 1.3-0.9 % for two nematodes from marine muds. Balch (1972) found a mean value of 0.78 % of total body carbon (or 1.89 % of non-lipid carbon) for the planktonic copepod Calanus jknarchicus. Thiel & Holm-Hansen (unpubl. data quoted by Hodson, Helm-Hansen & Azam, 1976) found that the carbon: ATP ratios for a variety of meiofaunal and macrofaunal animals (up to 3.0 mg dry weight) isolated from shallow and deep (1200 m) sediments, averaged close to the ratio of 250 found in microbial organisms. The present values for bivalves from the Scottish west coast give ratios of carbon: ATP varying from 31.7 to 15 1.5 with a mean of 57.9. Moreover, the distribution of deviations from the mean ratio is clearly not random (Fig. l), but correlated with taxonomic differences and showing a hierarchy in which closely-related species, e.g., from the same genus, generally show a greater resemblance than those species belonging to more remotely related taxonomic groups. Differences have also been found in the ATP content of individual organs (Table II) as would be expected, but the differences in ATP content of whole animals cannot be accounted for entirely in terms of differences in the relative proportions of the dfferent organs. Neither do the differences found show a clear correlation with differences in the oxygen consumption (Table II). The ATP values found here for whole-animals and for individual organs, are in good agreement with most of the limited number of values for the ATP of bivalve molluscs and mollusc tissues which have been published. The values for MytiZus edulis (whole animals) agree well with those given by Addink & Veenhof (1975) and Wijsman (1976a, b) but are higher than the values found by Zs.-Nagy & Ermini (1972) for M. gulloprovinciulis. Wijsman (1976a) gives values for the ATP of adductor muscle, digestive gland and mantle of M. edufis which are within the ranges found here for other bivalves, and which agree most closely with those of the related mytilid, Modiolus modiolus. Beis & Newsholme (1975) give similar values for the ATP of the posterior adductor muscle of Mytilus edulis, and their values for the “snap muscle” (fast adductor) of Pecten agree closely with those found here. Higher values of ATP in the bivalves examined seem to reflect the ability of the animal to use energy rapidly for short periods, i.e., in bursts of activity which would not normally affect the rates of oxygen consumption as measured. Bivalves may use
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energy in this way for locomotion, especially during normal burrowing after disturbance, for recovery of position in the sediment following burial, and in escape responses such as swimming or leaping (Ansell, 1968). These habits and activities are difficult to quantify, but the results of the ATP determinations seem to indicate that there is a correlation between the scope for such activity and the ATP content. For example, among the epifaunal species examined, the free-living Chlamys opercularis, which like other pectinids is able to swim actively, has a greater ATP content than the nestbuilding species Lima hiuns, which can also swim, and both have a greater ATP content than the two byssally-attached species Mytilus edulis and Modiolusphaseolinus, whose movements are more limited. Among the infauna, the very active razor shell, Cultellus pellucidus has the highest ATP content; like other solenids, this species can burrow rapidly to escape predation and, under certain conditions, makes leaping movements. The rapid and relatively deep-burrowing Abra and Tellina species contain greater amounts of ATP than sluggish, shallow-burrowing species such as Venus striatula, Corbula gibba, and Cardium edule, while these in turn have a greater ATP content than the very slow and shallow-burrowing Astarte species. In general, there is a clear relationship between a higher ATP content and a potential for more active movement. This relationship is supported by the distribution of high values of the ATP content in individual organs. The highest values were found in the organs which are immediately involved in such bursts of activity, notably the fast element of the adductor muscle in the pectinids examined, and in the foot and mantle of the solenid, Ensis arcuatus.
The difference in ATP content between the ‘fast’ and ‘slow’ elements of the adductor muscle also supports the conclusion that high levels of ATP are maintained in those muscles which are involved in bursts of activity. The fast element which, in the pectinids and in some other groups consists of transversely banded, striated muscle, contracts quickly and is involved in producing rapid adductions of the shell valves, for example, during burrowing in infaunal species such as Ensis (Trueman, 1967), and during swimming in pectinids (Yonge, 1936). The slow element consists of nonstriated muscle fibres which are responsible for the long-maintained contractions which keep the valves closed, for example, during exposure. The division is clear in some species, but, in others, one or other type of muscle only may be present, or the two types of fibre may intermingle (Hoyle, 1964; Morrison & Odense, 1973). The present results show that in certain tissues, the ATP content is maintained at a high level, but they provide no information on the rates at which ATP may be consumed or regenerated. In muscle systems, maintained high levels of ATP should provide a capacity for faster response, but continued activity must depend on the total energy immediately available, or on the capacity for the glycolytic pathways of the muscle to provide energy. The regeneration of the ATP used during the rapid contractions involved in burrowing or escape responses of the more active bivalves probably takes place in the first instance at the expense of the phosphagen, arginine
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phosphate, which acts as an immediately available energy store in molluscan muscles, while this store in turn is regenerated, at the expense of stored carbohydrates, by normal aerobic or in some species and under certain circumstances, by anaerobic metabolic pathways. For the fast adductor muscle of Pecten muximus, the arginine phosphate pool is known to be large (52.16 pmol/g of frozen muscle; Beis & Newsholme, 1975) compared with the posterior adductor muscle of Mytilus edulis (1.99 pmol/g). Pectinid muscles also contain high levels of total carbohydrate reserves (Ansell, 1974; Comely, 1974). The full significance of the maintained higher level of ATP in some bivalve muscle systems can be assessed only when more complete information is available on all the processes which provide energy for the normal operation of these systems. REFERENCES ADDINK, A. D. F. & P. R. VEENHOF,1975. Regulation of mitochondrial matrix enzymes in Mytilus edulis. L. In, Proc. 9th Europ. mar. biol. symp., edited by H. Barnes, The Aberdeen University Press, Aberdeen, pp. 109-l 19. ANSELL, A. D., 1968. Defensive adaptations to predation in the Mollusca. Proc. Symp. Mollusca, mar. biol. Ass. India, Pt. 2, pp. 487-512. ANSELL, A. D., 1973. Oxygen consumption by the bivalve Donax vittatus (da Costa). J. exp. mar. Biol. Ecol., Vol. 11, pp. 311-328. ANSELL,A. D., 1974. Seasonal changes in biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea Area. Mar. Biol., Vol. 25, pp. 85-99. BALCH, N., 1972. ATP content of Calanusjinmarchicus. Limnol. Oceanogr., Vol. 17, pp. 906-908. BEIS, I. & E. A. NEWSHOLME,1975. The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem, J.. Vol. 152, pp. 23-32. BERLAND,B. R., D. J. BONIN,P. L. LABORDE& S. Y. MAESTRINI,1972. Variations de quelque facteurs estimatifs de la biomasse, et en particulier de I’ATP, chez plusieur algues marines planktoniques. Mar. Biol., Vol. 13, pp. 338-345. COMELY, C. A., 1974. Seasonal variations in the flesh weights and biochemical contents of the scallop Pecten maximus (L.) in the Clyde Sea Area. J. Cons. perm. int. Explor. Mer, Vol. 35, pp. 281-295. ERNST, W., 1970. ATP als Indikator fur die Biomasse mariner Sedimente. Oecologia (Berl.) Bd 5, S. 56-60. GOERKE,H. & W. ERNST, 1975. ATP content of estuarine nematodes: contribution to the determination of meiofauna biomass by ATP measurements. In, Proc. 9th Europ. mar. biot. symp., edited by H. Barnes, The Aberdeen University Press, Aberdeen, pp. 683-691. HAMILTON,R. D. & 0. HOLM-HANSEN,1967. Adenosine triphosphate content of marine bacteria. Limnol. Oceanogr., Vol. 12, pp. 319-324. HODSON,R. E., I. HOLM-HANSEN& F. AZAM, 1976. Improved methodology for ATP determination in marine environments. Mar. Biol., Vol. 34, pp. 143-149. HOLM-HANSEN,O., 1969. Determination of microbial biomass in ocean profiles. Limnol. Oceanogr., Vol. 14, pp. 740-747. HOLM-HANSEN,O., 1970. ATP levels in algal cells as influenced by environmental conditions. PI. Cell Physiol., Tokyo, Vol. 11, pp. 689-700. HOLM-HANSEN,0. & C. R. BOOTH,1966. The measurement of adenosine triphosphate in the ocean and its ecological significance. Limnol. Oceanogr., Vol. 11, pp. 510-519. HOYLE, G., 1964. Muscle and neuromuscular physiology. In, Physiology of Mollusca, Vol. 1, edited by K. M. Wilbur & C. M. Yonge, Academic Press, New York, pp. 313-351. LEE, C. C., R. F. HARRIS,J. D. H. WILLIAMS,D. E. ARMSTRONG& K. J. SYERS,1971a. Adenosine triphosphate in lake sediments: I. Determination. Soil Sci. Sot. Am. Proc., Vol. 35, pp. 82-86.
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LEE, C. C., R. F. HARRIS,J. D. H. WILLIAMS,J. K. SYERS& D. E. ARMSTRONG,1971b. Adenosine triphosphate in lake sediments: II. Origin and significance. Soil Sci. Sot. Am. Proc., Vol. 35,
pp. 86-91. MOORE,R. C., 1969 (Editor). Treatise on invertebratepaleontology. Pt. N. Vol. 1. Mollusca 6, Bivalvia. The Geological Society of America Inc. and the University of Kansas Press, Kansas, 489 pp. MORRISON,C. M. &P. H. ODENSE,1973. Gross structure of the adductor muscles of some pelecypods. J. Fish. Res. Bd Can., Vol. 30, pp. 1583-1585. TRUEMAN,E. R., 1967. The dynamics of burrowing in Ensis (Bivalvia). Proc. R. Sot. B, Vol. 166, pp. 4599476. WIJSMAN,T. C. M., 1976a. Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. camp. Physiol., Vol. 107, pp. 129-140. WIJSMAN,T. C. M., 1976b. ATP content and mortality in Mytilus edulis from different habitats in relation to anaerobiosis. Neth. J. Sea Res., Vol. 10, pp. 140-148. YONGE, C. M., 1936. The evolution of the swimming habit in the Lamellibranchia. Mem. Mm. r. Hist. nut. Belg., T. 3, (Melanges Paul Pelseneer), pp. 77-100. ZS.-NAGY, I., & M. ERMINI, 1972. ATP production of the tissues of the bivalve Mytilus galloprovincialis (Pelecypoda) under normal and anoxic conditions. Comp. Biochem. Physiol., Vol. 43B, pp. 593-600.