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Free amino acids in three species of mollusc: Responses to factors associated with reduced salinity
Camp. Biochem. PrInted tn Great
Phwui.Vol70A.pp I7m 22.1981 Britam
0300-9629l81/090017-06802.00/O Copyright 0 1981 Pergamon Press Ltd
All rights reserved
FREE AMINO ACIDS IN THREE SPECIES OF MOLLUSC: RESPONSES TO FACTORS ASSOCIATED WITH REDUCED SALINITY M. IVANOVICI*, SEBASTIAN F. RAINER?~ and
ANGELA *Biochemistry tCSIR0
Division
V. A. WADLEY~
Department, of Fisheries
John Curtin School of Medical Research, P.O. Box 334. Canberra City, A.C.T. 2601 and and Oceanography, P.O. Box 21. Cronulla. N.S.W. 2230. Australia (Rrcrirrtl
7 Jam~ur~ 1981)
Concentrations of ninhydrin-positive substances (NPS), including free amino acids, in muscle tissue of estuarine molluscs (the oyster Saccostrea commercialis, the whelk Pyrazus ebeninus and of 35”“” salinity. the clam Anadara trapezia), were 145-168 pmol g- ’ wet weight under field conditions 2. Total NPS concentrations decreased with reduced salinity (lo”,,,,) only in P. ebeninus. 3. Concentrations of alanine, arginine and glutamic acid increased, while those of aspartic acid and glycine decreased, in molluscs kept at lo”,,,,, suggesting a shift to anaerobic metabolism. 4. Changes in the ratio of taurine to glycine occurred only in S. commerciulis.
Abstract-l.
INTRODUCTION
The concentration and distribution of ninhydrinpositive substances (NPS), including free amino acids (FAA), respond in many estuarine and marine invertebrates to changes in environmental conditions. These include salinity (Schoffeniels, 1976: Florkin, 1966; Bayne. 1975). temperature and nutritional levels (Bayne. 1973). oxygen availability (Abati & Reisch, 1972; Bayne, 1975). disease (Watts, 1971) and environmental stresses associated with pollution (Schafer. 1968; Jeffries, 1972). The increase of the ratio of taurine to glycine to more than 3.0 under stressful conditions in some species of bivalve indicates its possible use to identify a stress condition in marine invertebrates (Bayne et al., 1976; Roesijadi & Anderson, 1979). Its value as a general indicator of stress is questionable. however. because the response is not found in many invertebrate species (Webb et rrl., 1972: Baginski & Pierce, 1977). This study examined the NPS concentration, the taurine to glycine ratios and their responses to an environmental stress of reduced salinity in three estuarine species not previously reported. These were the intertidal gastropod P~tr:~rs rhenin~ (Bruguiire, 1792), the intertidal oyster Strccostrrrr cwmn~ercitrlis (Iredale & Roughley, 1933) and the subtidal clam Anudara trapezia (Deshayes, 1840). The observed responses were compared with that of adenylate energy charge (Atkinson, 1971). which was measured simultaneously (Rainer et rrl.. 1979). and whose application to stress assessment is described elsewhere (ivanovici, 1980a). This comparison was done to determine which method was most appropriate in the evaluation of stress in these species. ,MATERIALS AND Anirntrls
md
c~upwimmrtd
METHODS
c~oidirions
to S. F. Rainer. Laboratories Reprint
The pattern in untransformed data (pmol g-’ wet wt) obtained from 24 samples was determined by principal components analysis. The analysis of principal components was followed by back-correlation of component loadings (Harman. 1968) with each NPS. This was done to identify which NPS were important in grouping the samples. The significance of differences in the mean values of each NPS measured for every species in control and stressed conditions was determined by a I-test. Because of the small number of animals collected from low salinity conditions. data obtained from 24 and 48 hr were pooled. The final statistical rest was for the overall significance of differences between control and experimental animals. regardless of species. using -2C In p (Sokal & Rohlf, 1969). RESULTS Composition Twenty-one
Experimental conditions were constant reduced salinity (lO.l”,,,,) and constant temperature (21’C). The control was $ Reprint requests C.S.I.R.O. Marine
a collection of molluscs from a typical field habitat (Rainer et al., 1979). Measured salinity was 35”,,,, at low tide at the collection times throughout the experiment, while temperature and food availability varied tidally and diurnally. Two molluscs of each species were removed from experimental conditions or from the field, 24 and 48 hr after beginning the experiment. Muscle tissue was removed from the animals following the optimal procedures determined by Wadley et al. (1980). and supernatants extracted (Ivanovici, 1981). Supernatants were analysed with a JEOL amino acid autoanalyser (Model 6AH). after dilution (1: IO) with citrate buffer, pH 2.2 (Anon. 1971). Concentrations were determined by the peak height method of Hadden rt trl. (1971). Absorbances were read at 570 nm for all NPS except proline (440 nm). Preliminary investigation of analytical precision indicated that a single analysis per animal was adequate. with a between-sample coefficient of variation of 5”/:, or less. Internal standards added to supernatants from each species indicated no need for correction factors.
No. 1187.
of‘ the NPS pool NPS
were detected (Table 1) in the These were: 17 FAA, taurine, ammonia and two unidentified compounds (NPS-1, NPS-2). Of these, no more than 5 NPS in any species accounted for more than approx 80”,, of the NPS
muscle tissue examined.
& 2.8 i_ 0.08 f 0.73 +_ 1.3 * 0.15 + I.5 f 1.7 + 0.16 * 0.06 * 0.15 f 0.05 + 0.13 & 0.04 & 0.53 + 0.25 + 4.4 + 0.54 & 0.10 i: 0.17
and adenylate
_+ 6.1 * 0.57 + 1.1 + I.5 * 0.03 + 0.19 * 1.4 * 0.35 * 0.10 + 0.18 + 0.16 * 0.03 + 0.01 * 1.4 f 0.26 * 1.3 i 0.69 + 0.03 * 0.02
0.55 * 0.05
1.45
0.9 i 0.00 126.9 i 11.9
29.4 1.1 8.4 6.0 0.1 10.2 24.1 0.6 0.3 0.6 0.5 0.4 0.1 5.4 0.9 34.5 1.9 0.3 0.7
+ _t f f * & * f & + * 1 * & f * * + i
6.1 0.07 0.37 0.39 0.03 2.1 1.9 0.22 0.00 0.03 0.02 0.02 0.01 0.37 0.10 3.1 0.42 0.05 0.03
energy
samples
0.8 i 0.22 109.4 f 13.7 1.68 0.60 + 0.08
27.7 0.6 7.2 4.1 0.1 I I.6 18.4 0.4 0.2 0.3 0.4 0.1 0.1 3.6 0.X 31.0 I.5 0.2 0.5
rhenimi.s 48 hr 24 hr -_.___-
ratios
I SF. with Y = 4 for field (control)
I.0 * 0.15 153.8 * 9.0 1.32 0.87 * 0.01
34.1 0.7 7.8 8.2 0.2 12.4 29.8 0.8 0.3 0.6 OS 0.4 0.1 6.2 1.2 39.4 2.3 0.3 0.7
Field
Pytrm
of NPS. taurine:glycine
Values are giLen as mean t
Alaninc Ammonia Arginine Aspartic acid fi-alanine Glutamic acid Glycine Histidine Isoleucine Leucme Lysine .~ethionine Phenylalanine Proline Serine Taurrne Threonine Tyrosine Valine NPS No. I NPS No. 3 Total NPS Tau:Gly Energy charge
Ninhydrin-posttivc substance
Table I. Concentrations
Field
167.9 i 17.1 1.00 0.69 & 0.03
&- 2.7 k 0.25 i 1.25 + 1.9 + 3.4 & 2.1 & 7.9 i: 0.03 i_ 0.00 i 0.02 +_ 0.06 & 0.06 + 0.03 & O.li + 0.28 + 3.5 f 0.18 * 0.04 + 0.03 i_ 2.X * 0.10 * 1.2 2 1.7 & 0.92 + 0.38 + 0.34 + 0.10 2 0.07 + 0.10 + 0.02 * 0.01 + 0.08 i_ 2.1 kO.23 + 4.9 + 0.13 + 0.06 rf- 0.05
172.2 I_ 7.9 0.76 0.71 + 0.08
6,h 1.3 6.1 7.1 26.2 6.Y 59.3 1.0 0.3 0.3 0.5 0.1 0.2 7.2 1.1 45.1 1.1 0.6 0.4
48 hr
’ wet wt)
( absent).
(pmoi g
and .I’ = 2 for lO.l”,,,, samples.
16X.1 i 5.6 0.82 0.83 i_ 0.05
10.4 1.0 4.6 8.4 14.0 1.9 53.5 1.3 0.3 0.3 0.8 0.2 0.2 7.0 1.4 53.6 I.6 0.5 0.4
Concentration
in muscle tissue of P. rheninrr.\. .4. rrupku conditions
6.9 + 0.49 0.6 + 0.07 3.6 I 0.35 9.2 * 0.93 18.3 i 3.1 7.4 + 0.23 57.8 _t 4.8 1.i * 0.14 0.3 * 0.03 0.3 * 0.04 0.7 + 0.09 0.1 f 0.01 0.2 + 0.06 11.6 i 3.1 0.6 k 0.14 47.2 * 3.0 1.4 * 0.22 0.5 * 0.07 0.3 & 0.03 trace
---
charge
& 1.1 + 0.10 & 0.17 + 1.2 If: 6.9 2 0.22 + 1.5 i_ 0.06 2 0.02 + 0.05 jl 0.08 i: 0.04 i: 0.02 i 3.5 * 0.13 $ 2.6 i: 0.39 F 0.13 I: 0.07 k 0.29 144.9 + 9.0 2.25 0.66 i: 0.04
7.4 0.5 0.6 14.0 32.6 4.5 21.0 0.2 0.2 0.2 0.2 0.1 0.1 13.5 0.4 47.3 0.9 0.3 0.2 0.8
Field
commrrc~idis
144.0 rf: 10.4 4.78 0.56 + 0.09
+ 0.38 k 0.02 + 0.10 F 6.7 + 3.0 + 0.38 & 1.7 + 0.01 & 0.02 i: 0.03 & 0.19 k 0.12 + 0.04 + 0.40 + 0.19 i_ 2.4 ) 0.06 k 0.06 +_ 0.06 F 0.07
24 hr 11.8 0.6 0.8 10.8 30.6 5.4 11.1 0.3 0.2 0.3 0.5 0.2 0.1 17.0 0.5 51.9 0.7 0.3 0.3 0.7
Sowostruci
(10. I”,,,,
+ 1.4 & 0.33 + 0.44 5 0.54 +_ 3.3 t 0.67 + 2.0 & 0.17 I 0.04 i. 0.01 & 0.06 f 0.06 * 0.05 F 0.48 * 0.13 & 12.6 * 0.01 * 0.02 2 0.11 1 0.24 151.9 ?_ II.0 4.32 0.51 * 002
10.8 0.9 1.3 II.3 36.6 6.4 13.4 0.4 0.2 0.2 0.6 0.1 0.2 9.9 0.4 57.9 0.3 0.4 0.3 0.2
48 hr
and S. contmerc,ict/i.\ from field and low salinity
c 2 L 2 f: c; =y
3 f ;: : E
Molluscan
Saccostrea commercialis
free amino
acids: reduced
salinity
19
I’
I
(32.9%)
Pyrazus ebeninus
Anadara trapezia
‘, \,
Fig. I. P~rcwa chwinus. Antrdurtr trupezitr and Saccostrecr commertiulis: component loadings for 8 samples from animals of each species. for the three largest principal components. Different symbols are used for each species; the proportion of the variance extracted is given for each component. For each species. dotted lines enclose loadings for the control animals (closed symbols) and the experimental animals (open symbols) separately.
pool. Taurine and glycine were major NPS in all three species. Concentrations of alanine and glutamic acid were higher in P. ehvninlrs than either of the bivalve species. while /I-alanine and proline were higher in both bivalve species. Aspartic acid was a major component only in S. commercialis. Gene4
trends _fkom the NPS dutu
The three largest components obtained by principal components analysis accounted for 66.9”/(, of the total variance. Although most of the variability was due to differences between the species, small differences due to treatment effects were apparent for P. ebeninus and A. trapezitr (Fig. I). Back-correlation of the component loadings yielded three groups of NPS (Fig. 2). Each group consisted of NPS whose concentrations were higher for one species than for the other two. This indicated species-specific differences in the overall composition of the NPS pool between the three species. k’uriution in NPS-pool six and imfiridutrl components The size of the NPS pool responded significantly to stress only in P. ebeninus (Table 2). The NPS pool decreased from 154 pmol g- ’ wet tissue wt in control animals to an average of 119 pmol g- ’ in those kept at lO.l”,,,,. The relative concentrations of the individual NPS remained constant, except for arginine which increased significantly (P < 0.05). In the bivalve species, the NPS pool size ranged between 168 and
171 pmolg-’ in A. trapezia and between 145 and 152 pmol g -’ in S. commercialis. Concentrations of ammonia, arginine and serine increased, while aspartic acid decreased, in A. trapezia kept at low salinity. Glycine decreased in S. commercialis, while the relative amounts of alanine, ammonia, arginine, glutamic acid, histidine and lysine increased. Of the nine NPS whose relative concentrations changed significantly in at least one species (Table 2), some showed similar although nonsignificant trends in the other species. When significances for a particular NPS for the three species were combined (final column, Table 2), levels of alanine, ammonia, arginine and glutamic acid were generally higher in low-salinity animals, while levels of aspartic acid and glycine were generally reduced. Vuriutions in tuurine:glycine charge
and denylate
energy
The ratio of taurine to glycine increased to more than 3.0 only in S. commercidis at low salinity (from 2.25 to 4.50). No significant increases in this ratio occurred in the other two species. The reductions in adenylate energy charge, which occurred in the muscle tissue of all three species (Rainer et al., 1979) are shown in Table 2. DISCUSSION
The composition of the NPS pools in P. ebeninus, A. trapezia and S. commercialis was similar to that
20
Saccostrea commercialis
,‘---;,NPs_l \
,’
\
I’
I
,I
/I
//
I’
/
I
/’ /I
Anadara trapezia
, Is0
LYS
I
/
I
1 \
.Hi!s
‘$1~ ‘_ __________---
_/’
-1.0
-
Fig. 2. Ninhydrin-positive substances: back-correlations between NPS values and component scat-es for the three principal components of Fig. I. Dotted lines enclose correlations for NTS’s having their greatest concentration
found
in the species indicated.
in many estuarine and marine molluscs, with cies, such as the clam Mycl urmclritr (Virkar & Webb. 1970) and the oysters Cru.s.so.strecl Grginicu (Lynch & and glycine as major components (e.g. EmerWood, 1966) and Ostreu etldis (Schoffeniels & Gilles, son. 1969). Up to three additional NPS were major components of the NPS pool in muscle tissue. The 1972), where the NPS pool changed in size within 48 hr of changes in salinity. The absence of response NPS pool varied with species. Thus, alanine and glutamic acid were major components in P. ehcr~inu.s, in A. trupeziu and S. commerciulis suggests that intracellular osmotic regulation by the NPS may not bc while /I-alanine and proline were significant in the NPS pools of A. trupe-_irr and S. cotnmrrcirtlis. Asparimportant in these species at least for the first 48 hr tic acid was a major component only in S. CO~H~?ICY-of reduced salinity. Rather, osmotic change is temporarily resisted by closure of the shell valves during low ciulis. Although these differences in major comsalinity, as occurs in some species of bivalve (Robertponents seemed species-specific. the major NPS in other gastropods and bivalves show no such trends, son, 1964). Increases in arginine and ammonia suggest that even between similar tissues (Emerson, 1969; Du Paul reduced salinity conditions. by causing the molluscs & Webb, 1970: Bedford, 1971). The apparent lack of similarity among related species and similar tissues to withdraw from the stressful environment, were may be due to the variety of methods which have interfering with nitrogenous excretion. Several molbeen used for extraction and analysis of NPS (Bayne luscan species show an increase in ammonia in conditions of reduced salinity (Emerson, 1969). The inrt ul.. 1976: Baginski & Pierce, 1977). This variation remains to be investigated by comparing the available crease in arginine (a nitrogen-bearing precursor to procedures with a variety of species and tissues. urea) is consistent with a detoxification process under Many marine and estuarine gastropods and conditions which prevented normal excretion of ambivalves respond to changes in external salinity at the monia. intracellular level by altering the size of their NPS Anaerobic metabolism in many invertebrate species pool (Lange. 1963: Emerson, 1969: Pierce & Greenis characterized by higher levels of alanine and glutamic acid and lower levels of aspartic acid and glycine berg, 1973). In this study, only P. eheninus showed a (Stokes & Awapara. 1968; De Zwaan & Wijsman. significant reduction in the NPS pool after transfer to 1976; Fellbeck, 1980). Similar trends in the FAA of low salinity. The lack of response in A. trupeziu and S. the three species studied here indicated that the concommerciulis contrasts with findings for similar spetaurine
Moliuscan free amino acids: reduced salinity
21
Table 2. Relative concentrations (%) of NPS in muscle tissue of P. ebeninus, A. trapezia and S. commercialis from field and low salinity (lO.l%,) conditions Pyrazus eheninus
Anadara trupezia
Saccostraa
NPS
Field
Low sal.
Field
Low sal.
Field
Alanine Ammonia Arginine Aspartic acid Glutamic acid Glycine Histidine Lysine Serine
22.20 0.47 5.10 5.32 8.06 19.36 0.53 0.33 0.79
24.16 0.69 6.57* 4.29 9.22 17.97 0.44 0.36 0.74
4.13 0.34 2.17 5.46 4.39 34.37 0.64 0.43 0.35
5.01 0.53* 3.1_5* 4.56 4.36 33.30 0.66 0.37 0.73**
5.09 0.37 0.44 9.65 3.13 14.47 0.13 0.12 0.27
Total NPS (pm01 g- ’ wet wt)
153.8
118.2*** 168.1
144.9
168.0
commercialis
Low sal. 7.&5** 0.51 0.70 7.47 4.00** 8.28*** 0.25* 0.35** 0.29
Direction of change
-22
Increase Increase Increase Decrease Increase Decrease
In p
19.2** 1x.9** 22.6*** 15.8* 16.4* 14.4* 7.1 8.3 9.9
148.0
Tau : Gly
1.32
1.56
0.82
0.88
2.25
4.50
Energy charge
0.87
0.57***
0.83
0.70*
0.66
0.53*
NPS selected are those showing significant treatment effects in individual species or, after pooling, significances for all three species. Total NPS, mean energy charge and taurine:glycine ratio data are included for comparison. Taurine:glycine ratios were calculated for low salinity conditions as the mean of the 24 and 48 hr values. Significance levels are indicated by: *O.Ol < P < 0.05; **O.OOl< P < 0.01: ***p < 0.001.
ditions of reduced salinity had promoted anaerobic metabolism. The high concentration of alanine in P. eheninus relative to the other two species suggests that this gastropod may be a facultative anaerobe, relying on anaerobic metabolism even in the presence of oxygen (De Zwaan & Wijsman, 1976). Glycine was particularly reduced in S. ~~~l~~~r~~~l~js and may account for the higher glutamic acid level. The composition of the NPS pool of several marine invertebrates has been shown to vary with environmental harshness (Schafer, 1961, 1963, 1968). In this study, all the NPS present in control animals were also found in animals in the low salinity. The responses described by Schafer perhaps occur only when the duration of exposure to harsh conditions exceeds the 48 hr reported here. A marked decrease in glycine concentration, with a concomitant increase in the ratio of taurine to glycine, has been associated with stress conditions in a variety of molluscan species. Anndtlru trupeziu. as the species most stressed by the reduced salinity in this experiment (Rainer et III., 1979), was expected to show the greatest change in the ratio of taurine to glycine but did not. In contrast, S. commercialis, which tolerates conditions of low salinity for long periods (Rainer, unpublished data), showed a change in the ratio. P_vazus ebeni~s, which tolerates low salinities for at least 5 wk (Ivanovici, 198Ob), also showed no change in taurine to glycine ratio. These data. therefore, do not support the use of taurine to glycine ratios as a general indicator of metabolic change in molluscs as a group. The response did not occur in the most sensitive species and did not correlate with environmental harshness. The data also suggest that a response in the taurine to glycine ratio may be species-specific, as it occurred only in the oyster. The adenylate energy charge was the only measure which responded consistently in all three species under conditions associated with reduced salinity in
this experiment (Rainer rr tri., 1979). The changes in the NPS components indicated that metabolism was altered towards anaerobiosis in all three species, especially the bivalves. Although the response of the energy charge was consistent for the three species (i.e. a reduction below control levels), the possible contribution of anaerobic metabolism to the reduction of energy charge (as has been shown in Myrifus edulis by Wijsman, 1976), especially in the bivalves, prevents a final evaluation of energy charge.
The measured responses of the NPS pool (size and composition), taurine to glycine ratio and adenylate energy charge indicate that changes in the metabolism of P. ebeninus, A. trupeziu and S. commercialis occurred within 48 hr of reduction in salinity. The changes in composition of individual NPS indicated increased reliance on anaerobic metabolism in the molluscs under reduced salinity. The ratio of taurine to glycine did not increase consistently with increasing environmental harshness for the species in this experiment, and its use as an indicator of stressful conditions, at least for molluscs. is questioned. WVhile further studies are required to separate the response of AEC to anaerobic conditions and to low salinity, this method was the only one to show a consistent change and thus warrants further studies to evaluate its potential. Acknowlrdpvnenrs-We thank Mr Higginbottom and Dr Thomas of The University of Sydney for assistance in amino acid analysis. REFERENCES
(1971) Instruction hook j)r acid anal.vser mode/ JLC-hAH.
ANON
Laboratory. Tokyo, Japan.
a fu//y automatic
amino
Japan Electron Optics
ABAU J. L. & REISH D. J. (1972) The effect of lowered dissolved oxygen concentrations and salinity on the free amino acid pool of the polychaetous annelid Neunthes urenucrodmrartr. Bull. sot. Ctrl. AcUd. sci. 71. 32-39. ATKINSON D. E. (1971 I Adebine nucleotides as stoichlometric coupling agents in metabolism and as regulatoq modifier>: the adenylate energy charge. In Mcf&oli<~ Ptrrkwrrrs (Edited by VOC,I I H. J.). Vol V. 3rd Edn. pp. I ?I. Academic Press. Ncu York. BAC;IUSI\‘I R. M. & PIIR( I S K. JK (1977) The tImc course of Intracellular free amino acid accumulation in tissues of Mot&>/us t/w~i.\.su.\ during high salinity adaptation. Camp. Biochcm. Ph,Gi. 57A, 407~ 412. BAYER B. L. (1973) PhysIological changer in :!Jj,ri/u\ 41tli.t L. induced by temperature and nutritive stress. J. mcrr. hid. .4.w. U.K. 53, 39-58. BAYNI: B. L. (1975) .4spects of physiologIcal condition In Lf\~filu.t dulr L.. with special reference to the efrects of oxygen tension and salinlt). Prcw iir/~ E:‘rrr. ,Mdr. Bid syup. 21 3 73x. BAYU~ B. L.. LI~INGST~N~ D. R.. MOORE M. N. & WIDDOWS J. (1976) 4 cytochemical and biochemical index of stress in Mytilus et/u/is L. Mar. Pollut. Bull. 7, 221 124. BEI>FORI) J. J. (1971) Osmoregulation In Mc/trwp.sis triftrsc’itrtcr Grar 1843 -111. The intracellular nitrogenous compounds. Camp Biodwm. Phnrol. 40A, 899 910. Db Z~AAU A. & WIIS~IAN T C. M. (1976) Revieu. anacrobit metabolism in bivalvia (Mollusca): characteristics of anaerobic metabolism. Corny. Bioc~hum. Ph~siol. 54B, 313 314. Dr PAIIL W. D. & WEHI( K. L. (1970) The eR’ect of temperature on salinity-induced changes in the free amino acid pool of .%lw ~rrentrri~r. Camp. Biodwm. Ph~~.siol. 32, 785 X01. E’rlt.KSON D. N. (1969) Influence of salinity on ammonia sxcretion rates and tissue constituents of euryhaline In\crtcbratcs. C‘ornp, Biodwm. P/I~..\;~I/. 29, I I 15 I 133. FLLD~(.I( H. (1980) Investigations on the role of the amIno acids in anaerobic metabolism of the lugworm .Arcwic~olo mtlrlw L. .1. wnIp. PhJ,.tiol. 137. I83 192. FLORKIU M. (1966) Nitrogen metabolism. In P/~~~.~io/o~q~ o/ Moil~rwo (Edited by WILBI.K K. M. & Yowr C. M.). Vol. 1. pp. 309-352. Academic Press. New York HAI)DEN N.. Ba’himN F.. M.KDONAI.U F.. MLIUK M.. STI:V~NWN R.. GFR~ D.. ZAUARO\I F. & MAJOKS R. (I971 ) Basic, Liyuirl CI~~or,~~rro(/,.trp/~~,. pp. X I to 8 70. Variun Aerograph. (1.S.A. HARMAU H. H. (1968) Mo[/crrI F‘twror 4rrtrl~~ti.t. C’ni\crsItl of Chicago Press. Chicago. MI. IVAUOVIU A. M. (1980a) Adenylate energy charge: an evaluation of applicability to assessment of pollution effects and directions for future research. Rupp. P-r. R&n. Cons. int. Euplor. Mrr. 179, 23-28. IVANWICI A. M. (1980b) The adenylate energy charge in the estuarine mollusc. Pwuxs cheninus. Laboratory studies of responses to salinity and temperature. Conlp. Biochem. Phy.dl. 66A, 4% 55. I~ANOVIU A. M. (1981) A method for extraction and assay of adenine nucleotldes in molluscan trssue. C.S.I.R.O. .Au.d Dir,. Fish. O~~~umxqr. Rep. I IX.
Phwui.Vol70A.pp I7m 22.1981 Britam
0300-9629l81/090017-06802.00/O Copyright 0 1981 Pergamon Press Ltd
All rights reserved
FREE AMINO ACIDS IN THREE SPECIES OF MOLLUSC: RESPONSES TO FACTORS ASSOCIATED WITH REDUCED SALINITY M. IVANOVICI*, SEBASTIAN F. RAINER?~ and
ANGELA *Biochemistry tCSIR0
Division
V. A. WADLEY~
Department, of Fisheries
John Curtin School of Medical Research, P.O. Box 334. Canberra City, A.C.T. 2601 and and Oceanography, P.O. Box 21. Cronulla. N.S.W. 2230. Australia (Rrcrirrtl
7 Jam~ur~ 1981)
Concentrations of ninhydrin-positive substances (NPS), including free amino acids, in muscle tissue of estuarine molluscs (the oyster Saccostrea commercialis, the whelk Pyrazus ebeninus and of 35”“” salinity. the clam Anadara trapezia), were 145-168 pmol g- ’ wet weight under field conditions 2. Total NPS concentrations decreased with reduced salinity (lo”,,,,) only in P. ebeninus. 3. Concentrations of alanine, arginine and glutamic acid increased, while those of aspartic acid and glycine decreased, in molluscs kept at lo”,,,,, suggesting a shift to anaerobic metabolism. 4. Changes in the ratio of taurine to glycine occurred only in S. commerciulis.
Abstract-l.
INTRODUCTION
The concentration and distribution of ninhydrinpositive substances (NPS), including free amino acids (FAA), respond in many estuarine and marine invertebrates to changes in environmental conditions. These include salinity (Schoffeniels, 1976: Florkin, 1966; Bayne. 1975). temperature and nutritional levels (Bayne. 1973). oxygen availability (Abati & Reisch, 1972; Bayne, 1975). disease (Watts, 1971) and environmental stresses associated with pollution (Schafer. 1968; Jeffries, 1972). The increase of the ratio of taurine to glycine to more than 3.0 under stressful conditions in some species of bivalve indicates its possible use to identify a stress condition in marine invertebrates (Bayne et al., 1976; Roesijadi & Anderson, 1979). Its value as a general indicator of stress is questionable. however. because the response is not found in many invertebrate species (Webb et rrl., 1972: Baginski & Pierce, 1977). This study examined the NPS concentration, the taurine to glycine ratios and their responses to an environmental stress of reduced salinity in three estuarine species not previously reported. These were the intertidal gastropod P~tr:~rs rhenin~ (Bruguiire, 1792), the intertidal oyster Strccostrrrr cwmn~ercitrlis (Iredale & Roughley, 1933) and the subtidal clam Anudara trapezia (Deshayes, 1840). The observed responses were compared with that of adenylate energy charge (Atkinson, 1971). which was measured simultaneously (Rainer et rrl.. 1979). and whose application to stress assessment is described elsewhere (ivanovici, 1980a). This comparison was done to determine which method was most appropriate in the evaluation of stress in these species. ,MATERIALS AND Anirntrls
md
c~upwimmrtd
METHODS
c~oidirions
to S. F. Rainer. Laboratories Reprint
The pattern in untransformed data (pmol g-’ wet wt) obtained from 24 samples was determined by principal components analysis. The analysis of principal components was followed by back-correlation of component loadings (Harman. 1968) with each NPS. This was done to identify which NPS were important in grouping the samples. The significance of differences in the mean values of each NPS measured for every species in control and stressed conditions was determined by a I-test. Because of the small number of animals collected from low salinity conditions. data obtained from 24 and 48 hr were pooled. The final statistical rest was for the overall significance of differences between control and experimental animals. regardless of species. using -2C In p (Sokal & Rohlf, 1969). RESULTS Composition Twenty-one
Experimental conditions were constant reduced salinity (lO.l”,,,,) and constant temperature (21’C). The control was $ Reprint requests C.S.I.R.O. Marine
a collection of molluscs from a typical field habitat (Rainer et al., 1979). Measured salinity was 35”,,,, at low tide at the collection times throughout the experiment, while temperature and food availability varied tidally and diurnally. Two molluscs of each species were removed from experimental conditions or from the field, 24 and 48 hr after beginning the experiment. Muscle tissue was removed from the animals following the optimal procedures determined by Wadley et al. (1980). and supernatants extracted (Ivanovici, 1981). Supernatants were analysed with a JEOL amino acid autoanalyser (Model 6AH). after dilution (1: IO) with citrate buffer, pH 2.2 (Anon. 1971). Concentrations were determined by the peak height method of Hadden rt trl. (1971). Absorbances were read at 570 nm for all NPS except proline (440 nm). Preliminary investigation of analytical precision indicated that a single analysis per animal was adequate. with a between-sample coefficient of variation of 5”/:, or less. Internal standards added to supernatants from each species indicated no need for correction factors.
No. 1187.
of‘ the NPS pool NPS
were detected (Table 1) in the These were: 17 FAA, taurine, ammonia and two unidentified compounds (NPS-1, NPS-2). Of these, no more than 5 NPS in any species accounted for more than approx 80”,, of the NPS
muscle tissue examined.
& 2.8 i_ 0.08 f 0.73 +_ 1.3 * 0.15 + I.5 f 1.7 + 0.16 * 0.06 * 0.15 f 0.05 + 0.13 & 0.04 & 0.53 + 0.25 + 4.4 + 0.54 & 0.10 i: 0.17
and adenylate
_+ 6.1 * 0.57 + 1.1 + I.5 * 0.03 + 0.19 * 1.4 * 0.35 * 0.10 + 0.18 + 0.16 * 0.03 + 0.01 * 1.4 f 0.26 * 1.3 i 0.69 + 0.03 * 0.02
0.55 * 0.05
1.45
0.9 i 0.00 126.9 i 11.9
29.4 1.1 8.4 6.0 0.1 10.2 24.1 0.6 0.3 0.6 0.5 0.4 0.1 5.4 0.9 34.5 1.9 0.3 0.7
+ _t f f * & * f & + * 1 * & f * * + i
6.1 0.07 0.37 0.39 0.03 2.1 1.9 0.22 0.00 0.03 0.02 0.02 0.01 0.37 0.10 3.1 0.42 0.05 0.03
energy
samples
0.8 i 0.22 109.4 f 13.7 1.68 0.60 + 0.08
27.7 0.6 7.2 4.1 0.1 I I.6 18.4 0.4 0.2 0.3 0.4 0.1 0.1 3.6 0.X 31.0 I.5 0.2 0.5
rhenimi.s 48 hr 24 hr -_.___-
ratios
I SF. with Y = 4 for field (control)
I.0 * 0.15 153.8 * 9.0 1.32 0.87 * 0.01
34.1 0.7 7.8 8.2 0.2 12.4 29.8 0.8 0.3 0.6 OS 0.4 0.1 6.2 1.2 39.4 2.3 0.3 0.7
Field
Pytrm
of NPS. taurine:glycine
Values are giLen as mean t
Alaninc Ammonia Arginine Aspartic acid fi-alanine Glutamic acid Glycine Histidine Isoleucine Leucme Lysine .~ethionine Phenylalanine Proline Serine Taurrne Threonine Tyrosine Valine NPS No. I NPS No. 3 Total NPS Tau:Gly Energy charge
Ninhydrin-posttivc substance
Table I. Concentrations
Field
167.9 i 17.1 1.00 0.69 & 0.03
&- 2.7 k 0.25 i 1.25 + 1.9 + 3.4 & 2.1 & 7.9 i: 0.03 i_ 0.00 i 0.02 +_ 0.06 & 0.06 + 0.03 & O.li + 0.28 + 3.5 f 0.18 * 0.04 + 0.03 i_ 2.X * 0.10 * 1.2 2 1.7 & 0.92 + 0.38 + 0.34 + 0.10 2 0.07 + 0.10 + 0.02 * 0.01 + 0.08 i_ 2.1 kO.23 + 4.9 + 0.13 + 0.06 rf- 0.05
172.2 I_ 7.9 0.76 0.71 + 0.08
6,h 1.3 6.1 7.1 26.2 6.Y 59.3 1.0 0.3 0.3 0.5 0.1 0.2 7.2 1.1 45.1 1.1 0.6 0.4
48 hr
’ wet wt)
( absent).
(pmoi g
and .I’ = 2 for lO.l”,,,, samples.
16X.1 i 5.6 0.82 0.83 i_ 0.05
10.4 1.0 4.6 8.4 14.0 1.9 53.5 1.3 0.3 0.3 0.8 0.2 0.2 7.0 1.4 53.6 I.6 0.5 0.4
Concentration
in muscle tissue of P. rheninrr.\. .4. rrupku conditions
6.9 + 0.49 0.6 + 0.07 3.6 I 0.35 9.2 * 0.93 18.3 i 3.1 7.4 + 0.23 57.8 _t 4.8 1.i * 0.14 0.3 * 0.03 0.3 * 0.04 0.7 + 0.09 0.1 f 0.01 0.2 + 0.06 11.6 i 3.1 0.6 k 0.14 47.2 * 3.0 1.4 * 0.22 0.5 * 0.07 0.3 & 0.03 trace
---
charge
& 1.1 + 0.10 & 0.17 + 1.2 If: 6.9 2 0.22 + 1.5 i_ 0.06 2 0.02 + 0.05 jl 0.08 i: 0.04 i: 0.02 i 3.5 * 0.13 $ 2.6 i: 0.39 F 0.13 I: 0.07 k 0.29 144.9 + 9.0 2.25 0.66 i: 0.04
7.4 0.5 0.6 14.0 32.6 4.5 21.0 0.2 0.2 0.2 0.2 0.1 0.1 13.5 0.4 47.3 0.9 0.3 0.2 0.8
Field
commrrc~idis
144.0 rf: 10.4 4.78 0.56 + 0.09
+ 0.38 k 0.02 + 0.10 F 6.7 + 3.0 + 0.38 & 1.7 + 0.01 & 0.02 i: 0.03 & 0.19 k 0.12 + 0.04 + 0.40 + 0.19 i_ 2.4 ) 0.06 k 0.06 +_ 0.06 F 0.07
24 hr 11.8 0.6 0.8 10.8 30.6 5.4 11.1 0.3 0.2 0.3 0.5 0.2 0.1 17.0 0.5 51.9 0.7 0.3 0.3 0.7
Sowostruci
(10. I”,,,,
+ 1.4 & 0.33 + 0.44 5 0.54 +_ 3.3 t 0.67 + 2.0 & 0.17 I 0.04 i. 0.01 & 0.06 f 0.06 * 0.05 F 0.48 * 0.13 & 12.6 * 0.01 * 0.02 2 0.11 1 0.24 151.9 ?_ II.0 4.32 0.51 * 002
10.8 0.9 1.3 II.3 36.6 6.4 13.4 0.4 0.2 0.2 0.6 0.1 0.2 9.9 0.4 57.9 0.3 0.4 0.3 0.2
48 hr
and S. contmerc,ict/i.\ from field and low salinity
c 2 L 2 f: c; =y
3 f ;: : E
Molluscan
Saccostrea commercialis
free amino
acids: reduced
salinity
19
I’
I
(32.9%)
Pyrazus ebeninus
Anadara trapezia
‘, \,
Fig. I. P~rcwa chwinus. Antrdurtr trupezitr and Saccostrecr commertiulis: component loadings for 8 samples from animals of each species. for the three largest principal components. Different symbols are used for each species; the proportion of the variance extracted is given for each component. For each species. dotted lines enclose loadings for the control animals (closed symbols) and the experimental animals (open symbols) separately.
pool. Taurine and glycine were major NPS in all three species. Concentrations of alanine and glutamic acid were higher in P. ehvninlrs than either of the bivalve species. while /I-alanine and proline were higher in both bivalve species. Aspartic acid was a major component only in S. commercialis. Gene4
trends _fkom the NPS dutu
The three largest components obtained by principal components analysis accounted for 66.9”/(, of the total variance. Although most of the variability was due to differences between the species, small differences due to treatment effects were apparent for P. ebeninus and A. trapezitr (Fig. I). Back-correlation of the component loadings yielded three groups of NPS (Fig. 2). Each group consisted of NPS whose concentrations were higher for one species than for the other two. This indicated species-specific differences in the overall composition of the NPS pool between the three species. k’uriution in NPS-pool six and imfiridutrl components The size of the NPS pool responded significantly to stress only in P. ebeninus (Table 2). The NPS pool decreased from 154 pmol g- ’ wet tissue wt in control animals to an average of 119 pmol g- ’ in those kept at lO.l”,,,,. The relative concentrations of the individual NPS remained constant, except for arginine which increased significantly (P < 0.05). In the bivalve species, the NPS pool size ranged between 168 and
171 pmolg-’ in A. trapezia and between 145 and 152 pmol g -’ in S. commercialis. Concentrations of ammonia, arginine and serine increased, while aspartic acid decreased, in A. trapezia kept at low salinity. Glycine decreased in S. commercialis, while the relative amounts of alanine, ammonia, arginine, glutamic acid, histidine and lysine increased. Of the nine NPS whose relative concentrations changed significantly in at least one species (Table 2), some showed similar although nonsignificant trends in the other species. When significances for a particular NPS for the three species were combined (final column, Table 2), levels of alanine, ammonia, arginine and glutamic acid were generally higher in low-salinity animals, while levels of aspartic acid and glycine were generally reduced. Vuriutions in tuurine:glycine charge
and denylate
energy
The ratio of taurine to glycine increased to more than 3.0 only in S. commercidis at low salinity (from 2.25 to 4.50). No significant increases in this ratio occurred in the other two species. The reductions in adenylate energy charge, which occurred in the muscle tissue of all three species (Rainer et al., 1979) are shown in Table 2. DISCUSSION
The composition of the NPS pools in P. ebeninus, A. trapezia and S. commercialis was similar to that
20
Saccostrea commercialis
,‘---;,NPs_l \
,’
\
I’
I
,I
/I
//
I’
/
I
/’ /I
Anadara trapezia
, Is0
LYS
I
/
I
1 \
.Hi!s
‘$1~ ‘_ __________---
_/’
-1.0
-
Fig. 2. Ninhydrin-positive substances: back-correlations between NPS values and component scat-es for the three principal components of Fig. I. Dotted lines enclose correlations for NTS’s having their greatest concentration
found
in the species indicated.
in many estuarine and marine molluscs, with cies, such as the clam Mycl urmclritr (Virkar & Webb. 1970) and the oysters Cru.s.so.strecl Grginicu (Lynch & and glycine as major components (e.g. EmerWood, 1966) and Ostreu etldis (Schoffeniels & Gilles, son. 1969). Up to three additional NPS were major components of the NPS pool in muscle tissue. The 1972), where the NPS pool changed in size within 48 hr of changes in salinity. The absence of response NPS pool varied with species. Thus, alanine and glutamic acid were major components in P. ehcr~inu.s, in A. trupeziu and S. commerciulis suggests that intracellular osmotic regulation by the NPS may not bc while /I-alanine and proline were significant in the NPS pools of A. trupe-_irr and S. cotnmrrcirtlis. Asparimportant in these species at least for the first 48 hr tic acid was a major component only in S. CO~H~?ICY-of reduced salinity. Rather, osmotic change is temporarily resisted by closure of the shell valves during low ciulis. Although these differences in major comsalinity, as occurs in some species of bivalve (Robertponents seemed species-specific. the major NPS in other gastropods and bivalves show no such trends, son, 1964). Increases in arginine and ammonia suggest that even between similar tissues (Emerson, 1969; Du Paul reduced salinity conditions. by causing the molluscs & Webb, 1970: Bedford, 1971). The apparent lack of similarity among related species and similar tissues to withdraw from the stressful environment, were may be due to the variety of methods which have interfering with nitrogenous excretion. Several molbeen used for extraction and analysis of NPS (Bayne luscan species show an increase in ammonia in conditions of reduced salinity (Emerson, 1969). The inrt ul.. 1976: Baginski & Pierce, 1977). This variation remains to be investigated by comparing the available crease in arginine (a nitrogen-bearing precursor to procedures with a variety of species and tissues. urea) is consistent with a detoxification process under Many marine and estuarine gastropods and conditions which prevented normal excretion of ambivalves respond to changes in external salinity at the monia. intracellular level by altering the size of their NPS Anaerobic metabolism in many invertebrate species pool (Lange. 1963: Emerson, 1969: Pierce & Greenis characterized by higher levels of alanine and glutamic acid and lower levels of aspartic acid and glycine berg, 1973). In this study, only P. eheninus showed a (Stokes & Awapara. 1968; De Zwaan & Wijsman. significant reduction in the NPS pool after transfer to 1976; Fellbeck, 1980). Similar trends in the FAA of low salinity. The lack of response in A. trupeziu and S. the three species studied here indicated that the concommerciulis contrasts with findings for similar spetaurine
Moliuscan free amino acids: reduced salinity
21
Table 2. Relative concentrations (%) of NPS in muscle tissue of P. ebeninus, A. trapezia and S. commercialis from field and low salinity (lO.l%,) conditions Pyrazus eheninus
Anadara trupezia
Saccostraa
NPS
Field
Low sal.
Field
Low sal.
Field
Alanine Ammonia Arginine Aspartic acid Glutamic acid Glycine Histidine Lysine Serine
22.20 0.47 5.10 5.32 8.06 19.36 0.53 0.33 0.79
24.16 0.69 6.57* 4.29 9.22 17.97 0.44 0.36 0.74
4.13 0.34 2.17 5.46 4.39 34.37 0.64 0.43 0.35
5.01 0.53* 3.1_5* 4.56 4.36 33.30 0.66 0.37 0.73**
5.09 0.37 0.44 9.65 3.13 14.47 0.13 0.12 0.27
Total NPS (pm01 g- ’ wet wt)
153.8
118.2*** 168.1
144.9
168.0
commercialis
Low sal. 7.&5** 0.51 0.70 7.47 4.00** 8.28*** 0.25* 0.35** 0.29
Direction of change
-22
Increase Increase Increase Decrease Increase Decrease
In p
19.2** 1x.9** 22.6*** 15.8* 16.4* 14.4* 7.1 8.3 9.9
148.0
Tau : Gly
1.32
1.56
0.82
0.88
2.25
4.50
Energy charge
0.87
0.57***
0.83
0.70*
0.66
0.53*
NPS selected are those showing significant treatment effects in individual species or, after pooling, significances for all three species. Total NPS, mean energy charge and taurine:glycine ratio data are included for comparison. Taurine:glycine ratios were calculated for low salinity conditions as the mean of the 24 and 48 hr values. Significance levels are indicated by: *O.Ol < P < 0.05; **O.OOl< P < 0.01: ***p < 0.001.
ditions of reduced salinity had promoted anaerobic metabolism. The high concentration of alanine in P. eheninus relative to the other two species suggests that this gastropod may be a facultative anaerobe, relying on anaerobic metabolism even in the presence of oxygen (De Zwaan & Wijsman, 1976). Glycine was particularly reduced in S. ~~~l~~~r~~~l~js and may account for the higher glutamic acid level. The composition of the NPS pool of several marine invertebrates has been shown to vary with environmental harshness (Schafer, 1961, 1963, 1968). In this study, all the NPS present in control animals were also found in animals in the low salinity. The responses described by Schafer perhaps occur only when the duration of exposure to harsh conditions exceeds the 48 hr reported here. A marked decrease in glycine concentration, with a concomitant increase in the ratio of taurine to glycine, has been associated with stress conditions in a variety of molluscan species. Anndtlru trupeziu. as the species most stressed by the reduced salinity in this experiment (Rainer et III., 1979), was expected to show the greatest change in the ratio of taurine to glycine but did not. In contrast, S. commercialis, which tolerates conditions of low salinity for long periods (Rainer, unpublished data), showed a change in the ratio. P_vazus ebeni~s, which tolerates low salinities for at least 5 wk (Ivanovici, 198Ob), also showed no change in taurine to glycine ratio. These data. therefore, do not support the use of taurine to glycine ratios as a general indicator of metabolic change in molluscs as a group. The response did not occur in the most sensitive species and did not correlate with environmental harshness. The data also suggest that a response in the taurine to glycine ratio may be species-specific, as it occurred only in the oyster. The adenylate energy charge was the only measure which responded consistently in all three species under conditions associated with reduced salinity in
this experiment (Rainer rr tri., 1979). The changes in the NPS components indicated that metabolism was altered towards anaerobiosis in all three species, especially the bivalves. Although the response of the energy charge was consistent for the three species (i.e. a reduction below control levels), the possible contribution of anaerobic metabolism to the reduction of energy charge (as has been shown in Myrifus edulis by Wijsman, 1976), especially in the bivalves, prevents a final evaluation of energy charge.
The measured responses of the NPS pool (size and composition), taurine to glycine ratio and adenylate energy charge indicate that changes in the metabolism of P. ebeninus, A. trupeziu and S. commercialis occurred within 48 hr of reduction in salinity. The changes in composition of individual NPS indicated increased reliance on anaerobic metabolism in the molluscs under reduced salinity. The ratio of taurine to glycine did not increase consistently with increasing environmental harshness for the species in this experiment, and its use as an indicator of stressful conditions, at least for molluscs. is questioned. WVhile further studies are required to separate the response of AEC to anaerobic conditions and to low salinity, this method was the only one to show a consistent change and thus warrants further studies to evaluate its potential. Acknowlrdpvnenrs-We thank Mr Higginbottom and Dr Thomas of The University of Sydney for assistance in amino acid analysis. REFERENCES
(1971) Instruction hook j)r acid anal.vser mode/ JLC-hAH.
ANON
Laboratory. Tokyo, Japan.
a fu//y automatic
amino
Japan Electron Optics
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