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Identification of phaeopigments in the digestive gland of Mytilus edulis L. by microspectrofluorimetry
J. exp. mar. Biol. Ecol., ! 980, Vol. 43, pp. 281--292 © Elsevier/North-Holland Biomedical Press
IDENTIFICATION OF PHAEOPIGMENTS IN THE DIGESTIVE GLAND OF M Y T I L U S EDULIS L. BY MICROSPECTROFLUORIMETRY I
S. R. GELDER and W. E. ROBINSON Marine Science Institute, Northeastern UniversiO,, Nahant, MA 01908. U.S.A. Abstract: Epi-illuminated fluorescence microscopy was used to observe chlorophyll degradation products localized in lipid spherules within the digestive gland of Mytilus edulis L. These fluorescing materials, known collectively as phaeopigments, then were spectrally analysed in situ with a microspectrofluorimeter. The results were compared with acetone extracts of the digestive gland and chlorophyll a standard solutions. Emission wavelength peaks were 671.2 nm for chlorophyll a in 85'~/,; acetone. 672.8 nm for the acidified chlorophyll a solution, 666.3 nm for digestive gland extracts in 859~, acetone. and 677.1 nm for phaeopigment in tissue sections. Acid factors of 1.0 for digestive gland extracts and tissue sections confirmed the red fluorescing material to be phaeopigment. Quantities up to 167 ng for an I I-#m diameter lipid spherule were encountered. Results were compared with calculated concen-' trations of phaeopigment determined spectrophotometrically.
INTRODUCTION
The spectral analysis of fluorescence emission has provided a simple and sensitive method for studying chlorophylls and their degradation products (vide Smith & Benitez, 1955; Udenfriend, 1962, 1969; Mackas & Bohrer, 1976). Such methods have been used to identify chlorophyll derivatives in sediments (Gorham, 1960) and estimate algal productivity in open waters and laboratory cultures (Yentsch & Menzei, 1963; Holm-Hansen et al., 1965; Strickland & Parsons, 1972; Shuman & Lorenzen, 1975). These pigments have been demonstrated in situ with epi-illuminated fluorescent microscopy by Jones (1974) to estimate epilithic diatom production and by Wilde & Fliermans (1979) to distinguish between several types of unicellular algae. The ability to use chlorophylls and their degradation products as convenient natural markers enabled Williamson (1977) to study an algal-invertebrate symbiosis by differentiating zoochlorella from isomorphic bodies in the sponge, Spongilla lacustris. Similarly, the fate of algae has been traced by observing its fluorescing pigments during ingestion and digestion in veliger larvae of the oyster, Crassostrea virgblica (Babinchak & Ukeles, 1979). Smith & Benitez (1955) have shown that the degradation of chlorophylls is accompanied by a shift of a few nanomcters to a higher fluorescent emission wavelength and a change in the absorption spectra. Similarly, Currie (1962) compared i Contribution number 90 from Marine Science Institute, Northeastern University, Nahant, Massachusetts, U.S.A. 281
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S.R. GELDER AND W. E. ROBINSON
the emission peaks from acetone extracts of zooplankton faeces with extracts of the diatom, Skeletonema sp., and interpreted the wavelength shift as indicative of a rapid breakdown of the ingested chlorophyll a. Ansell (1974a, b) studied the absorption spectra of acetone extracted pigments from the "mantle" tissue (defined as the remaining viscera after removal of gonad and adductor muscle) of several species of bivalves. From this spectral data he construed the pigments to be similar to phaeophytins. Algal pigments are retained primarily within the digestive gland of bivalves following digestion and absorption of algal food (Morton, 1956; Ansell, 1974b). Seasonal fluctuations of phaeophytin levels in "mantle" extract of Abra alba and Chlamys septemradiata correspond to fluctuations in feeding activity (Ansell, 1974a, b). A similar correlation between carotenoid levels in whole animal homogenates and fluctuating densities of phytoplankton is reported for Mytilus edulis (Jensen & Sakshaug, 1970a, b). The relative fluorescence of acetone extracted pigments from Crassostrea gigas and Ostrea edulis is roughly proportional to the bivalves' nutritional state and can be expressed as a feeding index (Mann, 1977). Index values for individual bivalves are altered quickly by short-term bursts of feeding, which makes it difficult to establish the nutritional profile for an entire population. Use of the index is further complicated due to the inclusion of a residue of pigment which remains in the digestive gland of both these bivalves for up to three weeks after cessation of feeding. Chlorophylls and phaeopigments can be observed with greater intracellular precision and in smaller quantities by epi-illuminated fluorescent microscopy than by bright-field or phase-contrast illumination. This study was designed to demonstrate chlorophylls and their fluorescing degradation products in the digestive gland cells of M. e&dis by epi-illuminated fluorescent microscopy and provide a semiquantitative microspectrofluorimetric method for comparing the pigment complexes in situ with those extracted by acetone. MATERIAL AND METHODS
Specimens of Mytilus edulis L. were collected from the intertidal zone at East Point, Nahant, Massachusetts in June and July, 1979. The digestive gland was removed from recently collected specimens and cut into two pieces, one for histological sectioning and the other for pigment extraction. TISSUE PREPARATIONS
The larger piece of digestive gland was macerated in 10 ml of 85 °'j,, acetone (analytical grade) with approximately 1 mg of magnesium carbonate for 5 min, centrifuged for 10 min at 4750 rpm, and the supernatant decanted. The second piece of tissue was cooled rapidly with dry ice (solid carbon dioxide,
M YTIL US DIG ESTIVE (3 LAND PHA EOPIG M ENTS
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-78.5 °C) for 5 min, transferred to a microtome-cryostat chamber maintained at - 2 0 °C, affixed to a specimen stub with water, and left for 10 min. Serial, 10-~um thick sections were mounted directly onto cleaned microscope slides, immersed in Baker's 10~ calcium formalin for 5 min, and washed in three changes of distilled water. Slides then were stained either for lipid by the supersaturated isopropanol technique, using Oil Red 0 dye (Lillie & Fullmer, 1976) and mounted in Von Apathy gum syrup (Humason, 1979), or mounted directly for the subsequent examination of fluorescent pigments. Control slides involved the removal of lipid, chlorophyll, and chlorophyll degradation products from the sections by soaking them in either 85~'o acetone or 95~ ', ethanol for 10-30 min. Possible autofluorescent contamination of sections by the fixative or mountant were checked and proved unfounded. INSTRUMENTATION A N D CALIBRATION
Sections were examined under a Leitz Dialux 20 microscope by transmitted brightfield or phase-contrast illumination, or with epi-fluorescence illumination. The light source for the fluorescence methods consisted of an Osram mercury HBO 50-W arc lamp powered by the unstabilized 115-120 V supply. The light was passed through a Ploem illuminator 2.4 which contained filter cubes A and H2 for broad band ultraviolet and violet-blue light excitation, respectively. Conversion of the microscope into a microspectrofluorimeter involved the insertion of a Bausch and Lomb Czerney-Turner grating type excitation monochromator between the lamp and the Ploem illuminator for the selection of the 436-nm mercury line. The fluorescent emissions from the samples were spectrally analyzed in a darkened room using a x 40 Fluorotar objective and a Farrand Microscope Spectrum Analyzer (MSA). The MSA contained a Farrand, Czerney-Turner (205-780 nm) grating type monochromator, a range of slit and aperture widths, plus a R446 Hamamatsu photomultiplier tube. The emission trace was recorded on a chart recorder. Fluctuations in line voltage on the unstabilized mercury lamp were checked by monitoring the peak emission wavelength from uranyl glass (Schott, GG21) (PIoem, 1977). /o This showed that the maximum modulation caused by voltage variation was 0.5 ''j of the relative fluorescence during the longest (3 min) emission scan times. The organelles in the 10-/zm thick sections were critically focused, positioned with the 15-#m diameter measuring spot, and the resulting relative fluorescence spectrally analyzed by the MSF. The measuring spot diameter was chosen because it provided optimal operating conditions. The quantity of material excited, calculated as the volume of a cylinder [v = n(7.5/~m)-' (10 #m)], was 1.77 x 10-~,um 3. Solutions of pigments were placed in a cavity slide (2.5 mm deep, containing approximately 0.85 ml) and sealed with a coverslip. The x 40 objective was lowered until it rested on the coverslip so that the cone of excitation extended into the solution to a depth of 264/~m; the measuring spot remained at 15/~m in diameter so that the emission was analyzed from 4.66 x 104/~m-~of extract. To calibrate the wavelength scale of
284
S.R. GELDER AND W. E. ROBINSON
the MSF a chlorophyll a solution (Sigma C-5753) in 85~ acetone (analytical grade) was analyzed spectrally with the MSF and spectrofluorimeter. The uncorrected scans agreed. Quantitative calibration of the MSF was achieved by making serial dilutions of the chlorophyll a solutioI~, recording their relative fluorescence (Rf units) at the emission wavelength maximum, and then constructing a standard curve. Samples of acetone-extracted pigments from the digestive'gland were analyzed by the MSF and with a Bausch & Lomb Spectronic 700 spectrophotometer. MEASUREMENTS AND CALCULATIONS
After spectrally analyzing sections, acetone-extracted pigments of digestive gland, and chlorophyll a solutions, all of these preparations were acidified to convert any chlorophyll into the corresponding phaeopigments (Strickland & Parsons, 1972) and re-analyzed. From these data the standard dilution curves for chlorophyll a and phaeopigments were plotted, the acid factors (RfJRf,~jd) calculated (Yentsch & Menzel, 1963; Holm-Hansen et al.. 1965), and the concentration of phaeopigments in the digestive gland extracts and sections determined. A spectrophotometer was used to measure absorbance at 480, 510, 630, 647, 664, 665, and 750 nm in the nonacidified and at 665 and 750 nm in the acidified pigment extracts. Then these values were substituted in the equations of Jeffrey & Humphrey (1975) to calculate the amounts ofchlorophyll a, b and c. The equations of Richards and Lorenzen (in Strickland & Parsons, 1972) were used for the calculations of carotenoid and phaeopigment respectively. OBSERVATIONS
The histology of the digestive gland of M. edulis has been described by Owen (1972) and Langton (1975). The gland is a greenish-brown organ composed of a network of blind-ending tubules, ducts, and connective tissue which surround the stomach. Ciliated primary ducts, which arise from the stomach wall, branch into short, secondary ducts that terminate in blind-ending digestive tubules. These tubules are oval in transverse section1 with a unicellular epithelium composed largely of columnar digestive cells and conical basophil cells arranged along the folds or crypts. Under ultravi~)let or violet light excitation the digestive gland epithelium fluoresces red while the adjacent connective tissue and musculature autofluoresces a dull blue. Scattered throughout these tissues are pale yellow lipofuscin granules, 2-12 pm in diameter. The epithelium of the digestive tubules, and to a lesser extent the primary and secondary ducts, fluoresce a dull, uniform red while in the basal regions of the cells are bright orange-red fluorescing spherules. Some spherules also are found in the connective tissue adjacent to the tubules. The size of the spherules varies in the different regions: 2-11 /~m in the digestive tubule epithelium and neighboring con-
M Y T I L U S DIGESTIVE GLAND PHAEOPIGMENTS
285
nective tissue; 1-2 pm in the secondary; and 1--6/~m in the primary ducts. Spherules in comparable locations and of similar dimensions were demonstrated by the Oil Red 0 technique, indicating their lipid nature. Sections placed in 85% acetone or 95~ ethanol for 10 min showed no discernible red fluorescing pigment or lipid, but the yellow autofluorescing lipofuscin remained. MICROSPECTROFLUORIMETRY
A typical spectral analysis of the emission from a digestive cell containing red fluorescing pigment spherules and lipofuscin granules excited with violet light (436 nm) is presented in Fig. I. The lipofuscin emission is spread over a broad 40 677
3o
Rf units
546 20
10
O
•
500
II
600 nrN
ii
•
700
Fig. i. Spectral scan of the emission from an area of the digestive gland o| Mvtilus e~hdL~"which includes lipofuscin granules and phaeopigment-containing lipid spherules" microspectrofluorimeter excitation wavelength fixed at 436 nm.
wavelength band, peaking at 546 nm, followed by the red-fluorescing pigment peak at 677 nm. Scans specifically of pigment spherules (Fig. 2, trace a) confirmed that peak emission occurs at 677.1 nm (SD +2.5 nm; n = 12). Although no pigment fluorescence could be seen in the 10-min ethanol- or acetone-emersed control sections, a small amount of residual fluorescence could be detected in the cytoplasm by the MSF (Fig. 2, trace b). This was easily distinguished from background fluctuations when compared with the reference uranyl glass emission trace (Fig. 3). The emissions from acetone extracts of o;gments from the digestive glands were analyzed spectrally under the MSF (Fig. 2, trace c). The primary emission peak occurred at 666.3 nm (SD = + 2.8 nm; n = 12) with a shoulder evident from 690 to
286
S.R. G E L D E R A N D W. E. ROBINSON
720 nm. Aliquots of samples analyzed in a spectrofluorimeter (Fig. 4) confirmed the position of the primary peak at approximately 663 nm while the shoulder was resolved into two secondary peaks (709 and 725 nm). The shape of these traces and their wavelength characteristics indicate that the pigment is either chlorophyll a, a degradation product, or a combination of the two. ,C 40
30
20
Rf units IO
b
•
600
m..~_
~
~
'
'
__
- ~
;,so
nm Fig. 2. Traces of microspectrofluorimetric emission scans of areas from sections of M.vtih~s e&~lis digestive gland: a, before, and b, after a 10rain extraction in 85". acetone; a trace of the extracted pigment in a solution of 85"0 acetone (c); all measurements made with an excitation wavelength of 436 nm.
0
20
4b sees
go
8"0
Fig. 3. Portion of the emission trace from the uranyl glass calibration, excited at 436 nm, and recorded at 540 nm under the microspectrotluorimeter.
Microspectrofluorimetric analysis of the chlorophyll a standard solution showed an emission wavelength peak at 671.2 nm (SD= +2.3 nm; n = 11). Following acid hydrolysis of these solutions to the phaeopigment derivative, a slight increase in peak emission wavelength was found (672.8 nm; SD= +_2.6 nm; n = 10), but this shift was not statistically significant (paired t test; t = 1.528; n = 10). Similarly, the differences in wavelength emission peaks following acidification of both acetone extracts of the digestive gland (t test; t = 1.7340; d.f. = 18) and tissue sections (t test; t = 0.4662; d.f. = 20) were not statistically different from their preacidification values. A marked decrease in the relative fluorescence of the chlorophyll a standard solutions was recorded following acid hydrolysis (Fig. 5). The mean acid factor for
M YTILUS DIGESTIVE GLAND PHAEOPIGMENTS
287
,°] 70
Rf units
50
30
10 0
I
600
ii
iii
i i
7~0 nm
Fig. 4. Spectrofluorimetric emission trace of a digestive gland extract in 8';"_j. acetone excited at 436 nm' primary peak at 662.7 nm and two secon:dary peaks at 709 and 725 nm.
,/
8.0'5
Rf units
2
0
0.4
0.8
1.2
pg/ml Fig. 5. Standard curve of chlorophyll a in 85"~, acetone: before (O), ~g/ml - 0 . 0 2 Rfu + 0.04, r - 0.99, n = I I, and after acidification (A), pg/ml = 0.20 Rfu + 0.005, r - 0.98, n - 10.
288
S.R. G E L D E R AND W. E. ROBINSON
these solutions was 11.5 (SD-'- +0.72; i~ = 5). In contrast, acid factors of 1.0 were calculated for the tissue sections (~'Rf0/~Rfac~a) and digestive gland pigment extracts (SD -- + 0.025; n - 4). Hence, the chlorophyll derivatives within the digestive gland were shown to be predominantly phaeopigment. QUANTITATIVE DETERMINATION OF PHAEOPIGMENT
The amount of fluorescing pigment in the organelles of the tissue sections and in the digestive gland extracts can be determined from their relative fluorescence, despite the dissimilar composition of the samples and the different volumes measured. The cylinder measured differs it~ depth from 10 ,urn in a tissue section to 264 ,um in the fluid preparations. As the measuring spot diameter remains constant, the ratio of the volumes and, in turn, the relative fluorescence, is proportional (1:26.4). Therefore, the concentrations of pigment in the sections, as extrapolated from the acidified chlorophyll a standard curve (Fig. 5), were increased by a factor of 26.4 so that the concentrations in the tissues were directly comparable to those of the standard solution and digestive gland extracts. For example, the strongest fluorescing area in the digestive gland tissue section of mussel number one had an Rf value of 56. Only one lipid spherule, 11 ,um in diameter, filled approximately 85~,,~ of the measuring spot. Since the background fluorescence from the cytoplasm surrounding tile spherule was low, the concentration of pigment was proportional to the Rf value. Hence, the concentration of pigment within the spherule after extrapolating from the acidified standard curve (12.1 ,ug. ml -~) and correcting for volume ( x 26.4) was 319.4 ~ug. mi -~. By calculating the volume of the spherule [v =~ n(5.5)-~], the amount of pigment within the body was computed as 167 ng. A mean Rf value of 28.1 (SD = +11.1; n---24) was obtained for various spherule-containing areas of the digestive gland, equivalent to 159.1 ~g phaeopigment, ml-x. In contrast, the concentration of pigment in the extract from a piece of the same tissue (Rf value 35.0) was 7.5 ,ug.ml ....~. This illustrates the high concentration of phaeopigment in the spherules. TABI.F 1 Concentrations of plant pigments extracted from the digestive gland of Mvtilus e&dis" (I) calculated from formulas of Jeffrey & Humphrey (1975); (2) calculated from formulas of Richards (in, Strickland & Parsons, 1972); (3) calculated from formulas of Lorenzen (in, Strickland & Parsons, 1972); and (4) calculated from microspectrofluorimetric acidified chlorophyll a dilution series (Fig. 5); all values are expressed in ~ug pigment . g - I wet wt of digestive gland (D.G.).
Mussel
Wet wt D.G. (g)
Chlorophyll a (1)
Chlorophyll h (i)
Chlorophyll c (I)
Carolenoids (2)
Phaeopigment (3)
Phaeopigment (4)
I 2 3 4
0. 3419 0.1864 0.2682 0.3711
! 23.0 213.4 229.4 172.9
22.7 27.7 2 !.4 22.4
31. ! 43.8 3 !.0 28.9
178.2 314.5 305.5 259.8
1690.7 3095.4 3 ! 86.7 3333.3
303.4 48 !.8 485.4 332.8
M YTILUS DIGESTIVE G L A N D P H A E O P I G M E N T S
289
A more meaningful unit of concentration is/zg.g-J where factors such as the solvent dilution and sample wet weight are accommodated in the final figure. Therefore, 7.5 ,ug phaeopigment .ml -I in the digestive gland extract of mussel number one is equivalent to 303.4/~g phaeopigment • g-~. Concentrations of phaeopigment in the digestive gland extracts of three other mussels as determined by the MSF are given in Table I. For comparison, concentrations of phaeopigment and other plant pigments were determined spectrophotometrically on the same extracts and also presented. The calculations based on the spectrophotometric measurements indicate that the amount of all chlorophylls i:;, on the average, an order of magnitude smaller than the phaeopigments. A comparison of calculated phaeopigment values determined by the two methods differs by factors of 5.6 to 10.0. However, both sets of figures substantiate that the red fluorescing pigment in the digestive gland is predominantly phaeopigment. DISCUSSION Chlorophyll degradation products have been identified in faecal pellets from copepods, marine muds, and oceanic water samples by a number of workers (Currie, 1962; Yentsch & Menzel, 1963; Holm-Hansen et al., 1965; Jeffrey, 1974; Shuman & Lorenzen, 1975; Mackas & Bohrer, 1976) using spectrophotometric and/or the more sensitive spectrofluorimeter and fluorimetric procedures. By using a microspectrofluorimeter, this study has applied the sensitivity of spectrofluorimetry to analyze subcellulariy localized pigments in situ and pigment extracts in vitro. Hence, phaeopigments were identified in the lipid spherules of the digestive gland cells and quantified by comparing their relative fluorescen,,'.e with that of the acidified chlorophyll a standard curve. A number of conditions have been shown to degrade chlorophyll to a more stable form. Dilute hydrochloric acid appears to free the magnesium ion from chlorophyll a and leave the phytol chain intact (Holm-Hansen et al., 1965). Studies by Moreth & Yentsch (1970) indicate that photo-oxidation accounts for the majority of chlorophyll degradation, while Barrett and Jeffrey (1971) attributed phytol release to the action of chlorophyllase. More recently, Shuman & Lorenzen (1975) reported that chlorophyll a could be degraded to phaeophorbide a by exposure to hydrochloric acid. Whichever of the phaeopigments are formed, their emission spectra and relative intensities are almost identical (Smith & Benitez, 1955) and thus can be used for comparison with the acidified chlorophyll a standard curve. The observed peak emission from this solution (672.8 nm) compares favorably with values reported for phaeophytin: 672.5 nm (Smith & Benitez, 1955) and 673.0 nm (French et ai., 1956). However, the emission wavelength peak of phaeopigment measured in situ was 1! nm higher than that recorded in the acetone extract. This difference in emission wavelength may be explained by the pigments associated with other molecules, especially lipids, within the cells (Lascelles. 1964).
290
S.R. GELDER AND W. E. ROBINSON
The calculated acid factors from the chlorophyll a standard solution of 11.5 appear unreasonably high when compared with values of 1.7 (Yentsch & Menzel, 1963) and 1.85 to 1.95 (Shuman & Lorenzen, 1975). However, Saijo & Nishizawa (1969) note that the excitation wavelength peak fell from 430 to 415 nm following the degradation of chlorophyll a. By continuing to excite the acidified sample at 436 nm (or 430 nm) the relative fluorescence shows a marked decrease which results in an acid factor of I 1.5, identical with that found in this study. This phenomenon is automatically compensated for in the Turner fluorimeter by the balancing optical bridge system and a broad band-pass excitation filter. As a result, much smaller acid factors are obtained. The acid factor of 1.0 for both phaeopigments in sections and extracts is consistent with values obtained from copepod faecal pellets" 1.0 (Yentsch & Menzel, 1963); 1.05 (Holm-Hansen et al., 1965). Jeffrey (1974) has determined by chromatographic separation that phaeophorbide a is present in the faecal pellets of copepods and that it is the significant indicator of biological processes rather than phaeophytin a. Similarly, phaeophorbide is probably the pigment in the digestive gland of M. edulis. Both acid conditions and hydrolytic enzymes are present in this organ. A pH of 6.0 in the digestive gland lumen has been reported (Owen, 1974), although much lower pHs are probably present in the phagosomes of the digestive cells during intracellular digestion (Owen, 1972). If an enzymatic reaction is involved in chlorophyll degradation, particularly in the splitting of the ester bond connecting the phytol chain with the pyrrole ring, then the presence of nonspecific esterases in the digestive gland of bivalves (Reid, 1968; Palmer, 1979) could be involved. Further investigation is necessary using these and enzyme histochemical techniques for understanding the processes and degree of,:hlorophyll degradation during digestion, uptake, and storage of food material within the bivalve digestive gland. ACK NOW LEDG EM ENTS
The authors would like to acknowledge the assistance of Mr. R. Enders of E. Leitz, Inc., The Farrand Optical Company of New York, and Mr. R. E. Young of Boston. Thanks are extended to ~r. A. Halpern, Department of Chemistry, Northeastern University, Boston, Massachusetts and Dr. C.S. Yentsch, Bigelow Laboratory, Boothbay Harbor, Maine for reading and commenting on the manuscript. The project was supported in part by NIH Grant No. RR07143 from the Department of Health, Education, and Welfare. REFERENCES ANsrtt, A.D., 1974a. Seasonal changes in biochemical composition of the bivalve Abra aiba l¥om the Clyde Sea area. Mar. Biol., Vol. 25, pp. 13-21). A NSI~LL, A. D., 1974b. Seasonal changes in biochemical composition of the bivalve Chlamvs septemradiata from the Clyde Sea area. Mar. Biol., Vol. 25, pp. 85-99.
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W|LLIAMSON,C. E., 1977. Fluorescence identification of zoochlorellae: a rapid method for investigating alga-invertebrate symbioses. J. exp. Zool., Vol. 202, pp. 187-194. YENTSCll, C. S. & D.W. MENZFL, 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res., Vol. !0, pp. 221-231.
IDENTIFICATION OF PHAEOPIGMENTS IN THE DIGESTIVE GLAND OF M Y T I L U S EDULIS L. BY MICROSPECTROFLUORIMETRY I
S. R. GELDER and W. E. ROBINSON Marine Science Institute, Northeastern UniversiO,, Nahant, MA 01908. U.S.A. Abstract: Epi-illuminated fluorescence microscopy was used to observe chlorophyll degradation products localized in lipid spherules within the digestive gland of Mytilus edulis L. These fluorescing materials, known collectively as phaeopigments, then were spectrally analysed in situ with a microspectrofluorimeter. The results were compared with acetone extracts of the digestive gland and chlorophyll a standard solutions. Emission wavelength peaks were 671.2 nm for chlorophyll a in 85'~/,; acetone. 672.8 nm for the acidified chlorophyll a solution, 666.3 nm for digestive gland extracts in 859~, acetone. and 677.1 nm for phaeopigment in tissue sections. Acid factors of 1.0 for digestive gland extracts and tissue sections confirmed the red fluorescing material to be phaeopigment. Quantities up to 167 ng for an I I-#m diameter lipid spherule were encountered. Results were compared with calculated concen-' trations of phaeopigment determined spectrophotometrically.
INTRODUCTION
The spectral analysis of fluorescence emission has provided a simple and sensitive method for studying chlorophylls and their degradation products (vide Smith & Benitez, 1955; Udenfriend, 1962, 1969; Mackas & Bohrer, 1976). Such methods have been used to identify chlorophyll derivatives in sediments (Gorham, 1960) and estimate algal productivity in open waters and laboratory cultures (Yentsch & Menzei, 1963; Holm-Hansen et al., 1965; Strickland & Parsons, 1972; Shuman & Lorenzen, 1975). These pigments have been demonstrated in situ with epi-illuminated fluorescent microscopy by Jones (1974) to estimate epilithic diatom production and by Wilde & Fliermans (1979) to distinguish between several types of unicellular algae. The ability to use chlorophylls and their degradation products as convenient natural markers enabled Williamson (1977) to study an algal-invertebrate symbiosis by differentiating zoochlorella from isomorphic bodies in the sponge, Spongilla lacustris. Similarly, the fate of algae has been traced by observing its fluorescing pigments during ingestion and digestion in veliger larvae of the oyster, Crassostrea virgblica (Babinchak & Ukeles, 1979). Smith & Benitez (1955) have shown that the degradation of chlorophylls is accompanied by a shift of a few nanomcters to a higher fluorescent emission wavelength and a change in the absorption spectra. Similarly, Currie (1962) compared i Contribution number 90 from Marine Science Institute, Northeastern University, Nahant, Massachusetts, U.S.A. 281
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S.R. GELDER AND W. E. ROBINSON
the emission peaks from acetone extracts of zooplankton faeces with extracts of the diatom, Skeletonema sp., and interpreted the wavelength shift as indicative of a rapid breakdown of the ingested chlorophyll a. Ansell (1974a, b) studied the absorption spectra of acetone extracted pigments from the "mantle" tissue (defined as the remaining viscera after removal of gonad and adductor muscle) of several species of bivalves. From this spectral data he construed the pigments to be similar to phaeophytins. Algal pigments are retained primarily within the digestive gland of bivalves following digestion and absorption of algal food (Morton, 1956; Ansell, 1974b). Seasonal fluctuations of phaeophytin levels in "mantle" extract of Abra alba and Chlamys septemradiata correspond to fluctuations in feeding activity (Ansell, 1974a, b). A similar correlation between carotenoid levels in whole animal homogenates and fluctuating densities of phytoplankton is reported for Mytilus edulis (Jensen & Sakshaug, 1970a, b). The relative fluorescence of acetone extracted pigments from Crassostrea gigas and Ostrea edulis is roughly proportional to the bivalves' nutritional state and can be expressed as a feeding index (Mann, 1977). Index values for individual bivalves are altered quickly by short-term bursts of feeding, which makes it difficult to establish the nutritional profile for an entire population. Use of the index is further complicated due to the inclusion of a residue of pigment which remains in the digestive gland of both these bivalves for up to three weeks after cessation of feeding. Chlorophylls and phaeopigments can be observed with greater intracellular precision and in smaller quantities by epi-illuminated fluorescent microscopy than by bright-field or phase-contrast illumination. This study was designed to demonstrate chlorophylls and their fluorescing degradation products in the digestive gland cells of M. e&dis by epi-illuminated fluorescent microscopy and provide a semiquantitative microspectrofluorimetric method for comparing the pigment complexes in situ with those extracted by acetone. MATERIAL AND METHODS
Specimens of Mytilus edulis L. were collected from the intertidal zone at East Point, Nahant, Massachusetts in June and July, 1979. The digestive gland was removed from recently collected specimens and cut into two pieces, one for histological sectioning and the other for pigment extraction. TISSUE PREPARATIONS
The larger piece of digestive gland was macerated in 10 ml of 85 °'j,, acetone (analytical grade) with approximately 1 mg of magnesium carbonate for 5 min, centrifuged for 10 min at 4750 rpm, and the supernatant decanted. The second piece of tissue was cooled rapidly with dry ice (solid carbon dioxide,
M YTIL US DIG ESTIVE (3 LAND PHA EOPIG M ENTS
283
-78.5 °C) for 5 min, transferred to a microtome-cryostat chamber maintained at - 2 0 °C, affixed to a specimen stub with water, and left for 10 min. Serial, 10-~um thick sections were mounted directly onto cleaned microscope slides, immersed in Baker's 10~ calcium formalin for 5 min, and washed in three changes of distilled water. Slides then were stained either for lipid by the supersaturated isopropanol technique, using Oil Red 0 dye (Lillie & Fullmer, 1976) and mounted in Von Apathy gum syrup (Humason, 1979), or mounted directly for the subsequent examination of fluorescent pigments. Control slides involved the removal of lipid, chlorophyll, and chlorophyll degradation products from the sections by soaking them in either 85~'o acetone or 95~ ', ethanol for 10-30 min. Possible autofluorescent contamination of sections by the fixative or mountant were checked and proved unfounded. INSTRUMENTATION A N D CALIBRATION
Sections were examined under a Leitz Dialux 20 microscope by transmitted brightfield or phase-contrast illumination, or with epi-fluorescence illumination. The light source for the fluorescence methods consisted of an Osram mercury HBO 50-W arc lamp powered by the unstabilized 115-120 V supply. The light was passed through a Ploem illuminator 2.4 which contained filter cubes A and H2 for broad band ultraviolet and violet-blue light excitation, respectively. Conversion of the microscope into a microspectrofluorimeter involved the insertion of a Bausch and Lomb Czerney-Turner grating type excitation monochromator between the lamp and the Ploem illuminator for the selection of the 436-nm mercury line. The fluorescent emissions from the samples were spectrally analyzed in a darkened room using a x 40 Fluorotar objective and a Farrand Microscope Spectrum Analyzer (MSA). The MSA contained a Farrand, Czerney-Turner (205-780 nm) grating type monochromator, a range of slit and aperture widths, plus a R446 Hamamatsu photomultiplier tube. The emission trace was recorded on a chart recorder. Fluctuations in line voltage on the unstabilized mercury lamp were checked by monitoring the peak emission wavelength from uranyl glass (Schott, GG21) (PIoem, 1977). /o This showed that the maximum modulation caused by voltage variation was 0.5 ''j of the relative fluorescence during the longest (3 min) emission scan times. The organelles in the 10-/zm thick sections were critically focused, positioned with the 15-#m diameter measuring spot, and the resulting relative fluorescence spectrally analyzed by the MSF. The measuring spot diameter was chosen because it provided optimal operating conditions. The quantity of material excited, calculated as the volume of a cylinder [v = n(7.5/~m)-' (10 #m)], was 1.77 x 10-~,um 3. Solutions of pigments were placed in a cavity slide (2.5 mm deep, containing approximately 0.85 ml) and sealed with a coverslip. The x 40 objective was lowered until it rested on the coverslip so that the cone of excitation extended into the solution to a depth of 264/~m; the measuring spot remained at 15/~m in diameter so that the emission was analyzed from 4.66 x 104/~m-~of extract. To calibrate the wavelength scale of
284
S.R. GELDER AND W. E. ROBINSON
the MSF a chlorophyll a solution (Sigma C-5753) in 85~ acetone (analytical grade) was analyzed spectrally with the MSF and spectrofluorimeter. The uncorrected scans agreed. Quantitative calibration of the MSF was achieved by making serial dilutions of the chlorophyll a solutioI~, recording their relative fluorescence (Rf units) at the emission wavelength maximum, and then constructing a standard curve. Samples of acetone-extracted pigments from the digestive'gland were analyzed by the MSF and with a Bausch & Lomb Spectronic 700 spectrophotometer. MEASUREMENTS AND CALCULATIONS
After spectrally analyzing sections, acetone-extracted pigments of digestive gland, and chlorophyll a solutions, all of these preparations were acidified to convert any chlorophyll into the corresponding phaeopigments (Strickland & Parsons, 1972) and re-analyzed. From these data the standard dilution curves for chlorophyll a and phaeopigments were plotted, the acid factors (RfJRf,~jd) calculated (Yentsch & Menzel, 1963; Holm-Hansen et al.. 1965), and the concentration of phaeopigments in the digestive gland extracts and sections determined. A spectrophotometer was used to measure absorbance at 480, 510, 630, 647, 664, 665, and 750 nm in the nonacidified and at 665 and 750 nm in the acidified pigment extracts. Then these values were substituted in the equations of Jeffrey & Humphrey (1975) to calculate the amounts ofchlorophyll a, b and c. The equations of Richards and Lorenzen (in Strickland & Parsons, 1972) were used for the calculations of carotenoid and phaeopigment respectively. OBSERVATIONS
The histology of the digestive gland of M. edulis has been described by Owen (1972) and Langton (1975). The gland is a greenish-brown organ composed of a network of blind-ending tubules, ducts, and connective tissue which surround the stomach. Ciliated primary ducts, which arise from the stomach wall, branch into short, secondary ducts that terminate in blind-ending digestive tubules. These tubules are oval in transverse section1 with a unicellular epithelium composed largely of columnar digestive cells and conical basophil cells arranged along the folds or crypts. Under ultravi~)let or violet light excitation the digestive gland epithelium fluoresces red while the adjacent connective tissue and musculature autofluoresces a dull blue. Scattered throughout these tissues are pale yellow lipofuscin granules, 2-12 pm in diameter. The epithelium of the digestive tubules, and to a lesser extent the primary and secondary ducts, fluoresce a dull, uniform red while in the basal regions of the cells are bright orange-red fluorescing spherules. Some spherules also are found in the connective tissue adjacent to the tubules. The size of the spherules varies in the different regions: 2-11 /~m in the digestive tubule epithelium and neighboring con-
M Y T I L U S DIGESTIVE GLAND PHAEOPIGMENTS
285
nective tissue; 1-2 pm in the secondary; and 1--6/~m in the primary ducts. Spherules in comparable locations and of similar dimensions were demonstrated by the Oil Red 0 technique, indicating their lipid nature. Sections placed in 85% acetone or 95~ ethanol for 10 min showed no discernible red fluorescing pigment or lipid, but the yellow autofluorescing lipofuscin remained. MICROSPECTROFLUORIMETRY
A typical spectral analysis of the emission from a digestive cell containing red fluorescing pigment spherules and lipofuscin granules excited with violet light (436 nm) is presented in Fig. I. The lipofuscin emission is spread over a broad 40 677
3o
Rf units
546 20
10
O
•
500
II
600 nrN
ii
•
700
Fig. i. Spectral scan of the emission from an area of the digestive gland o| Mvtilus e~hdL~"which includes lipofuscin granules and phaeopigment-containing lipid spherules" microspectrofluorimeter excitation wavelength fixed at 436 nm.
wavelength band, peaking at 546 nm, followed by the red-fluorescing pigment peak at 677 nm. Scans specifically of pigment spherules (Fig. 2, trace a) confirmed that peak emission occurs at 677.1 nm (SD +2.5 nm; n = 12). Although no pigment fluorescence could be seen in the 10-min ethanol- or acetone-emersed control sections, a small amount of residual fluorescence could be detected in the cytoplasm by the MSF (Fig. 2, trace b). This was easily distinguished from background fluctuations when compared with the reference uranyl glass emission trace (Fig. 3). The emissions from acetone extracts of o;gments from the digestive glands were analyzed spectrally under the MSF (Fig. 2, trace c). The primary emission peak occurred at 666.3 nm (SD = + 2.8 nm; n = 12) with a shoulder evident from 690 to
286
S.R. G E L D E R A N D W. E. ROBINSON
720 nm. Aliquots of samples analyzed in a spectrofluorimeter (Fig. 4) confirmed the position of the primary peak at approximately 663 nm while the shoulder was resolved into two secondary peaks (709 and 725 nm). The shape of these traces and their wavelength characteristics indicate that the pigment is either chlorophyll a, a degradation product, or a combination of the two. ,C 40
30
20
Rf units IO
b
•
600
m..~_
~
~
'
'
__
- ~
;,so
nm Fig. 2. Traces of microspectrofluorimetric emission scans of areas from sections of M.vtih~s e&~lis digestive gland: a, before, and b, after a 10rain extraction in 85". acetone; a trace of the extracted pigment in a solution of 85"0 acetone (c); all measurements made with an excitation wavelength of 436 nm.
0
20
4b sees
go
8"0
Fig. 3. Portion of the emission trace from the uranyl glass calibration, excited at 436 nm, and recorded at 540 nm under the microspectrotluorimeter.
Microspectrofluorimetric analysis of the chlorophyll a standard solution showed an emission wavelength peak at 671.2 nm (SD= +2.3 nm; n = 11). Following acid hydrolysis of these solutions to the phaeopigment derivative, a slight increase in peak emission wavelength was found (672.8 nm; SD= +_2.6 nm; n = 10), but this shift was not statistically significant (paired t test; t = 1.528; n = 10). Similarly, the differences in wavelength emission peaks following acidification of both acetone extracts of the digestive gland (t test; t = 1.7340; d.f. = 18) and tissue sections (t test; t = 0.4662; d.f. = 20) were not statistically different from their preacidification values. A marked decrease in the relative fluorescence of the chlorophyll a standard solutions was recorded following acid hydrolysis (Fig. 5). The mean acid factor for
M YTILUS DIGESTIVE GLAND PHAEOPIGMENTS
287
,°] 70
Rf units
50
30
10 0
I
600
ii
iii
i i
7~0 nm
Fig. 4. Spectrofluorimetric emission trace of a digestive gland extract in 8';"_j. acetone excited at 436 nm' primary peak at 662.7 nm and two secon:dary peaks at 709 and 725 nm.
,/
8.0'5
Rf units
2
0
0.4
0.8
1.2
pg/ml Fig. 5. Standard curve of chlorophyll a in 85"~, acetone: before (O), ~g/ml - 0 . 0 2 Rfu + 0.04, r - 0.99, n = I I, and after acidification (A), pg/ml = 0.20 Rfu + 0.005, r - 0.98, n - 10.
288
S.R. G E L D E R AND W. E. ROBINSON
these solutions was 11.5 (SD-'- +0.72; i~ = 5). In contrast, acid factors of 1.0 were calculated for the tissue sections (~'Rf0/~Rfac~a) and digestive gland pigment extracts (SD -- + 0.025; n - 4). Hence, the chlorophyll derivatives within the digestive gland were shown to be predominantly phaeopigment. QUANTITATIVE DETERMINATION OF PHAEOPIGMENT
The amount of fluorescing pigment in the organelles of the tissue sections and in the digestive gland extracts can be determined from their relative fluorescence, despite the dissimilar composition of the samples and the different volumes measured. The cylinder measured differs it~ depth from 10 ,urn in a tissue section to 264 ,um in the fluid preparations. As the measuring spot diameter remains constant, the ratio of the volumes and, in turn, the relative fluorescence, is proportional (1:26.4). Therefore, the concentrations of pigment in the sections, as extrapolated from the acidified chlorophyll a standard curve (Fig. 5), were increased by a factor of 26.4 so that the concentrations in the tissues were directly comparable to those of the standard solution and digestive gland extracts. For example, the strongest fluorescing area in the digestive gland tissue section of mussel number one had an Rf value of 56. Only one lipid spherule, 11 ,um in diameter, filled approximately 85~,,~ of the measuring spot. Since the background fluorescence from the cytoplasm surrounding tile spherule was low, the concentration of pigment was proportional to the Rf value. Hence, the concentration of pigment within the spherule after extrapolating from the acidified standard curve (12.1 ,ug. ml -~) and correcting for volume ( x 26.4) was 319.4 ~ug. mi -~. By calculating the volume of the spherule [v =~ n(5.5)-~], the amount of pigment within the body was computed as 167 ng. A mean Rf value of 28.1 (SD = +11.1; n---24) was obtained for various spherule-containing areas of the digestive gland, equivalent to 159.1 ~g phaeopigment, ml-x. In contrast, the concentration of pigment in the extract from a piece of the same tissue (Rf value 35.0) was 7.5 ,ug.ml ....~. This illustrates the high concentration of phaeopigment in the spherules. TABI.F 1 Concentrations of plant pigments extracted from the digestive gland of Mvtilus e&dis" (I) calculated from formulas of Jeffrey & Humphrey (1975); (2) calculated from formulas of Richards (in, Strickland & Parsons, 1972); (3) calculated from formulas of Lorenzen (in, Strickland & Parsons, 1972); and (4) calculated from microspectrofluorimetric acidified chlorophyll a dilution series (Fig. 5); all values are expressed in ~ug pigment . g - I wet wt of digestive gland (D.G.).
Mussel
Wet wt D.G. (g)
Chlorophyll a (1)
Chlorophyll h (i)
Chlorophyll c (I)
Carolenoids (2)
Phaeopigment (3)
Phaeopigment (4)
I 2 3 4
0. 3419 0.1864 0.2682 0.3711
! 23.0 213.4 229.4 172.9
22.7 27.7 2 !.4 22.4
31. ! 43.8 3 !.0 28.9
178.2 314.5 305.5 259.8
1690.7 3095.4 3 ! 86.7 3333.3
303.4 48 !.8 485.4 332.8
M YTILUS DIGESTIVE G L A N D P H A E O P I G M E N T S
289
A more meaningful unit of concentration is/zg.g-J where factors such as the solvent dilution and sample wet weight are accommodated in the final figure. Therefore, 7.5 ,ug phaeopigment .ml -I in the digestive gland extract of mussel number one is equivalent to 303.4/~g phaeopigment • g-~. Concentrations of phaeopigment in the digestive gland extracts of three other mussels as determined by the MSF are given in Table I. For comparison, concentrations of phaeopigment and other plant pigments were determined spectrophotometrically on the same extracts and also presented. The calculations based on the spectrophotometric measurements indicate that the amount of all chlorophylls i:;, on the average, an order of magnitude smaller than the phaeopigments. A comparison of calculated phaeopigment values determined by the two methods differs by factors of 5.6 to 10.0. However, both sets of figures substantiate that the red fluorescing pigment in the digestive gland is predominantly phaeopigment. DISCUSSION Chlorophyll degradation products have been identified in faecal pellets from copepods, marine muds, and oceanic water samples by a number of workers (Currie, 1962; Yentsch & Menzel, 1963; Holm-Hansen et al., 1965; Jeffrey, 1974; Shuman & Lorenzen, 1975; Mackas & Bohrer, 1976) using spectrophotometric and/or the more sensitive spectrofluorimeter and fluorimetric procedures. By using a microspectrofluorimeter, this study has applied the sensitivity of spectrofluorimetry to analyze subcellulariy localized pigments in situ and pigment extracts in vitro. Hence, phaeopigments were identified in the lipid spherules of the digestive gland cells and quantified by comparing their relative fluorescen,,'.e with that of the acidified chlorophyll a standard curve. A number of conditions have been shown to degrade chlorophyll to a more stable form. Dilute hydrochloric acid appears to free the magnesium ion from chlorophyll a and leave the phytol chain intact (Holm-Hansen et al., 1965). Studies by Moreth & Yentsch (1970) indicate that photo-oxidation accounts for the majority of chlorophyll degradation, while Barrett and Jeffrey (1971) attributed phytol release to the action of chlorophyllase. More recently, Shuman & Lorenzen (1975) reported that chlorophyll a could be degraded to phaeophorbide a by exposure to hydrochloric acid. Whichever of the phaeopigments are formed, their emission spectra and relative intensities are almost identical (Smith & Benitez, 1955) and thus can be used for comparison with the acidified chlorophyll a standard curve. The observed peak emission from this solution (672.8 nm) compares favorably with values reported for phaeophytin: 672.5 nm (Smith & Benitez, 1955) and 673.0 nm (French et ai., 1956). However, the emission wavelength peak of phaeopigment measured in situ was 1! nm higher than that recorded in the acetone extract. This difference in emission wavelength may be explained by the pigments associated with other molecules, especially lipids, within the cells (Lascelles. 1964).
290
S.R. GELDER AND W. E. ROBINSON
The calculated acid factors from the chlorophyll a standard solution of 11.5 appear unreasonably high when compared with values of 1.7 (Yentsch & Menzel, 1963) and 1.85 to 1.95 (Shuman & Lorenzen, 1975). However, Saijo & Nishizawa (1969) note that the excitation wavelength peak fell from 430 to 415 nm following the degradation of chlorophyll a. By continuing to excite the acidified sample at 436 nm (or 430 nm) the relative fluorescence shows a marked decrease which results in an acid factor of I 1.5, identical with that found in this study. This phenomenon is automatically compensated for in the Turner fluorimeter by the balancing optical bridge system and a broad band-pass excitation filter. As a result, much smaller acid factors are obtained. The acid factor of 1.0 for both phaeopigments in sections and extracts is consistent with values obtained from copepod faecal pellets" 1.0 (Yentsch & Menzel, 1963); 1.05 (Holm-Hansen et al., 1965). Jeffrey (1974) has determined by chromatographic separation that phaeophorbide a is present in the faecal pellets of copepods and that it is the significant indicator of biological processes rather than phaeophytin a. Similarly, phaeophorbide is probably the pigment in the digestive gland of M. edulis. Both acid conditions and hydrolytic enzymes are present in this organ. A pH of 6.0 in the digestive gland lumen has been reported (Owen, 1974), although much lower pHs are probably present in the phagosomes of the digestive cells during intracellular digestion (Owen, 1972). If an enzymatic reaction is involved in chlorophyll degradation, particularly in the splitting of the ester bond connecting the phytol chain with the pyrrole ring, then the presence of nonspecific esterases in the digestive gland of bivalves (Reid, 1968; Palmer, 1979) could be involved. Further investigation is necessary using these and enzyme histochemical techniques for understanding the processes and degree of,:hlorophyll degradation during digestion, uptake, and storage of food material within the bivalve digestive gland. ACK NOW LEDG EM ENTS
The authors would like to acknowledge the assistance of Mr. R. Enders of E. Leitz, Inc., The Farrand Optical Company of New York, and Mr. R. E. Young of Boston. Thanks are extended to ~r. A. Halpern, Department of Chemistry, Northeastern University, Boston, Massachusetts and Dr. C.S. Yentsch, Bigelow Laboratory, Boothbay Harbor, Maine for reading and commenting on the manuscript. The project was supported in part by NIH Grant No. RR07143 from the Department of Health, Education, and Welfare. REFERENCES ANsrtt, A.D., 1974a. Seasonal changes in biochemical composition of the bivalve Abra aiba l¥om the Clyde Sea area. Mar. Biol., Vol. 25, pp. 13-21). A NSI~LL, A. D., 1974b. Seasonal changes in biochemical composition of the bivalve Chlamvs septemradiata from the Clyde Sea area. Mar. Biol., Vol. 25, pp. 85-99.
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BABINCHAK.J. • R. UKELES, 1979. Epifluorescence microscopy, a technique for the study of feeding in Crassostrea virginica veliger larvae. Mar. Biol., Vol. 51, pp. 69-76. BARRETT, J. & S. W. JEFFREY, 1971. A note on the occurrence of chlorophyllase in marine algae. J. exp. mar. Biol. Ecol., Vol. 7, pp. 255-262. CURRIL R. l., 1962. Pigments in zooplankton faeces. Nature, Lond., Voi. 193, pp. 956-957. FRENCH, C.S., J. H.C. SMITH, H.I. VIRGIN & R.L. AIRTH, 1956. Fluorescence spectrum curves of chlorophylls, pheophytins, phycoerythrins, phycocyanins and hypericin. Plant Physiol., Vol. 31, pp. 369-374. GOR:IAM, E., 1960. Chlorophyll derivatives in surface muds from the English Lakes. Limnoi. Oceanogr., Vol. 5, pp. 29-33. HOLM-HANSEN, O., C.J. LORENZEN, R.W. HOLMES& J. D. H. STRICKLAND, 1965. Fluorometric determination of chlorophyll. J. Cons. porto, int. Explor. Mer, Vol. 30, pp. 3-15. HUMASON, G. L., 1979. Animal lissue techniques, 4th edition, W. H. Freeman, San Francisco, 661 pp. JEFFREY, S. W., 1974. Profiles of photosynthetic pigments in the ocean using thin-layer chromatography. Mar. Biol., Vol. 26, pp. I 01- ! I O. JEFFREY,S. W. & G. F. HUMPHREY, 1975. New spectrophotometric equations for determining chlorophylls a, b, c I and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. p/1., Vol. 167, pp. 191-194. JENSEN, A. & E. SAKSHAt;ta, 1970a. Producer-consumer relationships in the sea. l. Preliminary studies on phytoplankton density and Mytilus pigmentation. J. exp. mar. Biol. Ecol., Vol. 5, pp. 180-186. JENSFN,A. & E. SAKSHAUG,1970b. Producer-consumer relationships in the sea. II. Correlation between M.rtilus pigmentation and the density and composition of phytoplanktonic populations in inshore waters. J. exp. mar. Biol. Ecol., Vol. 5, pp. 246-253. JONFS, J. G., 1974. A method for observation and enumeration ofepilithic algae directly on the surface of stones. Oecologia, Vol. 16, pp. I-8. LANt;TON, R. W., 1975. Synchrony in the digestive diverticula of Mytilus ethdis L. J. mar. biol. Ass. U.K., Vol. 55, pp. 221-229. LASCELLES,J., 1964. Tetrapyrrole biosynthesis and its regulation. Benjamin, New York, 132 pp. LILLIL R. D. & H. M. FULLMER, 1976. Histopathologic technic and practical histochemistry. 4th edition, McGraw-Hill, New York, 942 pp. MACKAS, O. & R. BOttRER, 1976. Fluorescence analysis of zooplankton gut contents and an investigation of diel feeding patterns. J. exp. mar. Biol. Ecol., Vol. 25, pp. 77-85. MANN, R.. 1977. An assessment of the use of pigment content as a feeding index in oysters. Aquaculture, Vol. !0, pp. 373-376. MORFrH, C. M. & C.S. YI-N'rS(:H, [970. The role of chlorophyllase and light in the decomposition of chlorophyll from marine phyte;:,lankton. J. exp. mar. Biol. Ecol., Vol. 4, pp. 238-249. MORTON, J. E., 1956. The tidal rhythm and action of the digestive system of the lamellibranch Lasaea rubra. J. mar. biol. Ass. U.K., Vol. 35, pp. 563-586. OwF~, G., 1972. Lysosomes, peroxisomes and bivalves. Sci. Progr., 0.,(/~, Vol. 60, pp. 299-318. OWEN, G., 1974. Feeding and digestion in the Bivalvia. Adv. comp. Physiol. Biochem., Vol. 5, pp. 1-35. PAI,MI~R, R. E., 1979. A histological and histochemical study of digestion in the bivalve Arclica L~'lamfica L. Biol. Bull., Vol. 156, pp. I 15- ! 29. PLOEM, J.S., 1977. Quantitative fluorescence microscopy. In. Analytical ami quantitative methods in microscopy, edited by G.A. Meek & H, Y. Elder. Cambridge University Press. Cambridge, U.K., pp. 55 -89. RND, R.G.B., 1968. The distribution of digestive tract enzymes in lamelhbranchiate bivalves. Comp. Biochem. Physiol., Vol. 24. pp. 727-744. SAIJO, Y. & S. NISHIZAWA, 1969. Excitation spectra in the fluorometric determination of chlorophyll-a and pheophytin-a. Mar. Biol., Vol. 2, pp. 135-136. SHt;MAN, F. R. & C. J. LORt~:NZEN,I975. Quantitative degradation of chlorophyll by a marine herbivore. Limnoi. Oceanogr., Vol. 20, pp. 580-586. SMITH, J. H. C. & A. Bf-NITEZ, 1955. Chlorophylls: analysis in plant materials. In. Modern nlelhods al plant analysis, Voi. 4, edited by K. Paech & M. V. Tracey, Springer-Verlag, Berlin, pp. 142-196. STRICKLAND,J. D. H. & T. R. PARSONS, 1972. A practical handbook of seawater analysis. BuF. Fish, Res. Bd Can., No. 167 (2nd edition), 310 pp.
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UDENFRIEND, S., 1962. Fluorescence assay #1 biology and medicine, Vol. 1. Academic Press, New York, 505 pp.
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WILDE, E. W. • C. B. FLIERMANS,1979. Fluorescence microscopy for algal studies. Trans. Am. microsc. Soc., Vol. 98, pp. 96-102.
W|LLIAMSON,C. E., 1977. Fluorescence identification of zoochlorellae: a rapid method for investigating alga-invertebrate symbioses. J. exp. Zool., Vol. 202, pp. 187-194. YENTSCll, C. S. & D.W. MENZFL, 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res., Vol. !0, pp. 221-231.