SHORT COMMUNICATION Improved technique for the gut fluorescence method in a feeding study of small zooplankton

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1 Journal of Plankton Research Vol.19 no.1 pp , 1997 SHORT COMMUNICATION Improved technique for the gut fluorescence method in a feeding study of small zooplankton H.Takatsuji, K.Hamasaki.T.Toda and S.Taguchi Laboratory of Biological Oceanography, Department oj Bio engineering, Faculty of Engineering, Soka University, Tangi-Cho, Hachioji, Tokyo 192, Japan Abstract. An increase in the sensitivity of chlorophyll pigment analysis was provided by small-volume quartz cuvettes with a special holder for the determination of gut chlorophyll pigment in an individual small zooplankton. Feeding studies of herbivorous copepods are imperative in order to understand the carbon cycle in the marine ecosystem. The gut fluorescence method has been widely used for feeding studies of copepods (Boyd et al., 1980; Tande and Bamstedt, 1985; Dam, 1986; Dam and Peterson, 1988; Tiselius, 1988; Durbin et al., 1990; Rodriguez and Durbin, 1992;Tsuda and Sugisaki, 1994; Saito and Taguchi, 1996; Saito and Hattori, 1996; and others) since Mackas and Bohrer (1976) applied this technique to a feeding study of zooplankton. The method in which the gut contents of copepods are estimated from phytoplankton chlorophyll a-equivalent phaeopigments in their guts (Conover et al., 1986) provides information on the feeding rates immediately prior to collection, and thus can be readily used in vivo. The significance of individual behaviour has been emphasized in zooplankton ecology in recent years (Mobley, 1987; Kleppel et al., 1988). However, for relatively small animals such as neritic copepods, fluorescence measurements are made using numbers of animals in order to have sufficient signals and make precise measurements. Measurements on numbers of individuals tend to mask individual differences in gut pigment contents. Individuals of similar appearance, but different species or stages, can also be introduced into the measurement (Tiselius, 1988; Saito and Hattori, 1996). Once the gut pigment contents are determined individually, the synchrony of feeding behaviour can be examined (Rodriguez and Durbin, 1992). In addition to the size of individuals, a ratio of individual numbers to the extraction volume is one of the components which can improve the sensitivity of gut fluorescence measurements. This ratio has not been explicitly considered in gut fluorescence analysis despite highly variable body sizes. A variety of this ratio has been employed in the previous studies which is summarized in Figure 1. The ratio ranges from 0.1 to 5 ind. ml" 1 for smaller copepods (prosome length <1.3 mm) such as Acartia spp., Paracalanus parvus, Pseudocalanus elongatus and Temora longicornis (Dam, 1986; Tiselius, 1988; Durbin et al., 1990; Bautista et al., 1992; Rodriguez and Durbin, 1992; Saito and Taguchi, 1996). The medium-sized copepods (2-3 mm prosome length) show less variability in the ratio, ranging from 0.6 to 3 ind. ml- 1 (Tande and Bamstedt, 1985;Tiselius, 1988; Bautista et al., 1992;Tsuda and Sugisaki, 1994). The copepods of prosome length >5 mm show the least Oxford University Press 159

2 H.TakatsnjIefai Prosome length (mm) (pboyd et al 1980 Bautista et al 1992 Tande and B&mstedt 1985 Rodriguez and Durbin 1992 Dam 1986 Tsuda and Sugisaki 1988 Saito and Taguchi 1996 DuibinetaL 1990 Fig. L Relationship between prosome length (mm) and the number of individuals per extraction volume (ind. mh 1 ) in the published study. variability in the ratio, ranging from 0.7 to 2 ind. ml" 1 (Boyd etal., 1980;Tsuda and Sugisaki, 1994). Although gut fluorescence is not exactly related to body size, the high ratio would be desirable for smaller copepods. The detection limit of a Turner Design fluorometer, Model 10-AU, is 0.05 ng chlorophyll a ml" 1 with a 13 mm diameter cuvette, which requires at least a 4 ml volume of extraction solvent. When individual gut pigment content is <0.2 ng, the conventional method cannot measure individual contents accurately. Therefore, a small-volume cuvette can be designed to increase the sensitivity of chlorophyll pigment analysis. The objective of the present study is to establish the method to measure gut pigment contents individually. This new technique makes it possible to identify the species and measure the size of the examined small zooplankton. All fluorescence analyses were made on a Turner Design fluorometer, Model 10-AU, with a Daylight White lamp, an excitation filter of nm ( R) and an emission filter of 665 nm (10-051R). All fluorescence readings were obtained by 10 s average after a 15 s delay. The sensitivity of the fluorometer was adjusted at the maximum with a reasonable stability for the present purpose. To decrease the extraction volume, a new cuvette, which required a smaller volume of extraction solvent than the conventional 13 mm diameter cuvette, was designed. In the present study, Pyrex and quartz tubes (6 mm diameter, 1 mm thick- 160

3 Improved gotfluorescencemethod ness) were made for the fluorescence measurement. Cuvette holders were designed and shaped from PVC pipe to hold the 6 mm cuvette. The slit of the holder was enlarged as much as possible to increase the sensitivity of fluorescence (Figure 2). A/.N-Dimethylformamide (DMF) was employed as the extraction solvent because it has a high extraction ability and less decay of extracted pigments, and DMF extract responded to acidification in the same way as acetone extract (Suzuki and Ishimaru, 1990). Since the values of blank fluorescence had been known to vary with the direction of the cuvette in a cuvette holder, the variability in the blank fluorescence was determined with different directions of each cuvette. The variability of DMF blanks in 10 Pyrex and 40 quartz cuvettes was determined to compare the characteristics of the quality of the two kinds of cuvettes. The coefficient of variation in the blank fluorescence with different directions of each cuvette ranged from 6.1% to 29% for the Pyrex cuvette and from 18% to 227% for the quartz cuvette, respectively. Quartz cuvettes had lower average blank fluorescence than Pyrex cuvettes (Figure 3). The coefficient of variation of the quartz cuvettes (94%) was higher than that of the Pyrex cuvettes (35%). Blank fluorescence readings were different between the Pyrex and quartz cuvettes. Average blank fluorescence readings of quartz cuvettes were 7.0% of that of the Pyrex cuvettes. Since the fluorometer was always corrected for the blank readings to determine fluorescence, the smaller standard deviations would give stable readings. The quartz cuvettes would give more reasonable fluorescence readings than Pyrex ones. The relationship between chlorophyll a concentration and fluorescence reading was determined by using the cuvette which had the lowest blank reading. After chlorophyll a provided by Sigma was dissolved in DMF and subsequently diluted 32 mm in height 4 mm in width 6 mm cuvette (Pyrex & Quartz) 6 mm cuvette holder (PVC: non-reflecting black) Fig. 2. Schematic diagram of the cuvette and cuvette holder. The 6 mm cuvette holder was inserted and aligned to the Turner Design 13 mm cuvette holder. 161

4 H.Takatsuji el al 200- I 150- g 100- ^ I Pyrex cuvette (n= 10) Quartz cuvette (n=40) Fig. 3. Blankfluorescenceunits of Pyrex and quartz cuvettes. Bars indicate standard deviations of the individual cuvette when itsfluorescencewas determined at different directions in the cuvette holder. to various concentrations, the fluorescence of each dilution was measured with a 13 mm cuvette. The range of concentration determined was ng ml" 1. Fluorescence before and after acidification with 20 ul of 5% HCl was read. The later reading was made after 60 s. The average and coefficient of variation of fluorescence before acidification were determined at each concentration of chlorophyll a based on five replicates. The relationship between chlorophyll a concentration and fluorescence was linear within the range of measured concentrations (Figure 4). The coefficient of variation was highly variable and decreased with the concentration of chlorophyll a to <2%. When the fluorescence readings with coefficients of variation lower than 10% are accepted as the reasonably confident estimates, they are 0.05 ng ml" 1 for the conventional method and 0.11 ng ml" 1 for the present 150o Average Fb = 20.6 CHL a+ 731 r* = E 50 ' Mol o c a> '3 1 O.I I l Concentration (ng CHL a wt l ) Fig. 4. Relationships among chlorophyll a concentration and fluorescence units before acidification (Fb, dots), and percent coefficient of variation of Fb (diamonds) determined with the 6 mm cuvette. Bars indicate standard deviations of replicate determinations. 162

5 Improved gut fluorescence method method, respectively. In contrast, the extraction volume was 4 ml for the conventional method and 1 ml for the present method, respectively. Therefore, the present method would provide four times higher concentrations of extracted pigment contents than the conventional method. Consequently, the overall sensitivity of the present method was two times better than that of the conventional method. Adult female Pseudodiaptomus marinus was used for the measurement in the present study. An oblique tow using a net of 180 urn mesh with a large-sized cod end was made from 5 m depth to the surface to collect the zooplankton sample in Manazuru Port, Kanagawa, Japan, at 12:00 and 24:00 h on 19 August The contents of the cod end were filtered onto a 180 um mesh filter under gravity and immediately frozen at -80 C. In the laboratory, the frozen samples were thawed and kept in an ice-water bath on the dissecting microscope under a dim red light. Pseudodiaptomus marinus were picked, placed in a DMF solution and refrigerated for 24 h. Individual gut pigment contents of adult female P. marinus were examined by the present method. The gut pigment contents were expressed as the sum of chlorophyll a and phaeopigments. The prosome length of the individuals after their fluorescence measurement was determined under the biological microscope. The prosome length ranged from 0.70 to 0.91 mm for all individuals examined. The average with one standard deviation of the prosome length was 0.82 ± 0.06 mm during the day and 0.84 ± 0.05 mm during the night, respectively. The difference in the average prosome lengths between day and night was not statistically significant by Mest (P > 0.3). Since the size distribution of the prosome length revealed little difference between day and night, a similar population was probably collected during the day and at night. Day and night individual gut pigment contents of adult female P. marinus were highly variable (Figure 5). The average with one standard deviation of gut pigment contents was 0.35 ± 0.36 ng indr 1 in the daytime and 0.58 ± 0.32 ng indr 1 at nighttime, respectively. The difference in the average between day and night was statistically significant by Mest (P = 0.03). The average night value was 1.7 times higher than the day value. These kinds of trends were reported for the numerous species shown in Figure 1. Although the coefficient of variation in the day was higher than that at night, most values in the day (85%) were <0.41 ng indr 1. However, night values were distributed more randomly than day values, except the three high day values. This result may indicate that feeding behaviour is more synchronized within individuals during the day than during the night, assuming balanced ingestion and defaecation processes (Wang and Conover, 1986). An alternative explanation would be semicontinuous pigment ingestion (Mackas and Burns, 1986; Rodriguez and Durbin, 1992) with a discrete and random defaecation of ingested pigments during the night (Runge, 1980; Kleppel et al., 1988). The gut fluorescence method requires prompt identification of the zooplankton sample under a dissecting microscope to avoid possible destruction of chlorophyll pigments during handling. Even experienced scientists with well-trained eyes may not distinguish instantaneously similar species or stages (Tiselius, 1988; Saito and Hattori, 1996). Once numbers of an individual are employed for the gut fluorescence analysis, no further taxonomic identification would provide meaningful information. In contrast to the conventional method with numbers of 163

6 H.Ttkatouji tt at O & 1.0 O Average: s.d.: DAY (n=20) NIGHT (n=30) Fig. 5. Day-night comparison of gut pigment contents of P.marinus adult female. specimens, the present method would provide a definite opportunity for taxonomic identification and size determination after gut fluorescence analysis of the same individual. Acknowledgements We wish to express our gratitude to DrT.Kikuchi who provided all necessary facilities at Manazuru Marine Laboratory, Yokohama National University. Our appreciation is also extended to K.Tobimatsu who calibrated the Turner Design fluorometer. References Bautista3., Harris.R.P., Tranter,P.R.G. and HarbourA (1992) In situ copepod feeding and grazing rates during a spring bloom dominated by Phacocystis sp. in the English Channel. /. Plankton Res., 14, Boyd,C.M., Smith,S.L. and Cowles,T_J. (1980) Grazing patterns of copepods in the upwelling system off Peru. LimnoL Oceanogr., 25, Conover,R J., Durvasula,R., Roy.S. and Wang^R. (1986) Probable loss of chlorophyll-derived pigments during passage through the gut of zooplankton, and some of the consequences. LimnoL Oceanogr., 31,

7 Improved got fluorescence method Dam^I. (1986) Short-term feeding of Temora longicomis Mflller in the laboratory and the field. /. Exp. Mar. BioL EcoL, 99, Dam,H. and Peterson.W.T. (1988) The effect of temperature on the gut clearance rate constant of planktonic copepods. /. Exp. Mar. BioL EcoL, 123,1-14. Durbin,A.G., Durbin,E.G. and WlodarczykJE. (1990) Diel feeding behavior in the marine copepod Acartia tonsa in relation to food availability. Mar. EcoL Prog. Ser., 68, Kleppel.G.S., Pieper^R.E. and Trager.G. (1988) Variability in the gut contents of individual Acartia tonsa from waters off Southern California. Mar. BioL, 97, MackasJ). and Bohrer.R. (1976) Fluorescence analysis of zooplankton gut contents and an investigation of diel feeding patterns. / Exp. Mar. BioL EcoL, 25, Mackas,D.L. and Burns,K.E. (1986) Poststarvation feeding and swimming activity in Calanus pacificus and Metridia pacifica. LimnoL Oceanogr., 31, Mobley.C.T. (1987) Time-series ingestion rate estimates on individual Calanus pacificus Brodsky: interactions with environmental and biological factors. /. Exp. Mar. BioL EcoL, 114, Rodriguez,V. and DurbinJE.G. (1992) Evaluation of synchrony of feeding behaviour in individual Acartia hudsonica (Copepoda, Calanoida). Mar. EcoL Prog. Ser., 87,7-13. RungeJ. A. (1980) Effects of hunger and season on the feeding behavior of Calanus pacificus. LimnoL Oceanogr., 25, Saito,H. and Hattori,H. (1996) Diel vertical migration and feeding rhythm of copepods under sea ice in the Saroma ko Lagoon. /. Mar. Syst., in press. Saito^H. and Taguchi,S. (1996) Diel feeding behavior of neritic copepods during spring and fall bloom in Akkeshi Bay, eastern coast of Hokkaido, Japan. Mar. BioL, 125, Suzuki,R. and Ishimaru,T. (1990) An improved method for the determination of phytoplankton chlorophyll using N,N-dimethylibrmamide. J. Oceanogr. Soc. Jpn, 46, Tande^C.S. and Bamstedt,U. (1985) Grazing rates of the copepods Calanus gladalis and Cfinmarchicus in arctic waters of the Barents Sea. Mar. BioL, 87, Tiselius,P. (1988) Effects of diurnal feeding rhythms, species composition and vertical migration on the grazing impact of calanoid copepods in the Skagerrak and Kattegat. Ophelia, 28, Tsuda^A. and Sugisaki.H. (1994) In situ grazing rate of the copepod population in the western subarctic North Pacific during spring. Mar. BioL, 120, Wang,R. and Conover^RJ. (1986) Dynamics of gut pigment in the copepod Temora longicomis and the determination of in situ grazing rates. LimnoL Oceanogr., 31, Received on May 1,1996; accepted on August 20,