in 1-h incubations. Incubation water volume (13 mL vs. 100 mL) did not significantly affect the excretion rate, and the overall mean excretion rate of C. sinicus ...
Plankton Benthos Res 10(1): 75–79, 2015
Plankton & Benthos Research © The Plankton Society of Japan
Note
Short-term variation in the Calanus sinicus ammonium excretion rate during the post-capture period TAKETOSHI KODAMA1,2,*, K AZUTAKA TAKAHASHI3, K EN-ICHI NAKAMURA3, SHINJI SHIMODE4, TAKAFUMI YAMAGUCHI4 & TADAFUMI ICHIKAWA1 1
National Research Institute of Fisheries Science, Fisheries Research Agency, 2–12–4, Fukuura, Kanazawa, Yokohama, Kanagawa 236–8648, Japan 2 Present Address: Japan Sea National Fisheries Research Institute, Fisheries Research Agency, 1–5939–22, Suido-cho, Chuou, Niigata, Niigata, 951–8121, Japan 3 Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1, Yayoi, Bunkyo, Tokyo, 113–8657, Japan 4 Manazuru Marine Center for Environmental Research and Education, Graduate School of Environment and Information Sciences, Yokohama National University, 61, Iwa, Manazuru, Kanagawa, 259–0202, Japan Received 4 December 2014; Accepted 15 December 2014
Abstract: Short-term variation in the ammonium excretion rate by Calanus sinicus during the post-capture period was evaluated in 1-h incubations. Incubation water volume (13 mL vs. 100 mL) did not significantly affect the excretion rate, and the overall mean excretion rate of C. sinicus was similar to reported values from congeneric species. However, the 24-h variations in the excretion rate denoted that acclimation stress decreased the excretion rates 1–2 h after introduction to a no-food condition. The effect of presence/absence of food was not significant up to 24 h after capture. Key words: acclimation, ammonium excretion, Calanus sinicus, short-term incubation, starvation
Nitrogen excreted by zooplankton is an important source for primary and bacterial production in the ocean (Harris 1959), comprising 5–40% of their requisite nitrogen (Hernández-León et al. 2008). Ammonium is the major form of excreted nitrogen, exceeding 50% of the total excreted nitrogen; thus, various studies have been conducted regarding the ammonium excretion process in zooplankton (Ikeda et al. 2000, Le Borgne 1986, Steinberg & Saba 2008). Most studies have reported a decrease in metabolic rates of zooplankton over time (Gardner & Paffenhöfer 1982, Ikeda et al. 2000, Le Borgne 1986). Since most metabolic measurements are conducted using the traditional sealed-chamber method under no-food conditions for relatively long incubation periods (∼24 h), starvation is considered to be the main cause of the observed reduction in metabolic rates (Atkinson & Whitehouse 2001, Ikeda et al. 2000). To overcome this problem, measurement immediately upon capture has been suggested as a more realistic approach to assess field metabolic rates (Ikeda et al. 2000). Although experiments with short incubation times are ideal to minimize the starvation effect, stress induced by the capture, handling, and acclimation process could also affect the metabolic rate, particularly dur* Corresponding author: Taketoshi Kodama; E-mail, takekodama@affrc. go.jp
ing the first hours of incubation (Le Borgne 1986). In this light, accurate understanding of short-term variation in the excretion rates during incubation is required in order to accurately assess the role of zooplankton in marine ecosystems, however the relatively low sensitivity of traditional analytical methods has hindered this attempt. Recently, highly sensitive ammonium analytical methods were successfully applied to estimate the ammonium excretion rate by Antarctic krill with short-term incubation times (＜3 h), while only a few similar attempts have been made thus far with respect to excretion by copepods. In the present study, we applied a highly sensitive analytical method to a planktonic copepod, Calanus sinicus, which is the major macrozooplankton component in Japanese coastal areas (e.g., Uye 2000), in order to examine short-term variations in their ammonium excretion rates under experimental conditions. The animals and seawater for determination of the ammonium excretion rate were collected in the morning (1000– 1100) of March 16, 2014 on R/V Tachibana of the Manazuru Marine Center for Environmental Research and Education (MMCER), Yokohama National University, at 35°08.9′N 139°10.5′E in the western part of the Sagami Bay in the south of Japan. The animals were collected by vertical hauls from 50 m to the surface using a 0.45-m-diameter plankton net (330-μm mesh, 3 L cod-end), which was towed slowly
76
T. Kodama et al.
(＜0.2 m sec−1). The collected animals were kept in an insulating container containing the in situ surface seawater until on-land sorting. The spring bloom of diatoms occurred at the study site, where chlorophyll a concentration was ＞5 μg L−1, and the copepods were allowed to feed on diatoms in the container until sorting on-land in the laboratory. The seawater used for incubations was collected from the surface and stored in acid-washed polyethylene tanks. Intact and healthy adult female C. sinicus individuals were sorted on-land in the laboratory close to the sampling station under dissecting microscopes within 1.8 h after collection. The sorted C. sinicus were transferred into ten 2-L bottles, which contained 1.5 L of 0.2-μm filtered surface seawater (FSW), and 5–6 individuals were pre-incubated in each bottle at the in situ sea surface temperature (13°C) in the darkness (Fig. 1), except for individuals that were used to determine the excretion rates under fresh conditions (t0, described in detail
Fig. 1. Experimental design of the present study. Pre-incubation: 5 or 6 copepods were incubated in 1.5 L of filtered seawater (FSW, starved) or 1.5 L of FSW with T. weissflogii (Fed) until just before sorting in the darkness at 13°C. Sorting: the pre-incubated individuals were poured into a large Petri dish and the copepod was picked up using a wide-bore pipette and washed twice with FSW. Bottle size experiment: the sorted, fed copepods were individually incubated in 13 mL or 100 mL of FSW for 1 h in the darkness at 13°C. Time course experiment: the sorted, starved or fed copepods were individually incubated in 13 mL of FSW for 1 or 3 h in the darkness at 13°C.
below). After 1–24 h of pre-incubation, C. sinicus individuals were gently transferred into a large Petri dish, individually picked up using a wide-bore pipette, washed twice with FSW, and placed into the experimental bottle for two types of excretion experiments (Fig. 1). After 1-h incubation in darkness at 13°C, seawater was sampled for the determination of ammonium concentration. All individuals were alive during the pre-incubation and excretion experiments. After the experiments, the individuals were fixed by 10% formaldehyde, and their prosome lengths (PLs) were measured under a microscope. The mean (±SD) PL of C. sinicus used in this study was 2.350±0.114 mm. Nitrogen contents of individual copepods were estimated on the basis of PL, according to Uye (1982). First, we evaluated the effect of bottle size on excretion rates. For this experiment, 15-mL (13 mL FSW) and 110-mL (100 mL FSW) bottles were prepared (Fig. 1). Copepods used in this experiment were fed the cultured diatom Thalassiosira weissflogii (∼2000 cells mL−1) for 2 h after pre-incubation (20 h) in the 2-L bottles. The pre-incubation bottles were gently shaken every 30 min so that the diatoms stayed suspended in the bottle. Five replicates, containing one individual each, and three controls (only FSW) were prepared for each bottle size. Second, a time course experiment was conducted using the 15-mL bottles to determine the effect of starvation and stress under the experimental conditions during the first 24 h after capture (Fig. 1). This measurement was conducted 6 times: at 1, 2, 3, 6, 12, and 24 h after the pre-incubation period (hereafter referred to as t1, t2, t3, t6, t12, and t24, respectively), and 4–6 replicates with 3 controls were prepared for each treatment. Before t6 and t12, a group of C. sinicus, fed T. weissflogii, was also prepared in the same manner as established for the bottle size-effect experiment, and the result of the bottle size-effect experiment was used as the excretion rate of fed C. sinicus at t24. The guts of the fed C. sinicus were filled with T. weissflogii-like substances just before the excretion experiments. The excretion rates of freshly collected individuals without pre-incubation were also measured. Copepods were immediately sorted from the container upon arrival at the laboratory and incubated for 1 h and 3 h in the same manner as described above (t0–1h and t0–3h, respectively). Ammonium concentration in the subsamples was determined soon after every experiment using a highly sensitive colorimetric system (Kodama et al. in press). The minimum and maximum detection limits were 84 and 1.40×105 ng N·L−1, respectively. The ammonium excretion rate was calculated by the difference in concentration between the incubated and control bottles. The mean weight-specific ammonium excretion rates by C. sinicus incubated in 13 mL and 100 mL water were 2.51± 0.47 and 2.65±0.92 ng N·μg body N−1·h−1 (54.5±12.1 and 66.7±23.8 ng N·ind−1·h−1), respectively (Fig. 2, both n＝5), although the difference was not significant (p＞0.1, Student s t-test). The ammonium excretion rate of freshly collected individ-
Ammonium excretion of Calanus sinicus
uals was 2.89±0.68 ng N·μg body N−1·h−1 (57.5±12.7 ng N·ind−1·h−1) for the first hour (t0–1h), and significantly decreased to 1.88±0.56 ng N·μg body N−1·h−1 (40.1±10.1 ng N·ind−1·h−1) in the 3-h incubation (t0–3h, Fig. 3) (both n＝6, ttest, p＜0.05). The ammonium excretion rate of starved C. sinicus (pre-incubated in FSW) was also low at t1 (1.73±0.61 ng N·μg body N−1·h−1, 38.3±15.7 ng N·ind−1·h−1, n＝5), but increased at t2, and varied between 2.94±0.87 and 4.01±1.01 ng N·μg body N−1·h−1 (70.4±22.2 and 89.7±22.8 ng N·ind−1·h−1) until t24. The excretion rate was not related to
Fig. 2. Excretion rates by C. sinicus estimated from 1-h incubations in 13 mL and 100 mL water. The error bars denote the standard deviation (n＝5).
77
body nitrogen content (F-test, p＞0.1), and pre-incubation period significantly affected the excretion rates (ANOVA, p＜0.05). Tukey s post-hoc test showed that the differences between the lowest rates (t1) and the highest (t6: 4.01±1.01 ng N·μg body N−1·h−1) or the second highest (t12: 3.92±1.00 ng N·μg body N−1·h−1) were significant (p＜0.01). The incubations at t6 and t12 corresponded to the local nighttime (Fig. 3), although the difference between day (t2, t3, and t24) and night (t6 and t12) was not significant (t-test, p＞0.1). The excretion rate in C. sinicus, fed diatoms, varied from 3.39±0.62 (t6) to 4.21±0.77 ng N·μg body N−1·h−1 (t12, Fig. 3) (from 70.8±13.2 to 91.2±16.6 ng N·ind−1·h−1), and no significant difference was observed from those of starved individuals at any time point (t-test, p＞0.1). The weight-specific excretion rates measured in the present study were 1.73±0.61–4.21±0.77 ng N·μg body N−1·h−1 (Fig. 3). To our knowledge, this is the first report of the excretion rate in C. sinicus; our data are comparable to rates evaluated in Calanus helgolandicus incubated at 15°C for 2–3 h after collection (1.6±0.53–4.3±1.4 ng N·μg body N−1·h−1; Harris and Malaj 1986), suggesting that our method could be used to accurately measure the ammonium excretion rate of a single individual with 1-h incubation. The excretion rate was not significantly different between C. sinicus incubated in different sized bottles (Fig. 2). The effect of crowding and confinement on metabolic rate measurements in copepods could vary depending on species-specific characteristics in their physiology and behavior (Ikeda et al. 2000). In agreement with our results, no appreciable effect of container size on metabolic rate was previously found in Calanus finmarchicus (Marshall & Orr 1958, Zeiss 1963). In the no-food condition, Calanus might be less sensitive to container size because of its rise/sink intermittent swimming activity (Krefft 1991), which does not require a large volume of
Fig. 3. Temporal variation of the ammonium excretion rate in C. sinicus. The rates of starved individuals (crosses), fed individuals (open circles), and freshly collected individuals incubated for 1 h (t0–1h; grey square) and 3 h (t0–3h; open square) are shown. The vertical bars denote the standard deviation. The nighttime period is indicated by the thick, black solid line along the x-axis. The horizontal widths of the symbols denote the start and end times of incubations.
78
T. Kodama et al.
water. Most previous studies that reported significant effects of animal density on metabolic rates were associated with crowding, as multiple individuals were incubated in a single container (Hargrave & Geen 1968, Nival et al. 1974). Even though the water volume available per specimen was limited in the present study, short-term measurement of metabolic rates of single individuals appears to be an advantageous approach to avoid unnatural individual interactions such as perception of stimuli, rapid depletion of oxygen, and associated accumulation of excreta (Ikeda & Skjoldal 1980, Ikeda et al. 2000). In the time course experiments, the excretion rates were clearly reduced within ∼1–2 h after the start of incubation. Some studies have noted decreases in the metabolic rates of zooplankton over time after capture, and capture stress and starvation have been suggested as the primary causes (Ikeda et al. 2000, Le Borgne 1986). However, neither capture stress nor starvation could explain the decrease observed in this study, since the rate increased again after this initial period. Therefore, we attributed the initial decrease of the excretion rates to the acclimatization stress of the animals to the nofood experimental condition. Båmstedt & Tande (1985) reported a similar temporal reduction in ammonium excretion rate by Calanus glacialis adult females 1–2 h after the start of excretion experiments, and Omori & Ikeda (1976) also reported a temporal reduction in metabolic activity soon after collection. Thereafter, the copepods seemed to become acclimated to the experimental condition, since the excretion rates recovered at 2–3 h after the start of incubation. No significant difference between the feeding and no-feeding incubations at t6 – t24 was observed. This result was consistent with that reported for Calanus pacificus (Miller & Landry 1984), although a number of authors reported higher excretion rates in fed animals than in non-fed animals (Atkinson & Whitehouse 2001, Gardner & Paffenhöfer 1982, Ikeda 1976, Lehette et al. 2012, Vanderploeg et al. 1986). Therefore, the contradictory results in this study may be attributed to the specific physiological characteristics of Calanus; for instance, the degree of starvation may not be evident at relatively low temperatures during 24 h in experimental animals that tend to store a large amount of lipid reserves under conditions of phytoplankton blooms (e.g. Gatten et al. 1980). In copepods or cladocerans, the ammonium excretion rate generally decreases ＜1.5 h after phytoplankton feeding (Gardner & Paffenhöfer 1982, Ikeda et al. 1982, Vanderploeg et al. 1986); therefore, Ikeda et al. (2000) suggested that the animals should be used immediately after capture in order to minimize the effect of starvation and accurately evaluate the natural excretion rate. On the basis of results from using a short-term incubation method, Lehette et al. (2012) found that the field rate of ammonium excretion in Antarctic krill was two times higher than previously thought, while the effect of stress by experimental manipulation during short incubation times was negligible. On the other hand, our results demonstrate that starvation did not affect the excretion rates in C.
sinicus at least for 24 h, while the initial acclimation process to experimental conditions could have a serious effect on the measurements, particularly in the case of short-term incubations. It thus appears that the short-term variation of zooplankton metabolism under experimental conditions is species-specific, and there are still many uncertainties related to their physiological condition, particularly at the initial phase of incubation. Therefore, establishment of an appropriate incubation period for each species, that takes into account the possible effects of capture, starvation, and manipulation stress is key to evaluate the natural excretion rate. Further research using the highly sensitive analysis with a short-term incubation method adopted in this study would be beneficial to discriminate the effects of such artifacts under captivity and to determine the excretion rate of zooplankton in the field. Acknowledgements We are grateful to the staff of the MMCER for their support to use the laboratory facilities. Prof. Ken Furuya allowed us to use the highly sensitive analysis system. Ms. Sachiko Horii provided the cultured T. weissflogii. This work was financially supported by the Fisheries Research Agency (T.K. and T.I.), and by grants from the Ministry of Education, Culture, Sports, Science and Technology to K.T. (24310007, 24121001, and 24121005) and to S.S. (25450255). Reference Atkinson A, Whitehouse MJ (2001) Ammonium regeneration by Antarctic mesozooplankton: an allometric approach. Mar Biol 139: 301–311. Båmstedt U, Tande KS (1985) Respiration and excretion rates of Calanus glacialis in arctic waters of the Barents Sea. Mar Biol 87: 259–266. Gardner WS, Paffenhöfer GA (1982) Nitrogen regeneration by the subtropical marine copepod Eucalanus pileatus. J Plankton Res 4: 725– 734. Gatten RR, Sargent JR, Forsberg TEV, Ohara SCM, Corner EDS (1980) On the nutrition and metabolism of zooplankton. XIV. Utilization of lipid by Calanus helgolandicus during maturation and reproduction. J Mar Biol Ass UK 60: 391–399. Hargrave BT, Geen GH (1968) Phosphorus excretion by zooplankton. Limnol Oceanogr 13: 332–342. Harris E (1959) The nitrogen cycle in Long Island sound. Bull Bingham Oceanogr Collect, 17: 31–65. Harris RP, Malej A (1986) Diel patterns of ammonium excretion and grazing rhythms in Calanus helgolandicus in surface stratified waters. Mar Ecol Prog Ser 31: 75–85. Hernández-León S, Fraga C, Ikeda T (2008) A global estimation of mesozooplankton ammonium excretion in the open ocean. J Plankton Res 30: 577–585. Ikeda, T (1976) The effect of laboratory conditions on the extrapolation of experimental measurements to the ecology of marine zooplankton. I. Effect of feeding conditions on the respiration rate. Bull Plankton Soc Jpn 23: 51–60. Ikeda, T, Skjoldal, R (1980) The effect of laboratory conditions on the extrapolation of experimental measurements to the ecology of marine zooplankton. Vl. Changes in physiological activities and biochemical components of Acartia australis and Acetes sibogae australis after capture. Mar Biol 58: 285–293. Ikeda T, Fay EH, Hutchinson SA, Boto GM (1982) Ammonia and inor-
Ammonium excretion of Calanus sinicus ganic phosphate excretion by zooplankton from inshore waters of the Great Barrier Reef, Queensland. 1. Relationship between excretion rates and body size. Aust J Mar Fresh Res 33: 55–70. Ikeda T, Torres JJ, Hernández-León S, Geiger SP (2000). Metabolism. In: ICES Zooplankton Methodology Manual (eds Harris R, Wiebe P, Lenz J et al.). Academic Press, San Diego, pp. 455–532. Kodama T, Ichikawa T, Hidaka K, Furuya K (in press) A highly sensitive and large concentration range colorimetric continuous flow analysis for ammonium concentration. J Oceanogr. DOI: 10.1007/s10872-0140260-6 Krefft KP (1991) The swimming behavior of the copepod Calanus marshallae under various food conditions. Master thesis. Oregon State University, USA. Le Borgne R (1986). The release of soluble end products of metabolism. In: The Biological Chemistry of Marine Copepods (eds Corner EDS and O Hara SCM). Clarendon Press, New York, pp. 109–164. Lehette P, Tovar-Sanchez A, Duarte CM, Hernández-León S (2012) Krill excretion and its effect on primary production. Mar Ecol Prog Ser 459: 29–38. Marshall SM, Orr AP (1958) Some uses of antibiotics in physiological experiments in sea water. J Mar Res 17: 341–348. Miller CA, Landry MR (1984) Ingestion-independent rates of ammo-
79
nium excretion by the copepod Calanus pacificus. Mar Biol, 78: 265– 270. Nival P, Malara G, Charra R, Palazzol.I, Nival S (1974) Study of respiration and excretion of some planktonic copepods (Crustacea) in upper layers along Morrocan coast line. J Exp Mar Biol Ecol 15: 231–260. Omori M, Ikeda T (1976) Doubutsu purankuton seitai kenkyuu hou [The methods of zooplankton ecology]. Vol. 5, Kyoritsu, Tokyo. (in Japanese) Steinberg DK, Saba GK (2008) Nitrogen consumption and metabolism in marine zooplankton. In: Nitrogen in the Marine Environment, 2nd Edition (eds Capone DG, Bronk DA, Mulholland MR et al.). Elsevier, Amsterdam, pp. 1135–1196. Uye S (1982) Length-weight relationships of important zooplankton from the Inland Sea of Japan. J Oceanogr Soc Jpn 38: 149–158. Uye S (2000) Why does Calanus sinicus prosper in the shelf ecosystem of the Northwest Pacific Ocean? ICES J Mar Sci 57: 1850–1855. Vanderploeg HA, Laird GA, Liebig JR, Gardner WS (1986) Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method. J Plankton Res 8: 341–352. Zeiss FR (1963) Effects of population densities on zooplankton respiration rates. Limnol Oceanogr 8: 110–115.
