Effect of tremata closures on the oxygen consumption rhythm of ezo abalone Haliotis discus hannai

Aquaculture 270 (2007) 312 – 320 www.elsevier.com/locate/aqua-online

Effect of tremata closures on the oxygen consumption rhythm of ezo abalone Haliotis discus hannai Jung Ah Lee, Jong Wook Kim, Wan Soo Kim ⁎ Marine Ecosystem and Conservation Research Division, Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul 425-600, South Korea Received 26 August 2006; received in revised form 2 April 2007; accepted 3 April 2007

Abstract Cultured adults of ezo abalone, Haliotis discus hannai (shell length, 89.3 ± 6.4 mm and flesh dry weight, 13.0 ± 1.6 g; n = 12) were exposed to different temperatures (10 and 25 °C) to determine whether respiration effects were induced by artificial closure of the second and third tremata (respiratory pores). A respirometer was used to determine the oxygen consumption rates (OCRs) as measures of metabolic activity. The closed tremata were the second and third of the four open tremata anterior to the head of the abalone. The OCRs of starved abalone were measured under constant conditions (CC: constant dark and constant temperature) during a 240-h period, consisting of 120 h before and 120 h after the closure of tremata. The endogenous rhythm of the OCRs in cultured ezo abalone exhibited a dominant circadian rhythm (unimodal rhythm) in the latter half of the experimental period and occasionally showed a weaker but similar circatidal rhythm (bimodal rhythm) in the first half of the experimental period regardless of temperature. The results from the present study indicate that the rhythmicity of the OCRs in starved abalone is not affected by closure of the second and third tremata. This study offers essential physiological information for utilizing tremata in developing a tagging technique in abalone. © 2007 Published by Elsevier B.V. Keywords: Ezo abalone; Haliotis discus hannai; Oxygen consumption; Tremata; Rhythm

1. Introduction Abalone are commercially important species cultured in Northeast Asia. In Korea, the ezo abalone Haliotis discus hannai is a particularly important high-value cultured species (Kang et al., 1996). However, abalone stocks have been decreasing due to overfishing, and some concerned countries are interested in restoring this resource (FAO, 1989). In Korea, several abalone seedlings have been discharged annually into coastal waters in the hope of increasing the recovery rate of this ⁎ Corresponding author. Tel.: +82 314006204; fax: +82 314085934. E-mail address: [email protected] (W.S. Kim). 0044-8486/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2007.04.007

valuable resource. It is necessary to develop effective marking and tagging methods to determine the effectiveness of discharging abalone seedlings into coastal waters (Kim et al., 2002a). Marking and tagging methods can also be used to acquire valuable information in biological studies of these target animals (i.e., growth, mortality, movement, and foraging behavior) (Poore, 1972; Lemarie et al., 2000). Various methods have been attempted to mark and tag abalone (Cox, 1962; Forster, 1967; Harison and Grant, 1971; Quayle, 1971; Poore, 1972; Sainsbury, 1982; Prince, 1991; Kang et al., 1996; Kim et al., 2002a), and these techniques fall into three types: making a hole on the shell; attaching tags to tremata (often called respiratory pores) with wire,

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metal split pins, fishing line, plastic rivets, or nuts and bolts; and attaching tags to the shell with adhesives. If these methods are safe to abalone, the use of any of these tremata techniques is preferable, as they use natural structures formed by the abalone. Good marking and tagging methods must have a minimal effect on the survival, growth, and behavior of tagged animals (Lemarie et al., 2000). When the tremata are used as sites to attach tags and marks, and the attachment is especially firm, parts of the tremata are frequently occluded. However, under natural conditions, the tremata of abalone are often occluded by various attached organisms (Prince, 1991; Voltzow and Collin, 1995). Tremata on shells are the most conspicuous feature of abalone. They are open in the right upper part of the mantle cavity, surrounding the gills and the outlets of various organs (i.e., gut, kidney, and reproductive gland), and are known to assist with respiration, reproduction, and waste removal (Fallu, 1991). It is assumed that these pores facilitate the creation of induced flow or enhance gamete dispersal, as well as maintain sanitation in the mantle cavity (Voltzow and Collin, 1995). Furthermore, elevated excurrent openings such as the tremata of abalone may contribute directly to an animal's reproductive success (Voltzow and Collin, 1995). However, the effects of tremata occlusion on the overall metabolism and condition of abalone were not clear. Generally, organisms possess endogenous rhythms, which are altered or broken down when exposed to severe environmental stress (Kim et al., 1996, 2001, 2004). Therefore, a detailed understanding of the effect of tremata closures was needed at the physiological level because marking and tagging techniques using tremata were potential tools for research and resource management. The purpose of the present study was to examine the effects of closing the second and third tremata on the endogenous rhythm of oxygen consumption in the ezo abalone within the range of its habitable temperature.

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aerated seawater was continuously supplied. They were then transferred into two smaller tanks (100 L) with two different temperatures (10 and 25 °C) for experimental temperature acclimation under laboratory conditions (12-h light [L]: 12-h dark [D]). As initial temperature has been known to influence the oxygen consumption rates (OCRs) of animals, individuals were kept for an additional week at each temperature prior to the experiment (Cox, 1974). Abalone were fed Laminaria sp., but were not fed for the final 2 days of the acclimation period to ensure a post-absorptive digestive state before their introduction into a test chamber. Experimental abalone was held in water with salinities of 31.3 to 32.0 psu. We selected the second and third tremata anterior to the head of the abalone, as their location and time of formation suggested that they were likely to play a major functional role. Two of four open tremata were artificially occluded using T Rubber (rubber septa, ϕ 4.8 mm; Sigma–Aldrich, St. Louis, MO, USA) approximately 120 h after the start of the experiment. Locations of the tremata closures and the T Rubber used for the closures are shown in Fig. 1. We pulled the rubber out through tremata from the inside of the shell, leaving the rubber's short branch hanging out inside of the shell in the experimental chamber. The tremata closures were performed carefully to avoid damaging the mantle of the abalone. All experiments were conducted in triplicate for each temperature regime. 2.2. Experimental design Experiments were carried out for two different experimental regimes based on temperature. In experiment 1, abalone were exposed to constant temperatures

2. Materials and methods 2.1. Abalone Twelve cultured ezo abalone (H. discus hannai ) were used in this study. They were 89.3 ± 6.4 mm (n = 12, mean ± SD) in shell length and 13.0 ± 1.6 g in flesh dry weight. After the epiphytes on their shells were culled by a chisel, the abalone were kept together for 1 week in a circular, indoor 500-L tank (15 °C) into which well-

Fig. 1. Locations of the tremata closures of abalone, Haliotis discus hannai, and the T Rubbers (rubber septa, ϕ 4.8 mm; Sigma–Aldrich, St. Louis, MO, USA) used for closures.

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(10 and 25 °C) under constant darkness to observe the endogenous rhythm in three replicate experiments for the duration (240 h) of the treatment. In experiment 2, abalone were kept at a low temperature (10 °C) and experienced closure of the second and third tremata to observe the effect of the closures; then the OCRs were measured in three replicate experiments for the duration (120–130 h) of the treatment. In experiment 3, these abalone were subjected to closure of the second and third tremata to demonstrate the effects of tremata closure at 25 °C under constant darkness; the OCRs were measured in three replicate experiments for the duration (120 h) of the treatment. The OCRs were measured over approximately 240 h by the automatic intermittent-flow respirometer (AIFR: one system with two chambers) following the procedures described by Kim et al. (1996). The OCRs of a pair of individuals (one system with two chambers) were monitored simultaneously by placing an abalone in each Plexiglas incubator chamber in a semi-circulatory system. Experimental water was filtered free of bacteria through sterile membrane filters (with two Saritorius capsule filters, input 0.2 μm and output 0.07 μm). Oxygen levels in the experimental chambers (1400 mL) were maintained between 85 (lowest) and 97% ( highest) saturation to minimize any physiological stress caused by hypoxia. No measurements were made while flushing the chamber with oxygen saturated seawater from a storage tank (20 L) to restore the oxygen saturation level to 97%. When the oxygen level dropped below the predetermined limit, the drive gear pump and the actuator valve (TX 350-1 DA-1/2, Ilyoung, Korea) supplied the system automatically with saturated seawater until the selected oxygen level was reached.

After each experiment, the chamber was rinsed with oxygen saturated water and the probe voltage was examined to ascertain whether it had deviated from the gauge voltage at the beginning of the experiment. After calibration of the oxygen probe (15 μm PO2, Eschweiler, Germany), the measuring system was computer program. The magnetic drive gear pump (MS-Z; Ismatec SA, Glattbrugg, Switzerland) produced horizontal water flow rates of 517.5 mL/min. Measurement were conducted in a darkened incubator (VS1203P5N, Vison Co., Seoul, Korea) with constant conditions (10 and 25 °C). More detailed descriptions of AIFR, including the location of the probe and a schematic of the apparatus, are provided by Kim et al. (1996, 2002b). 2.3. Statistics The OCRs were analyzed using the weighted smooth curve procedure at 2% individual error. The locally weighted least squares error method (KaleidaGraphy custom program for Macintosh; Synergy Software, Essex Junction, VT, USA) was used to plot a best-fit smooth curve through the center of the data. The values of 2 and 5% individual error obtained from the repeated tests yielded the best-fit curve. Statistical values were computed for each batch from the data points measured (Table 1). Significant differences in mean values were examined by Student's t-test. The OCR rhythm was determined from a maximum entropy spectral analysis (MESA) program using raw data transformed into 20– 40 min lag intervals. MESA was used to estimate the dominant periodicity peaks in the oxygen consumption time series (Dowse and Ringo, 1989). The values presented in this study are the means ± SD.

Table 1 Experimental conditions, parameters, and mean oxygen consumption rates of the unfed abalone (Haliotis discus hannai) used for each control group (no tremata closure during the experiment) and each experimental group (approximately 120 h of tremata closure) at 10 °C and 25 °C 10 °C

Number of experiments (n) Number of abalone per one experiment (N) Mean shell length (mm) Mean flesh dry weight (g ) Salinity (psu) Flow rate (ml min− 1) Oxygen saturation level (%) Duration of the experiment (h) Number of points measured Mean oxygen consumption rate (mL O2/gDW/h) Before the tremata closure After the tremata closure Values are the means ± SD.

25 °C

Control

Experiment

Control

Experiment

3 1 87.1 ± 10.6 12.8 ± 2.1 31.8–32.0 517.5 85.1–96.7 191–240 4303–6325

3 1 84.2 ± 7.1 12.4 ± 1.0 31.2–32.0 517.5 85.1–97.2 240 7527–8271

3 1 92.6 ± 2.1 12.1 ± 1.0 32.0 517.5 85.4–95.6 240 6159–6783

3 1 92.2 ± 1.9 14.3 ± 0.4 31.6–32.2 517.5 85.3–96.2 238–241 5618–6192

0.09 ± 0.01 0.09 ± 0.01

0.08 ± 0.01 0.09 ± 0.02

0.32 ± 0.04 0.35 ± 0.07

0.30 ± 0.02 0.29 ± 0.03

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3. Results 3.1. Experiment 1: constant temperature (at 10 and 25 °C) under darkness The OCR of the abalone at each experimental temperature exhibited rhythmicity throughout the entire experimental period (Fig. 2A). Observed OCRs were highly variable, ranging from 0.02 to 0.17 mL O2/gDW/h at 10 °C and from 0.10 to 0.67 mL O2/gDW/h at 25 °C. The mean OCR (mOCR), averaged separately over the duration of the first half of the experiment and the latter period, were 0.09 ± 0.04 and 0.09 ± 0.05 mL O2/gDW/h, respectively, at 10 °C and 0.22 ± 0.05 and 0.32 ± 0.08 mL O2/gDW/h at 25 °C, respectively. The abalone in the 25 °C control group showed particularly strong rhythmicity involving about five cycles during the latter 120-h period. Similar patterns of endogenous circadian and/or circatidal rhythmicity were observed in the other

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two replicates at each temperature. Total mean OCRs of abalone in the triplicate experiments are shown in Table 1. Fig. 2B and C are MESA presentations of the data from the first (a) and latter (b) period in the control group at 10 °C (Fig. 2A). The OCRs peaked at 12.2- and 24.3-h intervals during the first half of the experiment (120 h) (Fig. 2B) and at 24.2-h intervals during the latter (120 h) period of treatment (Fig. 2C). Fig. 3B and C are MESA presentations of the data from the first (a) and latter (b) period of the control group at 25 °C (Fig. 3A). The OCRs peaked at 12.1- and 24.1-h intervals during the first half (120 h) (Fig. 3B) and at 24.4-h intervals during the latter (120 h) period of treatment (Fig. 3C). 3.2. Experiment 2: effect of the second and third tremata closures at 10 °C The OCRs of abalone were measured for 120 h under constant darkness at 10 °C, and were then measured

Fig. 2. A: Time series of the oxygen consumption rate (OCR: mL O2/gDW/h) of an adult abalone (Haliotis discus hannai) without tremata closures under constant darkness at 10 °C over a 240-h period. Experimental data are divided into 120-h periods, which are represented by the letters a and b for maximum entropy spectral analysis (MESA). B and C: MESA spectra of data from part ‘a’ (the first 120 h) and part ‘b’ (the latter 120 h) presented in A. Period lengths (h) corresponding to the dominant peaks in the MESA plots are given in parentheses.

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Fig. 3. A: Time series of the oxygen consumption rate (OCR: mL O2/gDW/h) of an adult abalone (Haliotis discus hannai) without tremata closures under constant darkness at 25 °C over a 240-h period. Experimental data are divided into 120-h periods, which are represented by the letters a and b for maximum entropy spectral analysis (MESA). B and C: MESA spectra of data from part ‘a’ (the first 120 h) and part ‘b’ (the latter 120 h) presented in A. Period lengths (h) corresponding to the dominant peaks in the MESA plots are given in parentheses.

continuously for 120 h after the closure of the second and third tremata (Fig. 4A). The OCRs of a starved abalone (n = 1) were fitted to a weighted smooth curve of 2%. The OCRs of the abalone exhibited rhythmicity over each 120-h period before and after the tremata closures. The observed OCRs were highly variable, ranging from 0.02 to 0.17 mL O2/gDW/h before the tremata closures and from 0.02 to 0.19 mL O2/gDW/h after the tremata closures. The OCR values averaged for each period before and after the tremata closures were the same: 0.07 ± 0.04 mL O2/gDW/h. The endogenous rhythms of the OCRs were related to diurnal or tidal patterns over both periods before and after the tremata closures. Similar patterns of endogenous circadian and/ or circatidal rhythmicity were observed in the other two replicates. Total mean OCRs of abalone in triplicate experiments are shown in Table 1. Fig. 4B and C are MESA presentations of the data from the periods before (a) and after (b) the tremata closures in the experimental

group at 10 °C (Fig. 4A). The OCRs peaked at 8.9-, 12.1and 24.2-h intervals during the duration of the treatment before the closures (120 h) (Fig. 4B) and at 22.1h intervals during the duration of the treatment after the closures (120 h) (Fig. 4C). 3.3. Experiment 3: effect of the second and third tremata closures at 25 °C The OCRs of abalone were measured for 120 h under constant darkness at 25 °C, and were then measured continuously for 118 h under the closure of the second and third tremata (Fig. 5A). The OCRs of a starved abalone (n = 1) were fitted to a weighted smooth curve of 2%. The OCRs of this abalone exhibited rhythmicity over each period before and after tremata closures, showing particularly strong rhythmicity involving about six cycles during a 142-h period (from November to August) 1 day before the tremata closures. The observed

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Fig. 4. A: Time series of the oxygen consumption rate (OCR: mL O2/gDW/h) of an adult abalone (Haliotis discus hannai) in darkness at 10 °C for 120 h before and 120 h after tremata closures. Experimental data are divided into 120-h periods, which are represented by the letters a and b for maximum entropy spectral analysis (MESA). B and C: MESA spectra of data of part ‘a’ (the first 120 h) and part ‘b’ (the latter 120 h) presented in A. Period lengths (h) corresponding to the dominant peaks in the MESA plots are given in parentheses.

OCRs were highly variable, ranging from 0.15 to 0.75 mL O2/gDW/h before the tremata closures and from 0.15 to 0.75 mL O2/gDW/h after the tremata closures. The OCR values averaged over each period before and after the tremata closures were 0.33 ± 0.11 and 0.32 ± 0.11 mL O2/gDW/h, respectively. Similar patterns of endogenous circadian and/or circatidal rhythmicity were observed in the other two replicates. Total mean OCRs of abalone in the triplicate experiments are shown in Table 1. Fig. 5B and C are MESA presentations of the data recorded during the first 4 days (a) of the experiment and the latter period (b) from 1 day before the tremata closures until the end of the experiment in the experimental group at 25 °C (Fig. 5A). The OCR peaked at 12.2-h intervals during the first 4 days (96 h) (Fig. 5B) and at 14.5- and 24.0h intervals during the latter period (142 h) of the treatment (Fig. 5C).

4. Discussion In this study, the tested abalone was shielded from extrinsic factors such as light, food, temperature and tide, which could affect their rhythmic activities. The endogenous rhythms observed in the control groups showed circadian and/or circatidal rhythms regardless of the experimental temperature. In the wild, adult abalone sometimes graze on seaweed attached to the seabed and other seaweed drifting in the currents, which are important sources of food (Fallu, 1991). It is likely, therefore, that the circadian patterns of the OCRs have been influenced by species-specific sensitivity to light and all subsequent foraging behavior. Studies on certain abalone physiological variables, such as food intake, locomotive activity, and metabolism, have shown that these species are more sensitive to light than to various other environmental factors under natural diurnal cycles

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Fig. 5. A: Time series of the oxygen consumption rate (OCR: mL O2/gDW/h) of an adult abalone (Haliotis discus hannai) in darkness at 25 °C for 120 h before and 118 h after tremata closures. Experimental data are divided into the first 96 h and the latter 142 h, which are represented by the letters a and b for maximum entropy spectral analysis (MESA). B and C: MESA spectra of data for part ‘a’ (the first 96 h) and part ‘b’ (the later 142 h) presented in A. Period lengths (h) corresponding to the dominant peaks in the MESA plots are given in parentheses.

(Uki and Kikuchi, 1975; Jan et al., 1981; Barkai and Griffiths, 1987; Donovan and Carefoot, 1998). According to these studies, abalone species engage in the majority of their activities, such as foraging, at night rather than during the day, and thus show a circadian rhythm of night-accelerated metabolism. However, H. diversicolor supertexta showed no circadian metabolic rhythm under continuous light conditions (Jan et al., 1981). Furthermore, no significant differences in metabolic rates were observed between day and night in an experiment using H. tuberculata under a 12L:12D photoperiodicity, and the reason may have been related to an excess in available food (Peck et al., 1987). Species-specific differences and subtle methodological differences may explain such contradictory results (Chacón et al., 2003). The results of this study suggest that the latter seems to be more reasonable than the former. The behavioral activity of abalone will inevita-

bly be repressed under continuous light conditions, as they would be unfamiliar to such an environment. As in most other marine animals, the biorhythms of abalone are likely to be closely related to foraging. An example from other mollusk species showed that three carnivorous marine gastropods possess similar patterns of circadian respiratory biorhythms, i.e., maxima at diurnal high tide periods (Sivalingam, 1989). These species' respiratory rhythms were related to their feeding habitats. In this study, the tested abalone was cultured from an aquaculture field located on the coast, which experienced tidal currents twice daily. It is likely, therefore, that the circatidal patterns of the OCRs in the abalone were influenced by tidal currents and hydrostatic pressure, even though the abalone lived 2–5 m below the water surface. In this study, the tested abalone had two irregular but coexisting patterns (circatidal and circadian rhythm) in

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OCRs regardless of the experimental period. These are somewhat different results from the consistent results gained in experiments on the Pacific oyster Crassostrea gigas (Kim et al., 2002b) and Washington clam Saxidomus purpuratus (Kim et al., 2003) measured under the same conditions (constant condition: CC) using the same system. These two species were suggested to have two independent clocks that could shift endogenous OCRs from bimodal to unimodal as a means of energy conservation (N20% day− 1 of the energy normally spent on heat production) under constant conditions (Kim et al., 2003). It is generally accepted that a clock's main function is to alert an organism in advance of some periodic environmental event (Palmer, 1996). Although it was assumed to be due to a switch in the energy source of the fasting Washington clam, presumably from carbohydrates to others, as seen in perch (Mehner, 1994). Therefore, we can postulate that when marine organisms are exposed to constant conditions over the long term, their internal clock may alter the rhythmicity of their physiologic processes. The OCR rhythms of abalone in the first several days of this study showed a weak, low-amplitude rhythm contrasting with the strong and clear rhythm observed in previous studies (Kim et al., 1999, 2002b, 2003). However, this rhythm occasionally shifted to a strong and clear rhythm pattern on an individual basis at a certain point in the experiment. It is difficult to explain the weak rhythm shown in the first half of the experimental period in both the control and experimental groups at 25 °C, although the weak rhythms at 10 °C resulted from the inactivity of abalone metabolism under low temperatures. However, this phenomenon may have resulted from the species' cautious responses to sudden changes in their surrounding environment, and thus abalone may respond differently from bivalves. Therefore, further and detailed studies are needed to examine whether the observed phenomenon is a common response in Haliotis. When the OCR rhythms of abalone in the experimental group were compared with those of the control group at each temperature, and when the OCR rhythm after the closures was compared with that from before closures, no rhythm changes were observed to be caused by tremata closure. In this study, two experimental temperatures (10 and 25 °C) were selected to represent the range in water temperature experienced in situ by this species. If any effects of tremata closures occur in abalone, the physiological effects should be more clear at 25 °C than at 10 °C because of the high metabolic activity and the physiological burden caused by exposure to a somewhat high temperature for this species (Uki and Kikuchi, 1975;

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Lee, 2003). However, the rhythm did not change under either temperature. This is supported by the results represented in Fig. 5A, which show that a strong and clear rhythm pattern in the OCRs had already begun by the 4th day of the experiment (without closures) and that this continued during the period of tremata closures. No significant difference in mOCR was observed before and after the tremata closures (P N 0.05). The consistent circadian and/or circatidal rhythm shown by abalone after the tremata closures suggest that these closures did not appreciably affect the functioning of respiratory activity or even respiratory metabolism. According to Voltzow and Collin (1995), who examined the mantle cavity morphology and characterized the respiratory flow of the keyhole limpet Diodora aspera, in which the apical opening was naturally or experimentally blocked for 1 month, no evidence of damage to the mantle cavity or associated organs was observed. They concluded that the excurrent opening may not be necessary for respiratory function in animals with fissurellid pallial morphology but may enhance respiratory exchange by permitting an induced passive flow through the mantle cavity. Some studies have demonstrated induced flow in D. aspera and species of Haliotis (Murdock and Vogel, 1978; Voltzow, 1983; Tissot, 1992). Voltzow (1983) showed that for H. kamtschatkana, water enters passively either posteriorly to the left cephalic tentacle or through one of the anterior tremata, and leaves the mantle cavity via the more posterior tremata. Thus, the results of this study suggest that the closure of the second and third tremata do not cause problems for sanitation because of the open fourth tremata. Abalone can tolerate the effects of blocking tremata with a tag and can sustain a high level of natural interference with their tremata (Prince, 1991). A growth study did not detect any difference between the growth of tagged abalone in situ using a nylon rivet and untagged abalone (Prince, 1989). In conclusion, our results indicate that the rhythm of oxygen consumption in unfed ezo abalone is not affected by the closure of the second and third tremata. However, the effects of tremata closures over long periods, in situ, need to be determined by further studies on processes such as egestion, excretion, reproductive activity, and growth. No information exists about the effects of tremata closures on the dispersal of gametes during reproductive activity in abalone. The ezo abalone is one of the most commercially important species cultured in Northeast Asia, and its seedlings have been discharged annually into the coastal waters of Korea in an attempt to further the recovery of the species. Thus, for systematic resource management purposes, tagging techniques must be developed to

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