Short Report Difference in the hypoxia tolerance of the round crucian carp and largemouth bass: implications for physiological refugia in the macrophyte zone

Hiroki Yamanaka1**, Yukihiro Kohmatsu2, and Masahide Yuma3

1 Center for Ecological Research, Kyoto University, 2-509-3 Hirano, Otsu, Shiga 520-2113, Japan 2 Research Institute for Humanity and Nature, 457-4 Motoyama, Kamigamo, Kita-ku, Kyoto 603-8047, Japan 3 Department of Environmental Solution Technology, Faculty of Science & Technology, Ryukoku University, 1-5 Seta-Oe, Otsu, Shiga 520-2194, Japan * Present address: Research Institute for Humanity and Nature (RIHN), 457-4 Motoyama, Kamigamo, Kita-ku, Kyoto 603-8047, Japan (e-mail: [email protected])

Received: August 28, 2006 / Revised: November 28, 2006 / Accepted: December 1, 2006

Abstract The hypoxia tolerance of larval and juvenile round crucian carp, auratus gran- Ichthyological doculis, and largemouth bass, salmoides, was determined using respirometry to examine the potential of hypoxic areas in the macrophyte zone as physiological refugia for round crucian carp. Research The tolerance, which was measured as the critical oxygen concentration (Pc), was 1.32 mg O /l in the ©The Ichthyological Society of Japan 2007 2 round crucian carp and 1.93 mg O2/l in the largemouth bass. As the round crucian carp tolerated Ichthyol Res (2007) 54: 308–312 hypoxia better than the largemouth bass, hypoxic areas in the macrophyte zone might function as physiological refugia for round crucian carp. DOI 10.1007/s10228-006-0400-0

Key words Hypoxia tolerance · Critical oxygen concentration (Pc) · Physiological refugia · Carassius auratus grandoculis · Micropterus salmoides

or many larval and juvenile fi shes, the macrophyte early life history (Miura, 1966; Yuma et al., 1998). This Fzone is essential. It harbors large numbers of food observation suggests that cyprinid fi shes dwelling in the organisms (Petr, 2000; Grenouillet et al., 2002) and func- macrophyte zone have the physiological potential to endure tions as physical refugia in which the structural complexity hypoxic conditions. For example, Fujiwara et al. (1995) of macrophyte stands serves as a barrier to predators reported that the larvae of the round crucian carp Carassius (Savino and Stein, 1982; Werner and Hall, 1988; Warfe and auratus grandoculis use the innermost part (nearest to Barmuta, 2004). However, as the predator population shore) of the macrophyte zone, where hypoxia is severe. naturally contains individuals of various sizes, not all are Despite this ecological information, no physiological study excluded by macrophyte stands, and prey species in the has compared the hypoxia tolerance of cyprinid fi shes macrophyte zone are always exposed to the threat of small and their predators, including introduced largemouth bass, predators. Micropterus salmoides. The macrophyte zone tends to be hypoxic because of the In this study, the hypoxia tolerance of round crucian carp decomposition of macrophytes and oxygen consumption by and largemouth bass was measured experimentally using bacteria (Petr, 2000). Because dissolved oxygen (DO) is one respirometry and compared to examine the potential of the of the most important environmental factors affecting fi sh macrophyte zone as physiological refugia for indigenous habitat utilization, a difference may exist in the species or cyprinid fi sh. The largemouth bass was selected as a poten- size composition of fi sh between the inside and outside of tial predator of cyprinids. It has depleted the indigenous fi sh the macrophyte zone. Chapman et al. (1996) compared the community in many bodies of water in Japan (Maezono and fi sh community in the hypoxic macrophyte zone and a well- Miyashita, 2003; Yonekura et al., 2004), and thus it has been oxygenated open area in Lake Nabugabo, Uganda, and recognized as a serious problem. The round crucian carp found that the hypoxic conditions in the macrophyte zone was used to represent the potential prey of the largemouth might protect endemic species from the introduced Nile bass, as it spends much time in the macrophyte zone during perch, Lates niloticus. As hypoxia tolerance differs among its larval and juvenile stages, from early spring to late fi sh species (Davis, 1975), it is quite possible that the mac- summer (Miura, 1966). Because largemouth bass also rophyte zone provides physiological refugia for hypoxia- inhabit the shallow littoral zone (including the macrophyte tolerant prey. zone), these two species must be associated in a predator– Many fi shes in , Japan, inhabit the macrophyte prey relationship, making them suitable for a pilot study zone (Miura, 1966), and most of the indigenous cyprinid of physiological refugia in the macrophyte zone in Lake fi shes have a profound association with this zone in their Biwa. Implications for physiological refugia 309

Materials and Methods for round crucian carp and 2–7 days for largemouth bass. There is little information on the details of acclimation time Fish metabolism was measured as the routine metabolic for the larvae and juveniles of the two species with large rate (RMR; Fry, 1971), which is the mean rate for fi sh swim- variety of body size as used in the present study; thus, the ming voluntarily, i.e., normal activity, so it refl ects the acclimation time was decided by referring to Oikawa et al. oxygen consumption of fi sh under natural conditions (Fry, (1991), which set the period depending on the size of each 1971; Saint-Paul, 1984). Under normoxic conditions, fi sh individual of the sea bream Pagrus major for measuring consume oxygen at a constant rate (the RMR level in this oxygen consumption. All the fi sh were used for the experi- case), independent of the ambient DO. As the ambient DO ment in the fasting state, with an empty gut. Measurements starts to decrease, fi sh can maintain a constant level to some were made using a semiclosed respirometry system similar extent. However, below a specifi c DO level, oxygen con- to that described by Oikawa et al. (1991). Respiration sumption starts to decrease and becomes oxygen dependent chambers ranging in size from 30 to 4650 ml were prepared with further decrements in the DO (Fry, 1971). The thresh- for fi sh of different sizes. The system can operate four res- old DO level is called the critical oxygen concentration (Pc; piration chambers simultaneously. Each chamber was Ultsch et al., 1978; Saint-Paul, 1984). The Pc can be inter- equipped with an oxygen electrode (BRM-5; Iijima Elec- preted as the threshold level in the ambient DO below tronics, Gamagori, Japan). To avoid stimulating the fi sh, a which fi sh cannot maintain their normal activity. Pc and DO blind was placed around the respirometry system. are both expressed in mg O2/l, so Pc can be applied to fi eld After temperature acclimation, fi sh were carefully trans- DO distribution data directly to quantify the macrophyte ferred into each chamber. The number of fi sh placed in a zone as physiological refugia. Round crucian carp probably single chamber was determined so as to result in a constant −1 −1 survives under more severe hypoxic conditions, less than oxygen depletion speed of 1 mg O2 l h across the experi- the Pc, by adopting anaerobic metabolism, as does the ments. To facilitate acclimation to the chamber, the fi sh European crucian carp Carassius auratus (see Nilsson and were left in the chamber overnight. During this period, Renshaw, 2004), which is a related species. However, the chamber was supplied with oxygen-equilibrated water. anaerobic metabolism is an emergent way to withstand Then, the water supply was stopped and the DO in the anoxia, and metabolic condition will be depressed consider- chambers was measured at 5-min intervals. Three of the ably (Smith et al., 1996). Pc, used in the present study, is an four chambers were used to measure fi sh oxygen consump- indicator of hypoxia tolerance within the aerobic metabolism tion and the last one was kept as a control to measure the and a threshold DO level above which the normal background oxygen consumption by bacteria in the water. metabolism can be maintained. Therefore, the Pc can be a The result for the empty chamber was used to calibrate the suitable indicator to judge the specifi c DO condition as to fi sh oxygen consumption. The DO in the chambers decreased whether it is physiologically preferable and is comparable with fi sh respiration, and the difference between the initial among aquatic animals regardless of whether the animal and fi nal DO values for each measurement was noted as can employ anaerobic metabolism. the mass-specifi c oxygen consumption (mg O2/g-ww/h). The Round crucian carp were obtained from a fi sh farm in mean of the decrease in DO during an interval was consid- Takashima, , Japan. They were the fi rst ered to approximate the DO at which oxygen consumption fi lial generation of wild round crucian carp from Lake Biwa. occurred. During the experiment, fi sh condition was moni- Largemouth bass were caught from a reservoir in Otsu, tored through a slit in the blind, and the experiment was Shiga Prefecture, Japan, by angling and cast netting. The continued until either the fi sh lost its equilibrium or no body weight (BW) range of the round crucian carp used was more DO decrease was detected during a measurement 0.025–5.115 g wet weight (ww) and the range of standard interval. With the experimental system used in the present length (SL) was 9.75–51.42 mm; included were postlarval to study, the each chamber was closed during the measurement young-of-the-year juvenile fi sh found in the macrophyte of oxygen consumption; therefore, the partial pressure of zone (ca. 50 mm; Miura, 1966). In contrast, the BW range CO2 (Pco2) was probably increased by fi sh respiration. Thus of largemouth bass was 1.085–34.350 g ww and the SL range the result of the oxygen consumption might be affected by was 36.08–116.05 mm. The smallest size chosen was the size the Pco2; however, the ranking of Pc among the two species at which largemouth bass start to prey on fi sh. The largest would not be affected. size selected had larger gape than the body depth of the A graph of oxygen consumption was generated for the largest round crucian carp used. Therefore, it could prey on DO data from each chamber, and Pc was determined using all the round crucian carp used in this experiment. Because the method of Yeager and Ultsch (1989). All data points this study focused on physiological refugia in the macro- were fi tted using the two best-fi t regression lines that had phyte zone, the small individuals of largemouth bass used the smallest sum of the residual sums of squares of each could enter the macrophyte zone without being blocked by line, and the intersection of the two lines was regarded as the structural complexity of the macrophyte stands. Pc. All Pc and BW data were transformed to the common

Oxygen consumption by the fi sh was measured at 30°C, log, and log10Pc was regressed against log10BW for round which is the usual water temperature in the macrophyte crucian carp and largemouth bass separately because the zone during midsummer in Lake Biwa (Yamanaka, per- hypoxia tolerance of fi sh depends on body size in several sonal observation). Before the experiments, the fi sh were species (Almeida-Val et al., 2000; Robb and Abrahams, acclimated to the experimental temperature for 1–7 days 2003). Next, analysis of covariance (ANCOVA) was pre- 310 H. Yamanaka et al.

tions at or near the Pc of largemouth bass, they likely cannot prey because bass are actively swimming predators that need a great deal of energy to seek and attack their prey. In fact, largemouth bass even reduced their maximum sustained swimming speed at a relatively high DO of 5–

6 mg O2/l at 25°C (Dahlburg et al., 1968), far higher than the Pc we measured. Moreover, Burleson et al. (2001) indicated

that largemouth bass avoid a DO below about 2.24 mg O2/l at 23.7°C. Consequently, the hypoxic area with DO levels between the Pc values of the two species probably serves as potential physiological refugia for round crucian carp that only the prey can use. Few studies have examined the DO distribution with fi ne resolution in the macrophyte zone of Lake Biwa, although Fujiwara et al. (1995) observed the DO distribution in a macrophyte zone dominated by the reed austra- lis along the shore in Lake Biwa. In all observations, DO was very low (sometimes nearly normoxia) in the innermost part of the zone (near the shore) and gradually increased Fig. 1. Relationship between log10-transformed body weight (BW) and toward the edge of the zone (the offshore side), forming a critical oxygen concentration (Pc) in round crucian carp (black DO gradient. The DO at the edge was at least more than symbols) and largemouth bass (white symbols). Developmental stage approximately 50% of the air saturation level (ca. 4 mg O2/l of round crucian carp is indicated along with BW as additional at 30°C). Therefore, a DO range between the Pc values of information round crucian carp and largemouth bass, 1.32–1.93 mg O2/l, exists in the macrophyte zone. The difference in the hypoxia formed to determine whether log10Pc differed signifi cantly tolerances of the two species measured in this study proba- between the two species. bly creates physiological refugia in the fi eld. A conceptual diagram of physiological refugia in the macrophyte zone is illustrated in Fig. 2. By applying the Pc of prey and predator Results to the DO gradient (Fig. 2a), the area in the macrophyte zone can be divided into three parts (Fig. 2b): the area

In each species, log10Pc showed a signifi cant fi t to log10BW, unavailable to both the prey and predator, physiological and a negative relationship was observed between log10Pc refugia for prey, and the area available to both the prey and and log10BW in both species (Fig. 1; round crucian carp: predator. = = 2 = = log10Pc 0.087–0.090 log10BW, n 12, r 0.381, P 0.033; Structural complexity alone cannot keep out all preda- = = 2 = largemouth bass: log10Pc 0.357–0.089 log10BW, n 6, r tors; small individuals, such as used in the present study, can = 0.847, P 0.009). Although the rate of change in log10Pc probably enter the macrophyte zone. However, even these relative to log10BW did not differ between the species, selected individuals are not completely free to predate

ANCOVA revealed that log10Pc differed signifi cantly be- on their prey because they will be again affected by the = = tween the species (F1,14 16.668, P 0.001) and the mean structural complexity to be forced to reduce their of log10BW-adjusted log10Pc was lower in round crucian carp effi ciency (Bartholomew et al., 2000). Moreover, the inter- ± ± than in largemouth bass [0.119 0.134 vs. 0.286 0.074 mg O2/l ference effect to the predator’s activity is more intensive (mean ± SD)]; that is, the antilog of the mean was 1.32 and in larger individuals (Bartholomew et al., 2000); i.e., the

1.93 mg O2/l in round crucian carp and largemouth bass, physical effect of the macrophyte stands is less on smaller respectively. predators. Conversely, the effect of hypoxia at banning predators is greater in small individuals than larger ones, because the Pc of largemouth bass was higher in smaller Discussion individuals than in larger ones (Fig. 1); i.e., the physiological effect of the hypoxic condition is greater in smaller The Pc of round crucian carp was lower than that of large- predators. Therefore, the intensity of the physical and mouth bass, suggesting that round crucian carp better toler- physiological effect will change inversely in accordance with ate hypoxia and that they have a physiological advantage the increment of the body size of the predator. Consequently, over largemouth bass under hypoxic conditions. Predators the two effects contribute to the function of the macrophyte that tolerate hypoxia less well will manifest more profound zone by complimenting each other. For instance, large behavioral changes than more tolerant prey (Robb and individuals of largemouth bass would have enough hypoxia Abrahams, 2002). Consequently, prey will have a large tolerance to exploit the macrophyte zone; however, they advantage by inhabiting the hypoxic area that is stressful to could not attain high predation effi ciency because of their the predator, reducing the magnitude of the predation pres- body size, and as a result they are most likely not to select sure (Robb and Abrahams, 2002). Under hypoxic condi- the hypoxic and structurally complex habitat. The relative Implications for physiological refugia 311

level and ground slope, is clarifi ed, it might be possible to enlarge the potential size of refugia for indigenous fi shes by changing the water management of a lake or creating a lakeshore with suitable landforms.

Acknowledgments We sincerely thank Mr. Makoto Kobayashi (Shiga Prefecture Federation of Fishermen’s Co-operative Association) for supplying the round crucian carp for this study and Dr. Shin Oikawa (Fishery Research Laboratory, Kyushu University) for providing essential information concerning the fi sh respirometry experiment. We appreciate the helpful comments of Drs. Toshiya Yamamoto (Yahagi River Institute, Toyota) and Atsushi Maruyama (Ryukoku University). This research was partly supported by a Grant for the Biodiversity Research of the 21st Century COE (A14) and Ministry of the Environ- ment, Environmental Policy Bureau, Grant in Aid for Scientifi c Research, “Design of ecological networks for the conservation of endemic freshwater fi sh,” 2006 (2006–2008). We declare that all experi- ments in this study comply with the current laws of Japan.

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