Journal of Fish Biology (2003) 63, 580–592 doi:10.1046/j.1095-8649.2003.00172.x, available online at http://www.blackwell-synergy.com
Hypoxia tolerance of the mummichog: the role of access to the water surface K. L. S T I E R H O F F *, T. E. T A R G E T T *†
AND
P. A. G R E C A Y ‡
*University of Delaware, Graduate College of Marine Studies, Lewes, DE 19958,U.S.A. and ‡Salisbury University, Department of Biological Sciences, Salisbury, MD 21801, U.S.A. (Received 19 May 2002, Accepted 10 June 2003) Low dissolved oxygen (DO) had a significant effect on specific growth rate (GS), length increment (IL) and haematocrit (Hct) of the mummichog Fundulus heteroclitus. Regardless of access to the water surface, F. heteroclitus maintained high growth rates (GS and IL) at DO concentrations as low as 3 mg O2 l1. With access to the water surface, both GS and IL of F. heteroclitus decreased by c. 60% at 10 mg O2 l1 compared to all higher DO treatments. When denied access to the water surface, a further decrease in GS (c. 90%) and IL (c. 75%) was observed at 1 mg O2 l1. There was no effect of diel-cycling DO (1–11 mg O2 l1) with or without surface access on GS, IL or Hct of F. heteroclitus. Similar trends between GS and faecal production across DO treatments suggest that decreased feeding contributed significantly to the observed decrease in growth rate. Haematocrit was significantly elevated at 1 mg O2 l1 for fish with and without access to the water surface. Increased Hct, however, was not sufficient to maintain high GS or IL at severely low DO. When permitted to respire in the surface layer, however, F. heteroclitus was capable of maintaining moderate growth rates at DO concentrations of 1 mg O2 l1 (c. 15% saturation). Although aquatic surface respiration (ASR) was not quantified in this study, F. heteroclitus routinely swam in contact with the water surface and performed ASR at DO concentrations 3 mg O2 l1. No hypoxia-related mortality was observed in any DO or surface access treatment for as long as 9 days. This study demonstrates that surface access, and thus potential for ASR, plays an important role in providing F. heteroclitus substantial independence of growth rate over a wide range of low DO conditions commonly encountered in shallow estuarine environments. # 2003 The Fisheries Society of the British Isles Key words: aquatic surface respiration; dissolved oxygen; feeding rate; Fundulus heteroclitus; growth; haematocrit.
INTRODUCTION Estuaries are highly productive ecosystems that serve as nursery habitats for a variety of fishes (Weinstein, 1979; Miller et al., 1985; Day et al., 1989). Favourable physico-chemical properties, high abundance of suitable prey (Miller et al., 1985; Day et al., 1989) and relatively low predation (Joseph, 1973) are generally thought to contribute to the nursery value of these habitats. The dynamic and
†Author to whom correspondence should be addressed. Tel.: þ1 302 645 4396; fax: þ1 302 645 4028; email:
[email protected]
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rapidly changing nature of physical and chemical properties within estuaries, however, have the potential to constrain survival and growth of resident fishes. Therefore the success, in terms of feeding, growth and reproduction, of species that utilize estuaries is largely determined by their ability to tolerate changes in these physico-chemical variables. Hypoxia, or low dissolved oxygen (DO), is one physico-chemical factor that can potentially diminish the physiological performance of fishes. Hypoxia is a widespread phenomenon in estuaries and coastal bays, particularly during spring and summer months (Diaz & Rosenberg, 1995; Bricker et al., 1999; Diaz, 2000). Chronic hypoxia (days to weeks) is a common result of net respiration below the pycnocline in a density stratified water column (Paerl et al., 1998). In contrast, DO fluctuations between hypoxia and hyperoxia over the diel cycle are driven by the photosynthesis and respiration cycles of phytoplankton and macroalgae in nutrient enriched coastal waters (D’Avanzo & Kremer, 1994; R.A. Tyler, pers. comm.). The minimum oxygen requirements for survival and growth have been documented for a number of freshwater, estuarine and marine fishes (Davis, 1975; USEPA, 2000). In general, feeding (Weber & Kramer, 1983; Bejda et al., 1992; Pichavant et al., 2000) and growth (Cech et al., 1984; Bejda et al., 1992; Hales & Able, 1995; Secor & Gunderson, 1998; Taylor & Miller, 2001) are reduced in fishes when exposed to chronic hypoxia 30 mg O2 l1. Dissolved oxygen that cycles between air-saturation and hypoxia typically results in growth intermediate between that at constant saturated and hypoxic levels (Carlson et al., 1980; Bejda et al., 1992; Thetmeyer et al., 1999; Taylor & Miller, 2001). Despite these general observations, the effects of low DO on feeding, growth and survival are species-specific. Within the Cyprinodontoidei, the families Fundulidae (killifishes), Poeciliidae (poeciliids) and Cyprinodontidae (pupfishes) all contain species that are tolerant to low DO. These fishes share several morphological adaptations, including dorsally orientated mouths and dorso-ventrally flattened heads, which facilitate the use of aquatic surface respiration (ASR) (Lewis, 1970). Aquatic surface respiration is a behavioural adaptation to environmental hypoxia used by many cyprinodontoids to augment oxygen uptake from the more air-saturated surface layer. It has been shown to reduce mortality (Kramer & Mehegan, 1981; Kramer & McClure, 1982; Weber & Kramer, 1983) and to mitigate the effects of hypoxia on growth (Weber & Kramer, 1983) in the guppy Poecilia reticulata (Peters). Although studies exist that describe specific responses (e.g. growth, haematology and behaviour) to hypoxia for several hypoxia-tolerant fishes from tropical and temperate freshwater and marine systems, a comprehensive examination of the physiological and behavioural responses to hypoxia and their relationships to growth and mortality, as far as is known, does not exist for any species. The mummichog Fundulus heteroclitus (L.) is a euryhaline fish that inhabits shallow estuarine and freshwater habitats from south-western Newfoundland and Prince Edward Island (Canada) to Florida (U.S.A) (Able & Fahay, 1998). This species is known to perform ASR (Greaney et al., 1980; Wannamaker & Rice, 2000). Greaney et al. (1980) observed increased haematocrit (Hct) and increases in the specific activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH) #
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and glucophosphate isomerase (GPI) in F. heteroclitus exposed to chronic hypoxia (15–25 mg O2 l1), all of which were suggestive of anaerobic stress. The effects of hypoxia on growth and feeding rate of F. heteroclitus, and the role of access to the water surface in determining the effects of hypoxia on these processes, however, have not been investigated. The objectives of this study were to describe the physiological tolerance of F. heteroclitus to several levels of chronic hypoxia and diel-cycling hypoxia, and to determine the extent to which access to the water surface contributes to its tolerance of low DO. Specifically, the interaction between low DO and access to the water surface on the growth rate, feeding rate, Hct and mortality of F. heteroclitus was investigated. MATERIALS AND METHODS C O L L E C T I O N A N D H O L D I N G O F F I SH Fundulus heteroclitus were collected from Canary Creek (38 80’ N; 75 20’ W) (Lewes, DE, U.S.A) in February 2001. Fish were acclimated in the laboratory in 350 l fiberglass tanks at 25 C for >14 days and a salinity of 25 for >7 days prior to the experiments. A 14L : 10D photoperiod was maintained throughout the acclimation and experiments. During acclimation, fish were fed ad libitum on frozen mysid shrimp Mysis relicta twice daily.
EXPERIMENTAL DESIGN The effects of five DO treatments and access to the water surface were investigated on (a) growth and Hct and (b) feeding and growth of F. heteroclitus using a fully crossed 2 5 factorial design, with equal replication. The fish were grown at five nominal DO treatments: normoxia (7 mg O2 l1), three levels of constant hypoxia (1, 3 and 5 mg O2 l1) and diel-cycling hypoxia (1–11 mg O2 l1) at 25 C and a salinity of 25. Minimum (1 mg O2 l1) and maximum (11 mg O2 l1) DO concentrations in the diel-cycling treatments coincided with the beginning of the light (0700 hours) and dark (2100 hours) periods, respectively. Floating clear polyethylene lids were fitted to the surface of the experimental containers to prevent fish from accessing the water surface in treatments without surface access. Temperature, salinity and DO treatments in these experiments were representative of summer field conditions in Delaware estuaries.
D I S S O L V ED O X Y G E N R E G U L A T I O N Dissolved oxygen levels were regulated using a device described in detail by Grecay & Stierhoff (2002). Levels in each of five recirculating aquarium systems (c. 500 l each) were monitored and controlled simultaneously using a LabVIEWTM (National Instruments Co.) virtual instrument interfaced with a single Strathkelvin Instruments Model 781 DO meter. Levels in each treatment were checked by the device every 10 min, and adjusted as necessary by the addition of either compressed O2 or N2 gas to the head reservoir. Each aquarium system contained 10 replicate 18 l tanks. Levels in the replicate tanks of each system were verified twice daily using a Yellow Springs Instruments Model 55 DO meter. This device maintained stable DO levels throughout the experiments (Table I).
G R O W T H A N D H A E M A T O C R I T E X P E R IM E N T One fish was acclimated in each experimental tank for 72 h prior to the start of the experiment. Fish were 393 32 mm standard length (LS) (146 034 g wet body mass) (mean S.D., n ¼ 10 fish per treatment). Reduction of DO to treatment levels occurred during the final 24 h of acclimation. Fundulus heteroclitus were deprived of food for 15 h
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TABLE I. Sample size (n) and DO (mean S.E.) data from Fundulus heteroclitus experiments. Mean DO in each constant treatment was calculated from triplicate DO values measured twice daily in each system using a handheld DO meter. Mean DO in the diel-cycling treatments were calculated from triplicate DO values measured at 0700 (minimum DO) and 2100 hours (maximum DO)
Growth and haematocrit experiment Surface access
No surface access
Feeding and growth experiment Surface access
No surface access
Treatment (mg O2 l1)
n
Mean DO (mg O2 l1)
1 3 5 7 Diel-cycling (0700 hours) Diel-cycling (2100 hours) 1 3 5 7 Diel-cycling (0700 hours) Diel-cycling (2100 hours)
10 10 10 10 10
119 002 297 006 496 007 687 008 124 001 1103 022 114 004 300 005 484 003 681 002 352 045 1006 038
1 3 5 7 Diel-cycling (0700 hours) Diel-cycling (2100 hours) 1 3 5 7 Diel-cycling (0700 hours) Diel-cycling (2100 hours)
9 8 8 10 9
5 5 5 5 4 5 5 4 4 5
147 012 331 010 511 009 704 004 215 032 1042 046 141 006 321 009 503 008 705 004 218 031 1039 060
prior to being weighed (001 g) and measured (001 mm LS) at 0900 hours on day 0. Preliminary experiments demonstrated that deprivation of food for 15 h is sufficient for complete gut evacuation of F. heteroclitus at 25 C. Fundulus heteroclitus were held at treatment DO levels for 9 days (day 0–8). Fish were fed ad libitum on frozen M. relicta three times daily (0900, 1400 and 2000 hours) and were permitted to feed continuously for the duration of the experiment. Uneaten food was removed during the 0900 and 2000 hours feeding times to preserve food and water quality. At the final feeding on day 8, fish were fed only at 0900 and 1400 hours. Uneaten food was removed from containers at 1800 hours, 15 h prior to final weighing and measuring at 0900 hours on day 9. Experimental tanks were checked for mortalities three times daily during feeding. Fish for Hct analyses were killed by severing the vertebral column just behind the skull, and c. 10–30 ml of blood was collected from the severed caudal peduncle of each fish immediately after the final weighing. Blood was collected using heparinized micro-haematocrit capillary tubes, which were then centrifuged (4775 g) for 10 min.
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Total processing time of Hct samples was <20 min from the time of collection to minimize clotting and swelling of erythrocytes (Korstrom et al., 1996). After blood removal, each fish was dissected for sex determination. The following variables were calculated for each fish: daily specific growth rate (GS, % body mass day1) was calculated from the instantaneous growth coefficient (G) as: GS ¼ 100 (eG 1), where G ¼ (ln M2 ln M1) (t2 t1)1, and M2 and M1 were the masses at times t2 and t1 (t2 t1 ¼ the duration of the experiment in days) (Ricker, 1979). Linear growth increment (IL, mm LS day1) was calculated from IL ¼ (LS2 LS1) (t2 t1)1, where LS2 and LS1 were standard lengths at times t2 and t1. Haematocrit (Hct, % red blood cells) was measured as: Hct ¼ 100 (packed cell volume) (total blood volume)1.
FEEDING AND GROWTH EXPERIMENT Acclimation, feeding, weighing, measuring and sex determination followed the same procedures as the growth and haematocrit experiment, with the following differences: F. heteroclitus 395 28 mm LS (156 032 g wet body mass) (mean S.D., n ¼ 5 fish per treatment) were fed at treatment DO levels for 3 days (day 0–2). On the final feeding day (day 2), fish were fed only at 0900 and 1400 hours. Uneaten food was removed from containers at 1800 hours, 15 h prior to final mass and LS determinations at 0900 hours on day 3. No Hct samples were collected during this experiment. The feeding behaviour of F. heteroclitus, which included the rejection of partially ingested mysids, prohibited the precise quantification of uneaten food and thus direct measurement of mass specific feeding rate (% body mass day1). Targett & Targett (1990) successfully employed a method described by Crisp (1984) to calculate feeding rate (dry mass of macrophytes consumed) in parrotfish Sparisoma radians (Valenciennes) based on the dry mass of faeces produced (MF) and the proportion of ash in the faeces v. food (absorption efficiency, AE). In a preliminary experiment using this method, however, feeding rates of F. heteroclitus fed known masses of M. relicta were consistently underestimated. Although low flow rates of water and 500 mm screens covering the tank outflow minimized the loss of faeces, some was apparently lost. Therefore, since loss of faeces was presumably equal across DO treatments, and since AE was not statistically different across DO treatments (75–82%) (GLM, P ¼ 066), mass-specific faecal production (FP) was used to represent food intake. Faecal material produced by each fish during the 3 day experiment was removed by pipette daily (day 1–3) at 0900 hours and frozen at 80 C. Final faecal samples were collected just prior to the final mass and LS determinations on day 3. Faecal samples (n ¼ 3) were pooled to calculate AE and MF for each fish. Dry and ash mass of faecal material for these calculations were obtained after being dried at 50 C (24 h) and oxidized at 450 C (24 h). In addition to GS, the following variables were calculated for each fish: absorption efficiency (AE, %) was calculated as: AE ¼ 100 [1 (ash mass in food ash mass in faeces) (ash mass in food)1]. Mass-specific faecal production (FP, g faeces g fish1) over the 3 day experiment was calculated as: FP ¼ MF (mean fish mass)1, where MF ¼ total dry mass of faeces produced and mean fish mass was calculated as the sum of individual fish masses on day 0 to day 3 divided by the number of days. Fish masses on days 1 and 2 were calculated from Mt ¼ M0eGt, where M0 is mass on day 0 and t is time in days (Ricker, 1979). GS was calculated as in the growth and haematocrit experiment.
ST A T I S T I C A L A N A L Y S E S Statistical analyses were performed using SYSTAT (SPSS, 1996). Data used in GLM analyses were normally distributed (determined by one-sample Kolmogorov-Smirnov tests using the Lilliefors option). All GS data were logarithmically (log10) transformed to reduce heteroscedasticity. There were no statistical differences among the initial masses of fish used in experimental treatments. In the growth and haematocrit experiment, the effects of DO, surface access and sex on GS, IL and Hct of F. heteroclitus were determined using a three-way GLM. Additional #
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GLMs were performed to explore further those groups of F. heteroclitus identified by the full GLM analysis as belonging to homogeneous subsets of means. Sex was included as a factor in the model because of significant differences in GS (GLM; P < 005) between male and female F. heteroclitus. These differences were attributed to the presence of hydrated oocytes observed in mature females. In the feeding and growth experiment, the effect of DO and surface access on AE and FP of F. heteroclitus was determined using a two-way GLM. The effect of DO and surface access on GS was determined using a two-way GLM, with FP included as a covariate. Additional GLMs were performed to explore further those DO treatments identified by the full GLM as belonging to homogeneous subsets of means. The effect of sex on GS in the feeding and growth experiment was not significant, and was therefore eliminated from the analysis. A significance level of a¼ 005 was used in all analyses.
RESULTS GROWTH AND HAEMATOCRIT EXPERIMENT
Mean DO concentration and number of fish (n) for the growth and haematocrit, and feeding and growth experiments are presented in Table I. Several fish in no surface access treatments escaped from their tanks and were eliminated from the analyses. No hypoxia-related mortality in F. heteroclitus was observed at any DO level, with or without access to the water surface. Mean GS of F. heteroclitus ranged from 31 to 40% body mass day1 in constant DO treatments 3 mg O2 l1, and in the diel-cycling treatment, with and without surface access [Fig. 1(a)]. There was a significant overall effect of surface access, DO treatment and sex on GS (Table II). Furthermore, the GLM identified two homogeneous subsets of means according to GS: (a) low growth (DO ¼ 10 mg O2 l1) and (b) high growth (DO 30 mg O2 l1 and diel-cycling DO) [Fig. 1(a)]. At DO 30 mg O2 l1, and diel-cycling DO, there were no significant differences in GS between F. heteroclitus with or without access to the water surface. Within the low growth subset (10 mg O2 l1), however, GS was significantly reduced (GLM, P < 001) when access to the surface was denied [Fig. 1(a)]. Mean IL of F. heteroclitus ranged from 028 to 041 mm LS day1 in constant DO treatments 3 mg O2 l1 and in the diel-cycling treatment, with and without surface access [Fig. 1(b)]. There was a significant overall effect of DO on IL (Table II). As in the GS analysis, two homogeneous subsets of means were identified according to IL: (a) low growth (DO ¼ 10 mg O2 l1) and (b) high growth (DO 30 mg O2 l1 and diel-cycling DO) [Fig. 1(b)]. Again, there were no differences in IL between F. heteroclitus with or without access to the water surface at DO 30 mg O2 l1 and diel-cycling DO. Within the low growth subset (10 mg O2 l1), however, IL was significantly reduced (GLM, P < 005) when access to the surface was denied [Fig. 1(b)]. Mean Hct values for F. heteroclitus ranged from 27 to 33% in constant DO treatments 3 mg O2 l1 and in the diel-cycling treatment, with and without surface access [Fig. 1(c)]. Dissolved oxygen had a significant effect on Hct (Table II), with values of 37 and 40% at 10 mg O2 l1 for fish with and without surface access, respectively. High Hct (10 mg O2 l1) and low Hct (DO 30 mg O2 l1 and diel-cycling DO) homogeneous subsets were identified, but there were no significant differences in the Hct of F. heteroclitus within #
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6.0 (a) a
GS (% body mass day–1)
5.0
b
4.0 3.0 2.0 1.0
**
0.0
1.0
3.0
5.0
7.0
1.0–11.0
7.0
1.0–11.0
7.0
1.0–11.0
0.6 (b)
IL (mm day–1)
0.5
a
b
0.4 0.3 0.2 *
0.1 0.0
1.0
3.0
5.0
60 (c)
Hct (% red blood cells)
50
a
b
40 30 20 10 0
1.0
3.0
5.0 DO treatment (mg O2 l–1)
FIG. 1. Mean S.E. (a) specific growth rate, (b) linear growth increment and (c) haematocrit of Fundulus heteroclitus exposed to constant (1, 3, 5 and 7 mg O2 l1) and diel-cycling (1–11 mg O2 l1) dissolved oxygen treatments. Fish had access (&) or no access ( ) to the water surface. Lower case letters indicate those groups belonging to homogeneous subsets. *¼ P < 005 and ** ¼ P < 001 (significant differences between groups within homogeneous subsets).
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TABLE II. Summary statistics (three-way GLM) for the main effects of surface access, DO treatment and sex on specific growth rate (GS), linear growth increment (IL), and haematocrit (Hct) of Fundulus heteroclitus in the growth and haematocrit experiment. Each GLM included all two-way and three-way interactions. Since none were significant the values are not included in the table. * ¼ P < 005; ** ¼ P < 001 (main effects) Source GS Surface access DO Sex Error IL Surface access DO Sex Error Hct Surface access DO Sex Error
d.f.
MS
1 4 1 73
0101 0257 0124 0020
4948* 11474** 8944*
1 4 1 74
0077 0140 <0001 0021
3083 4035** 0016
1 4 1 73
1707 298289 11427 35994
0047 8287** 0318
F
either homogeneous subset [Fig. 1(c)]. Therefore, surface access did not have an effect on Hct at any DO level. Sex had a significant effect on GS, but not on IL or Hct (Table II). Female F. heteroclitus had significantly higher GS (GLM; P < 005) than males in the same DO treatments. No significant interaction, however, existed between DO and sex, or surface access and sex, illustrating a similar growth response of both male and female F. heteroclitus to hypoxia (Table II). FE E D I N G A N D G R O W T H E X P E R I M E N T
There was a significant overall effect of DO and FP on GS of F. heteroclitus (Table III). Similar to the growth and haematocrit experiment, two homogeneous TABLE III. Summary statistics (two-way GLM) for the effects of surface access, DO treatment and the covariate faecal production (FP) on specific growth rate (GS) of Fundulus heteroclitus in the feeding and growth experiment. * ¼ P < 005 (main effects) Source
d.f.
MS
F
GS Surface access DO FP Surface access DO Error
1 4 1 4 33
0011 0073 0155 0015 0026
0446 2784* 5945* 0563
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subsets of means were identified according to GS: (a) low growth (DO 30 mg O2 l1) and (b) high growth (DO 30 mg O2 l1 and diel-cycling DO) [Fig. 2(a)]. There were no significant differences in GS of F. heteroclitus within either homogeneous subset. There was no significant effect of either DO or surface access on FP [Fig. 2(b)]. Although no detectable difference in FP existed across DO treatments, the similar trends seen in mean FP and GS [Fig. 2(a), (b)] suggest that decreased feeding rate contributes significantly to the pattern seen in GS. Mean AE values ranged from 75 to 82%, with no significant differences across DO treatments (GLM; P ¼ 066).
8.0
GS (% body mass day–1)
(a)
b a
6.0
4.0
2.0
0.0 1.0
FP (g dry mass feces g wet mass fish–1)
0.006
3.0
5.0
7.0
1.0–11.0
3.0
5.0
7.0
1.0–11.0
(b)
0.005 0.004 0.003 0.002 0.001 0.000
1.0
DO treatment (mg O2 l–1) FIG. 2. Mean S.E. (a) specific growth rate and (b) faecal production of Fundulus heteroclitus exposed to constant (1, 3, 5 and 7 mg O2 l1) and diel-cycling (1–11 mg O2 l1) dissolved oxygen treatments. Fish had access (&) or no access ( ) to the water surface. Lower case letters indicate those groups belonging to homogeneous subsets.
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DISCUSSION Although ASR was not quantified in this study, F. heteroclitus was routinely observed in contact with the water surface performing ASR at DO concentrations 3 mg O2 l1 (unpubl. data). The design of the experimental device used to regulate DO precluded the accurate quantification of ASR. Initial attempts to quantify ASR during these experiments resulted in the disruption of ‘normal’ behaviour, and were therefore discontinued. Previous studies, however, report the use of ASR by F. heteroclitus exposed to hypoxia. Greaney et al. (1980) noted that F. heteroclitus were visibly stressed (i.e. had increased ventilation rate and decreased feeding relative to fish held at normoxia) and performed ASR for the first 21 days of a 35 days exposure to low DO (15–25 mg O2 l1). Moreover, when given a choice between 1 mg O2 l1 and any of three higher DO treatments (2, 4 and 6 mg O2 l1), F. heteroclitus did not choose the higher DO treatment, but merely increased the proportion of time spent performing ASR (60% at 1 mg O2 l1 v. 16% at 6 mg O2 l1) (Wannamaker & Rice, 2000). The present data show that F. heteroclitus is remarkably tolerant of severely low DO for up to 9 days regardless of whether they have access to the water surface. No hypoxia-related mortality was observed over the 9 days exposure to 1 mg O2 l1 (c. 15% air-saturation at 25 C), for fish with or without access to the water surface. This DO concentration approaches the 6 h LC50 (the concentration that is lethal to 50% of individuals after 6 h) (074–089 mg O2 l1 at 20 C) described previously for adult F. heteroclitus (Voyer & Hennekey, 1972). In a study by Weber & Kramer (1983), P. reticulata, another species that uses ASR, experienced 12% mortality during 11 days exposure to DO as low as 050 mg O2 l1 (25 C) when they had access to the water surface. Furthermore, P. reticulata experienced significant mortality (27–33%) at DO 2 mg O2 l1 and 100% mortality at 05 mg O2 l1 within 2–4 h when access to the water surface was denied. Therefore, in terms of survival, F. heteroclitus appears more tolerant of extreme hypoxia than is P. reticulata, when denied surface access. In addition to high survival at low DO, F. heteroclitus in this study were able to maintain moderate growth rates down to 10 mg O2 l1, and were unaffected by diel-cycling DO, when provided access to the water surface. These results were consistent between the growth and haematocrit experiment and the feeding and growth experiment. Although GS and IL in the growth and haematocrit experiment were reduced c. 60% in fish at 10 mg O2 l1 with surface access, the most striking reduction in GS (c. 90%) and IL (c. 75%) occurred when access to the water surface was denied. This growth reduction was apparent within 3 days of exposure in the feeding and growth experiment. In this experiment, mean GS of F. heteroclitus at 10 mg O2 l1 was reduced from 28% (surface access) to 04% body mass day1 (without surface access), despite the short experimental duration and small sample size. Poecilia reticulata exhibited a similar, but less substantial (41%) decrease in growth rate at a constant DO concentration of 1 mg O2 l1 for 11 days when denied access to the water surface (Weber & Kramer, 1983). In comparison, growth rate of F. heteroclitus was more sensitive to low DO than that of P. reticulata. A comparison of growth at 10 mg O2 l1 between F. heteroclitus and other species not adapted to perform ASR would be
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interesting, but data are not available since most species do not survive long periods at 10 mg O2 l1. In the present experiments, Hct of F. heteroclitus increased significantly within 9 days of exposure to low DO (1 mg O2 l1), with mean Hct values ranging from 27% (7 mg O2 l1 without surface access) to 40% (1 mg O2 l1 without surface access). These values are comparable to Hct values previously reported for F. heteroclitus exposed to normoxia (238% at 8–9 mg O2 l1) and hypoxia (366% at 15–25 mg O2 l1) for 35 days (Greaney et al., 1980). Increased Hct is a widespread adaptive response of teleosts to low DO, which augments the uptake of oxygen by increasing blood haemoglobin concentration through either the production of new erythrocytes (erythropoiesis) or the release of erythrocytes from the spleen. Red blood cell swelling could also result in increased Hct, but not necessarily in the improvement of respiratory capacity (Gilmour, 1998). It is unclear, however, which mechanisms were responsible for the observed increase in Hct in either the present study or in Greaney et al. (1980). Elevated Hct and any associated improvement in respiratory efficiency of F. heteroclitus at 10 mg O2 l1 is probably partially responsible for the maintenance of moderate GS and IL in fish that had access to the water surface at this DO level. Neither increases in Hct (this study) nor the potential increase in the specific activity of lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and glucophosphate isomerase (GPI) (Greaney et al., 1980), however, were sufficient to maintain even moderate growth under severely hypoxic conditions (10 mg O2 l1) when access to the water surface was denied. It appears that the observed decreases in GS and IL at 10 mg O2 l1 in the present study are at least partially due to decreased feeding rate. Several studies have attributed reduced growth to hypoxia-induced decreases in feeding (Thetmeyer et al., 1999; Pichavant et al., 2000). Thus, the combination of decreased feeding rate and metabolic efficiency at the lowest DO treatment (1 mg O2 l1 without surface access) could explain the observed decrease in growth rate. These data demonstrate that access to the water surface, and thus the potential for ASR, plays an important role in the short-term (days to weeks) hypoxia tolerance of F. heteroclitus by providing at least partial independence of growth rate over a range of low DO conditions common in shallow estuarine environments. Further investigation is required to determine what potential haematological adaptations (e.g. erythropoiesis and haemoglobin isoforms) and morphological adaptations (e.g. changes in respiratory surface area of the gill lamellae) may contribute to the apparent physiological (reduction of Hct and the specific activity of several metabolic enzymes) and behavioural (decreased ASR) acclimation in F. heteroclitus after 28 days exposure to severe hypoxia, as reported by Greaney et al. (1980). We thank U. Howson, R. Wong and J. Nye for field and laboratory assistance, and P. Gaffney for extensive statistical advice. In addition, we thank R. Tyler, D. Brady and D. Kirchman (as a part of MAST 607: Writing Papers in the Marine Sciences) for reviews of earlier drafts of this manuscript. The helpful comments of two anonymous reviewers are also appreciated. This research was supported by funding from the National Sea Grant Office, NOAA, U.S. Department of Commerce, under grant No. NA96RG0029 (Project R/F-17 to T.E. Targett).
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