- Email: [email protected]
Hypoxia-induced physiological changes in two mangrove swamp fishes: Sheepshead minnow, Cyprinodon variegatus lacepede and sailfin molly, Poecilia latipinna (lesueur)
Camp.
Eiochem.
Physiol.
Vol.
9lA,
No.
I, pp.
17-21,
1990
0300~9629/90
Printed in Great Britain
0
1990
%3.00
+ 0.00
Pergamon Press plc
HYPOXIA-INDUCED PHYSIOLOGICAL CHANGES IN TWO MANGROVE SWAMP FISHES: SHEEPSHEAD MINNOW, CYPRZNODON I/ARIEGATUS LACEPEDE AND SAILFIN MOLLY, POECILIA LATIPINNA (LESUEUR) MARKS. Harbor
Branch
PETERSON*
Oceanographic Institution, Inc. Division of Marine Science 5600 Old Dixie Highway Ft. Pierce, FL 34946, USA. Telephone: (601) 325-3120 (Received 19 December
1989)
Abstract-l. Laboratory measurements (3o’C and 300/w salinity) were made of plasma osmolality, plasma chloride ion concentration, hematocrit, oxygen consumption and survival of sheepshead minnow, Cyprinodon uariegatus Lacepede and sailfin molly, Poecilia luripinna (Lesueur) under normoxic (I 50 mm Hg) and hypoxic (40 mm Hg) conditions. 2. Significant increases in hematocrit and reductions in oxygen consumption were documented for both species. Plasma osmolality increased in sheepshead minnows while in hypoxic conditions but plasma chloride did not change from values in 150mm Hg in either species. There was no mortality in either
species during the 24 hr hypoxia survival tests. 3. Results suggest a strong tolerance of hypoxia in both species and use of aquatic
surface respiration (ASR) by P. latipinna. 4. Low-level mortality occurs in both species but severe mortality occurs only in C. uariegurus and may be due to synergistic environmental effects typical of mangrove swamp habitats.
INTRODUCTION
Mangrove swamps provide critical habitats for numerous resident and transient fishes (Odum et al., 1982; Thayer et al., 1987). These habitats, however, are being modified for mosquito control or waterfowl management in a number of southeastern USA estuaries (Whitman and Meredith, 1987) with resulting water quality deterioration and, at least in some areas, a decrease in fish species richness (Harrington and Harrington, 1982; Gilmore et al., 1982). The species that use these habitats must be able to endure fluctuating and stressful environmental conditions for extended periods of time. For example, sheepshead minnow and sailfin molly have been reported in hypersaline habitats (Gilmore et al., 1982) but are good osmoregulators (Gustafson, 1981; Nordlie, 1987). These two species and the mosquitofish, Gum busia afinis are also able to tolerate low-oxygen tensions (Cech et al., 1985) while mosquitofish, other mollies (Poulin et al., 1987), and striped mullet, Mugil cephufus (Moore, 1976) can behaviorally adjust their oxygen uptake by aquatic surface respiration (ASR). The comparative eco-physiology of sheepshead minnow, Cyprinodon vuriegutus, and sailfin molly, Poeciliu lutipinna, from impounded mangrove swamps is unknown. The objective of this study was to compare and contrast physiological changes between two closely related resident species that differ in their use of habitat. To do this, I documented hypoxia-induced changes in hematocrit, plasma os-
*Present address: Department of Biological Sciences, PO Drawer GY, Mississippi State University, Mississippi State, MS 39762, USA.
molality, plasma chloride ion concentration and oxygen consumption. Finally, I investigated survival in low-oxygen tensions in these two resident species. MATERIALSANDMETHODS Field collections and general laboratory protocol Fishes were collected from impounded mangrove swamps in the Indian River Lagoon, Florida, USA. They were transported to the laboratory in Styrofoam coolers containing impoundment water where they were held at 25°C overnight under high aeration or transferred directly into outdoor concrete vaults (900 1) when environmental temperatures approached experimental temperatures. Experimental animals were then transferred to 76 1 aquaria equipped with individual filters, aerators and heaters. They were held in 30 k 1960 salinity and 30 f 1°C under a 12L: 12D photoperiod centered at 1230 hr for at least 7 days. Experimental animals were fed Tetramin flake food ad libitum twice daily but were fasted for 24 hr prior to testing (except the survival experiments). In all experiments, sex of the fishes was not considered. Experimental salinities were produced using filtered (5 pm) Atlantic Ocean seawater diluted with aged reverse osmosis water. Salinities were checked daily. Normoxicjhypoxic
blood
constituents
experimental
prorocol
Fish were netted from their experimental aquaria and immediately measured to the nearest mm standard length (SL). All blood samples were obtained by first blotting each individual dry and severing its caudal fin. The incision was immediately blotted and blood from the caudal artery was drawn into a heparinized micro-capillary tube and centrifuged for 4 min at 13,460 g in an International Microcapillary Centrifuge (Model MB) for hematocrit (%) determination. All fish were processed within a 30min period and individual blood collection was completed within 1 min to reduce handling effects on blood constituents (Robertson et al., 1987). All individuals were killed between
MARK S. PETERSON
18
0800 and 0900 hr (Peterson and Gilmore, 1988). Plasma osmolality (mOsm/kg) was determined on a 10 11 sample with a Wescor Vapor Pressure Osmometer (Model 5500). Plasma chloride ion concentration (meq/l) was determined from a 10~1 sample on a Buchler Digital Chloridometer (Model 4-2500). Sheepshead minnows (n = 50) were held under the above-mentioned conditions for 11.5 + 0.5 days and sailfin mollies (n = 30) for 7.0 days prior to experimentation. Hypoxic conditions were produced by bubbling nitrogen gas directly into the aquarium for 3.0 hr, thus gradually reducing the oxygen tension to hypoxic conditions (38.5 + 2.6 mm Hg; about 26% saturation; 1.6 ppm). Fish were kept at the new PoZ tension for 2.5 hr. Experimental PO2 tensions were determined and monitored by intecting a single water sample into a calibrated Radiometer PHM73/D6161E5046 oxygen analyzer system. The experimental aquaria contained floating Styrofoam slabs in order to reduce surface breathing by sailfin mollies. Oxygen
consumption
Slatistical
lrealments
Fish size, osmolality and chloride ion concentration (all log10 transformed) and hematocrits (arcsine transformed) were analysed by Po, treatment by analysis of variance (ANOVA). Oxygen consumption rates were analvsed bv a paired r-test (&ha = 0.05) _whereas oxygen consumption rates vs wet weight were examined by linear regresion. D is the mean difference between paired values and only applies to the paired t-test.
rates
Small flow-through respirometers (modified 125 ml Erlenmeyer flasks) equipped with two glass tubes, one that allowed inflowing water to enter near the bottom of the flask while the other tube allowed water to pass out of the flask near the bottom of the stopper (top of flask) were used. Fifteen respirometers were connected by vinyl tubing to a PVC manifold that was equipped with nylon valves for controlling water flow rates (26.7 f 12.4ml/min for minnows; 21.6 + 8.7 for mollies). Due to flow rates and respirometer size. steady state was achieved rapidly (Propp Ed al.. 1982). Water of the desired oxygen tension entered the manifold from a 456 I headbox via an acrylic chamber (300 ml) which vvas used to obtain an initial water sample with a needle and syringe. Final water samples were taken with a needle and syringe from a piece of vinyl tubing attached to the glass tube leaving each respirometer. Desired oxygen tensions (mm Hg) were produced by passing water from a headbox through an in-line oxygen stripper (Cameron, 1986) made of PVC (152.4cm x 7.94 cm i.d.) and filled with marbles to increase surface area for nitrogern gas diffusion. Regulation of the counter flows brought about the desired oxygen levels. Routine oxygen consumption rates (mg O,/g/hr) were calculated by the equation VO, = (PO:, - PO,,) (a) (1.428) V!ww (g) (Lampert, 1984), where (Po,,) = oxygen tension of inhalent water; (Pole) = oxygen tension of exhalent water; a = solubility coefficient (from Cameron, 1986); I.428 converts ml/l to mg:l oxygen; V = flow rate (ml/hr); and ww = wet weight (g). Each experiment took a 43.5 hr period. An individual was placed within each of the respirometers at 1330 hr and held in the chamber for 18.5 hr in normoxic water (minnows = 150.1 mm Hg: mollies = 165.6 mm Hg). Between 0800 and 0900 hr, initial and final Po, readings were taken as well as a flow rate for each respirometer. A respirometer without fish was used to adjust for microbial respiration. The fish remained in the chamber for another 18.5 hr and then nitrogen was used to gradually reduce the partial pressure of oxygen (over the next 3.0 hr) flowing into the respirometers (minnows = 42.2 mm Hg; mollies = 42.8 mm Hg). Once the desired oxygen tension was obtained, the fish were allowed 2.5 hr to adjust to the lower oxygen levels. The initial and final hypoxic readings were taken between 0800 and 0900 hr. Each sheepshead minnow (n = 14) and sailfin molly (n = 14) was used only once. Surrirul
monitored at 1, 2, 4, 6, 8, 10, 12 and 24 hr intervals. Death was established when the fish did not move its operculum for 1 min. Throughout this portion of the experiments, water flow rates (121.1 k 9.0ml/min) flushed the 0.85 I tubes, on average, every 7.02min. Sheepshead minnows (n = 5) were held in the experimental conditions for 19.0 + 1.0 days and sailfin mollies (n = 5) for 31 .O k 2.1 days.
e.~prrimmrs
In order to more adequately address the effects of hypoxia on these species. survival experiments were set up at 60 and 40 mm Hg. Individual fish were placed in a 0.85 1 respirometer and were held for 18.5 hr under flowing, normoxic (162.3 + 13.6) water. Subsequently, between 0730 and 0800 hr. the oxygen tension was rapidly reduced to either 60 (59.6 f I .2) or 40 (39.6 + 1.2) mm Hg and death was
RESULTS
Normoxicjhypoxic Individual
blood constituents
sheepshead
minnow
sizes
ranged
from
24 to 40 and 31 to 45 mm SL for the normoxic and hypoxic experiments, respectively. Sailfin molly ranged from 36 to 54 and 32 to 39 mm SL. There were no significant size differences between the normoxic and hypoxic individuals (ANOVA; P > 0.05). Significantly (ANOVA; P < 0.05) elevated hematocrits were documented for both species under hypoxic conditions (Fig. 1). Only sheepshead minnows exhibited significantly (ANOVA; P < 0.01) elevated plasma osmolality, whereas there were no differences in plasma chloride ion concentration for either species (P < 0.05) (Fig. 1). Oxygen
consumption rates
For the size range examined, there was no statistically significant relationship between wet weight and oxygen consumption rates of sheepshead minnow (1.63-3.21 g ww) in either normoxic or hypoxic conditions (ANOVA; P > 0.05). However, sailfin molly (0.75-1.84 g ww) exhibited significant (P < 0.05) weight-dependent respiration in normoxic but not in hypoxic conditions (P > 0.05). This normoxic relationship, however, did not explain much of the variance (Y = 0.41) and thus the data for all sizes were pooled and analysed using a paired t-test. Significant reductions in weight-specific oxygen consumption were documented for sheepshead minnow (paired t = 10.70; df = 13: D = 0.8634; P < 0.001) and sailfin molly (paired t = 6.56; df = 13; D = 0.5391; P < 0.001) under hypoxic conditions (Fig. 2). Surcical experiments For oxygen
both sailfin molly and sheepshead tensions of 60 and 40mm Hg
minnow,
(2.6 and 1.6 ppm) were not lethal over a 24-hr period (Fig. 3). DISCUSSION
Sheepshead minnow and sailfin molly exhibit stress under hypoxic conditions, as indicated by a suite of secondary stress-response measures. Both species showed significant increases in hematocrit under hypoxic conditions. An increase in hematocrit has also been documented in other species in similar
Hypoxia-induced
physiological changes in two fishes
19
1.2, Cyprincdon variegotus -1 .o-
T
0.8-
0 6-
E’; 6 5
A c
04O.Z-
A %
‘; Q
0.0
-k
1
l
1
-II 1
T .
360
1
-
Poecdia latipinno
320
T
I4I 40 OXYGEN
TENSION
(mmHg)
Fig. 2. Comparison of oxygen consumption rates for sheepshead minnow and sailfin molly in normoxic (150 mm Hg) and hypoxic (40 mm Hg) conditions. Circle = significant difference (P < 0.05). Values are R & SD. sheepshead minnows reduced the energy associated with osmoregulation and spent more energy maintaining a higher gradient by increasing its ventilation
w
mm
-m
8
Fig. 1. Comparison of hematocrit, osmolality and chloride ion concentration in normoxic (150 mm Hg) and hypoxic (40 mm Hg) conditions for sheepshead minnow and sailfin molly. Circle = significant difference (P < 0.05) between oxygen tensions. Values are 8 & SD. hypoxic conditions and has been indicated as an initial response to hypoxia (Fievet et al., 1987). For example, Swift (1981) documented a significant increase in hematocrit for rainbow trout, Salmo gairdneri ( = Oncorhyncus mykiss: Smith and Stearley, 1989) exposed to 2.3 mg/l dissolved oxygen (or lower) for periods longer than 3 hr. Swift (1982) also suggested that increased hematocrit might pre-adapt fishes to hypoxic conditions, thus allowing them to bind more available oxygen when it is environmentally low. It is thought that elevated hematocrit is caused by an increased production of erythrocytes, fluid loss to the tissues with a subsequent decrease in plasma volume and/or swelling of the erythrocytes (Swift, 1981; Fievet et al., 1987). Although the values obtained in 30%0 salinity and normoxic conditions for both species were similar to those reported by Gustafson (1981) and Nordlie (1987), there are no osmolality and/or chloride ion data available for sailfin molly and sheepshead minnow in hypoxic conditions. The significant increase in plasma osmolality under hypoxic conditions in sheepshead minnow suggests that as the Paz gradient across the gills decreased under hypoxia,
Cyprinodon
20
variegatus
1
01
16 h
100
m
It\
m
20
6 Poecilio
0 0
.“,..‘,“.,.‘.,,.‘,‘.., 4
em W 8
12
TIME
24
,e%
16
latipinna
6OmmHq 40 mmtlg 20
24
(hrs)
Fig. 3. Comparison of survival data over a 24-hr period for sheepshead minnow and sailfin molly in two oxygen tenslons.
MARK S PETERSON
20
rate. Although I was not able to empirically measure ventilation rates in either species, rates were visibly higher under hypoxic conditions, a phenomena documented in numerous other teleosts under hypoxic conditions (Lomholt and Johansen, 1979; Boese, 1988). In contrast, there were no significant changes in either plasma osmolality or plasma chloride for sailfin molly, suggesting that this species may have taken oxygen from the relatively rich surface water surrounding the Styrofoam slabs, as has been documented in other mollies under hypoxic conditions (Poulin et al., 1987) and noticed in this study. Since this species is morphologically well adapted to using the oxygen rich layer of water near the surface (Lewis, 1970) they can survive even low oxygen tensions in nature without significant osmoregulatory disfunction. Although a significant decrease in metabolic rate with increased weight was seen for sailfin mollies in normoxia, Subrahmanyam (1980) and Gustafson (1981) documented slight but insignificant reductions in weight-specific oxygen consumption with increased weight in the same species. In contrast, there was no significant relationship between metabolic rate and weight in hypoxia. Such variations in oxygen consumption rates have been documented in a number of fish studies (Eccles, 1985; Boese, 1988). Significant weight-related differences in oxygen consumption rates in the sheepshead minnow as reported by Barton and Barton (1987) were not seen in this study. This discrepancy may have been due to differences in the size ranges examined among the studies (Table 1) and/or the techniques used. For example, Barton and Barton (1987) examined sheepshead minnows between 0.08 and 0.78 g ww, whereas minnows between 1.63 and 3.21 g ww were used in this study. This suggests that sheepshead minnows at sizes above 1.0 g ww may not have significant size-related oxygen consumption differences. The metabolic rates calculated by SubrahTable
I.
Comparison
of oxygen consumption ww
(C)
(g)
35 IO SW
? ? 25
20 20 I
SW
25
I
1.3
SW
25
I
2.4
30
30
14
2.14 k 0.44
SW
35 30
_=
N
25
25 30
0.084.78 0.09~.81 0.6
I
I .4
I
3.7
I
5.6
I8 I4
0.75-3.00 1.24kO.31
manyam (1980) for 1.45.6 g ww fish were not conclusive in terms of weight. Additionally, Barton and Barton (1987) calculated routine metabolic rates using a closed, oxygen depletion technique which introduces a series of problems not associated with flow-through respirometry (Lampert, 1984). These problems may have affected large individuals to a greater extent than small individuals. The significant reductions in oxygen consumption rates for sheepshead minnow and sailfin molly are typical for other fishes held under similar conditions (Lomholt and Johansen, 1979; Cech et al., 1985; Boese, 1988). The only previous documentation of the effects of hypoxia on oxygen consumption in these two species was a preliminary study by Subrahmanyam (1980) in which only three individuals of each species were tested. Although both species examined in this study exhibited reduced oxygen consumption rates in hypoxia, differences in activity between the two species were marked. While neither exhibited mortality in oxygen tensions as low as 40 mm Hg, sailfin molly activity was markedly increased in hypoxia while sheepshead minnow showed decreased activity. In summary, hypoxia-induced physiological changes in the sheepshead minnow and the sailfin molly were documented. The ability to tolerate hypoxic conditions depends upon physiological and behavioral mechanisms, which differ between these two species. Sailfin molly are morphologically adapted to use ASR whereas sheepshead minnow are not; however, minnows can tolerate significant fluctuations in their plasma osmolality while mollies can not. Both species are residents of mangrove swamp habitats, where major changes in dissolved oxygen occur on time scales of hours and thus these species are required to utilize both physiological and behavioral mechanisms to deal with hypoxia. Evidence from mangrove swamp habitats in east-central Florida indicates that when environmental conditions rates for sheepshead
Oxygen tension (mm Hg)
minnow
Oxygen consumption (mg 0, lgihr)
Cyprinodon cariegatus Saturation Saturation -78 -44 -85 -45 -81 -43 150. I 42.2
0.279XkO78 0.432-O. I55 - I .55 -0.23 -0.69 -0.77 -0.39 -0.18 0.88 * 0.30 0.14+0.10
Poecilia latipinna -85 -58 -84 -43 -84 -41
-0.36 -0.10 -0.33 - 0.08 -0.27 -0.08
90-155 165.6 42.8
- 0.47 0.55 * 0.31 0.13 +0.09
and sailtin molly
Authority Barton and Barton Subrahmanyam
(I 987)
(1980)
Present study ~. Subrahmanyam
(1980)
Gustafson (1981) Present study
Approximate values (from graphs); S = salinity; SW = seawater; T = temperature; ? = temperature not reported; N = sample size; ww = wet weight. Subrahmanyam (1980) used the same individual within a weight for the low and high oxygen tension experiments. Present study values are reported as X f SD.
Hypoxia-induced
physiological changes in two fishes
deteriorate, C. uariegatus undergoes severe mortality while P. fatipinna does not (Peterson, personal observation). Causal factors other than hypoxia that are common in mangrove swamp habitats and affect fishes are high hydrogen sulfide concentrations in bottom water and high temperatures. Differential mortality between these two species may be more a function of their typical habitat use pattern (epibenthic versus surface orientated) than physiological adaptations to hypoxic stress. Thus, synergistic environmental effects come into play in mangrove swamps that affect fish survival. Acknowledgements-I wish to thank K. Bird for the use of laboratory space and R. Laughlin and G. Gilmore for the use of equipment. Nancy Brown-Peterson and Drs F. G. Nordlie, T. G. Bailey and R. G. Gilmore reviewed this manuscript. Doug Scheidt, R. Brockmeyer and J. Luczkovich aided with field work. Funding was orovided bv a post-doctoral fellowship to MSP by th; Ha;bor Branch Institution. Inc. and a grant (CZM-194) to G. Gilmore from the Indian River Mosquito Control District. This is contribution No. 758 of the Harbor Branch Oceanographic Institution. Inc. REFERENCES
Barton M. and Barton A. C. (1987) Effects of salinity on oxygen consumption of Cyprinodon variegatus. Copeia 1987, 230-232. Boese B. L. (1988) Hypoxia-induced respiratory changes in english sole (Parophrys vetulus Girard). Comp. Biochem. Physiol. 89A, 257-260. Cameron J. (1986) Principles of Physiological Measurement. Academic Press, New York. Cech J. J., Massingill M. J., Vondracek B. and Linden A. L. (1985) Respiratory metabolism of mosquitofish, Gambusia a&is: effects of temperature, dissolved oxygen, and sex difference. Env. Biol. Fishes 13, 297-307. Eccles D. H. (1985) The effect of temperature and mass on routine oxygen consumption in the South African cyprinid fish Barbus aeneus Burchell. J. Fish Biol. 27, 155.-165. Fievet B., Motais R. and Thomas S. (1987) Role of adrenergic-dependent H+ release from red cells in acidosis induced by hynoxia in trout. Am. J. Phvsiol. R269-R275. Gilmore R. G.: Cooke D. W. and Donohoe C. J. (1982) A comparison of the fish populations and habitat in open and closed salt marsh impoundments in east-central Florida. Northeast Gulf Sci. 5, 25-37. Gustafson D. L. (1981) The influence of salinity on plasma osmolality and routine oxygen consumption in the sailfin molly, Poecilia latipinna (Lesueur), from a freshwater and an estuarine population. MS Thesis, University of Florida. Gainsville. FL. Harrington R. W. Jr and Harrington E. S. (1982) Effects on ^
tishes and their forage organisms of impounding a Florida
21
salt marsh to prevent breeding by salt marsh mosquitos. Bull. Mar. Sci. 32, 523-531. Lampert W. (1984) The measurement of respiration. In A Manual on Methods for the Assessment-of Secondary Productivity in Fresh Waters (Edited by Downing J. A. and Rigler F. H.), pp. 4lti68. Blackwell, Oxford. Lewis W. M. Jr (1970) Morphological adaptations of cyprinodontoids for inhabiting oxygen deficient waters. Copeia 1970, 319-326. _Lomholt J. P. and Johansen K. (1979) Hypoxia acclimation in carp-How it affects 0, uptake, ventilation. and 0, extraction from water. Physiol. ZooI. 52, 3849. Moore R. H. (1976) Seasonal oatterns in the resniratory metabolism of the mullets Mugil cephalus and Mugil curema. Contr. Mar. Sci. 20, 1333146. Nordlie F. G. (1987) Plasma osmotic, Na+ and Cl- regulation under euryhaline conditions in Cvprinodon variegatus Lacepede. Camp. Biochem. Physioi.*86A, 5761. Odum W. E., M&or C. C. and Smith T. J. III. (1982) The ecology of the mangroves of south Florida: a community profile: U.S. Fish and Wildl. Serv., Office of Biol. Services, Washinnton. D.C. FWSIOBS-81124. Peterson M. S. and Gilmore R. G. Jr (1988) Hematocrit, osmolality and ion concentration in fishes: consideration of circadian patterns in the experimental design. J. exp. Mar. Biol. Ecol. 121, 73-78. Poulin R., Wolf N. G. and Kramer D. L. (1987) The effect of hypoxia on the vulnerability of guppies (Poecilia reticulata, Poeciliidae) to an aquatic predator (Astronotus ocellatus, Cichlidae). Env. Biol. Fishes 20, 2855292. Propp M., Garber M. and Ryabuscko V. (1982) Unstable processes in the metabolic rate measurements in flowthrough systems. Mar. Biol. 67, 4749. Robertson L., Thomas P., Arnold C. R. and Trant J. (1987) Plasma cortisol and secondary stress responses of red drum to handling, transport, rearing density, and a disease outbreak. Prog. Fish-Cult. 49, I-12. Smith G. R. and Stearley R. F. (1989) The classification and scientific names of rainbow and cutthroat trouts. Fisheries 14, 410. Subrahmanyam C. B. (1980) Oxygen consumption of estuarine fish in relation to external oxygen tension. Comp. Biochem. Physiol. 67A, 1299133. Swift D. J. (1981) Changes in selected blood component concentrations of rainbow trout, Salmo gairdneri, exposed to hypoxia or sublethal concentrations of phenol or ammonia. J. Fish Biol. 19, 4561. Swift D. J. (1982) Changes in selected blood component concentrations of rainbow trout, Salmo gairdneri, following the blocking of the cortisol stress response with betamethasone and subsequent exposure to phenol or hypoxia. J. Fish Biol. 21, 2699277. Thayer G. W., Colby D. R. and Hettler W. F. Jr (1987) Utilization of the red mangrove prop root habitat by fishes in south Florida. Mar. Eeol. Prog. Ser. 35, 25-38. Whitman W. R. and Meredith W. H. (1987) Waterfowl and Wetlands Symposium. Delaware Coastal Manag. Prog., Delaware Dept. Nat. Res. Env. Control, Dover.
Eiochem.
Physiol.
Vol.
9lA,
No.
I, pp.
17-21,
1990
0300~9629/90
Printed in Great Britain
0
1990
%3.00
+ 0.00
Pergamon Press plc
HYPOXIA-INDUCED PHYSIOLOGICAL CHANGES IN TWO MANGROVE SWAMP FISHES: SHEEPSHEAD MINNOW, CYPRZNODON I/ARIEGATUS LACEPEDE AND SAILFIN MOLLY, POECILIA LATIPINNA (LESUEUR) MARKS. Harbor
Branch
PETERSON*
Oceanographic Institution, Inc. Division of Marine Science 5600 Old Dixie Highway Ft. Pierce, FL 34946, USA. Telephone: (601) 325-3120 (Received 19 December
1989)
Abstract-l. Laboratory measurements (3o’C and 300/w salinity) were made of plasma osmolality, plasma chloride ion concentration, hematocrit, oxygen consumption and survival of sheepshead minnow, Cyprinodon uariegatus Lacepede and sailfin molly, Poecilia luripinna (Lesueur) under normoxic (I 50 mm Hg) and hypoxic (40 mm Hg) conditions. 2. Significant increases in hematocrit and reductions in oxygen consumption were documented for both species. Plasma osmolality increased in sheepshead minnows while in hypoxic conditions but plasma chloride did not change from values in 150mm Hg in either species. There was no mortality in either
species during the 24 hr hypoxia survival tests. 3. Results suggest a strong tolerance of hypoxia in both species and use of aquatic
surface respiration (ASR) by P. latipinna. 4. Low-level mortality occurs in both species but severe mortality occurs only in C. uariegurus and may be due to synergistic environmental effects typical of mangrove swamp habitats.
INTRODUCTION
Mangrove swamps provide critical habitats for numerous resident and transient fishes (Odum et al., 1982; Thayer et al., 1987). These habitats, however, are being modified for mosquito control or waterfowl management in a number of southeastern USA estuaries (Whitman and Meredith, 1987) with resulting water quality deterioration and, at least in some areas, a decrease in fish species richness (Harrington and Harrington, 1982; Gilmore et al., 1982). The species that use these habitats must be able to endure fluctuating and stressful environmental conditions for extended periods of time. For example, sheepshead minnow and sailfin molly have been reported in hypersaline habitats (Gilmore et al., 1982) but are good osmoregulators (Gustafson, 1981; Nordlie, 1987). These two species and the mosquitofish, Gum busia afinis are also able to tolerate low-oxygen tensions (Cech et al., 1985) while mosquitofish, other mollies (Poulin et al., 1987), and striped mullet, Mugil cephufus (Moore, 1976) can behaviorally adjust their oxygen uptake by aquatic surface respiration (ASR). The comparative eco-physiology of sheepshead minnow, Cyprinodon vuriegutus, and sailfin molly, Poeciliu lutipinna, from impounded mangrove swamps is unknown. The objective of this study was to compare and contrast physiological changes between two closely related resident species that differ in their use of habitat. To do this, I documented hypoxia-induced changes in hematocrit, plasma os-
*Present address: Department of Biological Sciences, PO Drawer GY, Mississippi State University, Mississippi State, MS 39762, USA.
molality, plasma chloride ion concentration and oxygen consumption. Finally, I investigated survival in low-oxygen tensions in these two resident species. MATERIALSANDMETHODS Field collections and general laboratory protocol Fishes were collected from impounded mangrove swamps in the Indian River Lagoon, Florida, USA. They were transported to the laboratory in Styrofoam coolers containing impoundment water where they were held at 25°C overnight under high aeration or transferred directly into outdoor concrete vaults (900 1) when environmental temperatures approached experimental temperatures. Experimental animals were then transferred to 76 1 aquaria equipped with individual filters, aerators and heaters. They were held in 30 k 1960 salinity and 30 f 1°C under a 12L: 12D photoperiod centered at 1230 hr for at least 7 days. Experimental animals were fed Tetramin flake food ad libitum twice daily but were fasted for 24 hr prior to testing (except the survival experiments). In all experiments, sex of the fishes was not considered. Experimental salinities were produced using filtered (5 pm) Atlantic Ocean seawater diluted with aged reverse osmosis water. Salinities were checked daily. Normoxicjhypoxic
blood
constituents
experimental
prorocol
Fish were netted from their experimental aquaria and immediately measured to the nearest mm standard length (SL). All blood samples were obtained by first blotting each individual dry and severing its caudal fin. The incision was immediately blotted and blood from the caudal artery was drawn into a heparinized micro-capillary tube and centrifuged for 4 min at 13,460 g in an International Microcapillary Centrifuge (Model MB) for hematocrit (%) determination. All fish were processed within a 30min period and individual blood collection was completed within 1 min to reduce handling effects on blood constituents (Robertson et al., 1987). All individuals were killed between
MARK S. PETERSON
18
0800 and 0900 hr (Peterson and Gilmore, 1988). Plasma osmolality (mOsm/kg) was determined on a 10 11 sample with a Wescor Vapor Pressure Osmometer (Model 5500). Plasma chloride ion concentration (meq/l) was determined from a 10~1 sample on a Buchler Digital Chloridometer (Model 4-2500). Sheepshead minnows (n = 50) were held under the above-mentioned conditions for 11.5 + 0.5 days and sailfin mollies (n = 30) for 7.0 days prior to experimentation. Hypoxic conditions were produced by bubbling nitrogen gas directly into the aquarium for 3.0 hr, thus gradually reducing the oxygen tension to hypoxic conditions (38.5 + 2.6 mm Hg; about 26% saturation; 1.6 ppm). Fish were kept at the new PoZ tension for 2.5 hr. Experimental PO2 tensions were determined and monitored by intecting a single water sample into a calibrated Radiometer PHM73/D6161E5046 oxygen analyzer system. The experimental aquaria contained floating Styrofoam slabs in order to reduce surface breathing by sailfin mollies. Oxygen
consumption
Slatistical
lrealments
Fish size, osmolality and chloride ion concentration (all log10 transformed) and hematocrits (arcsine transformed) were analysed by Po, treatment by analysis of variance (ANOVA). Oxygen consumption rates were analvsed bv a paired r-test (&ha = 0.05) _whereas oxygen consumption rates vs wet weight were examined by linear regresion. D is the mean difference between paired values and only applies to the paired t-test.
rates
Small flow-through respirometers (modified 125 ml Erlenmeyer flasks) equipped with two glass tubes, one that allowed inflowing water to enter near the bottom of the flask while the other tube allowed water to pass out of the flask near the bottom of the stopper (top of flask) were used. Fifteen respirometers were connected by vinyl tubing to a PVC manifold that was equipped with nylon valves for controlling water flow rates (26.7 f 12.4ml/min for minnows; 21.6 + 8.7 for mollies). Due to flow rates and respirometer size. steady state was achieved rapidly (Propp Ed al.. 1982). Water of the desired oxygen tension entered the manifold from a 456 I headbox via an acrylic chamber (300 ml) which vvas used to obtain an initial water sample with a needle and syringe. Final water samples were taken with a needle and syringe from a piece of vinyl tubing attached to the glass tube leaving each respirometer. Desired oxygen tensions (mm Hg) were produced by passing water from a headbox through an in-line oxygen stripper (Cameron, 1986) made of PVC (152.4cm x 7.94 cm i.d.) and filled with marbles to increase surface area for nitrogern gas diffusion. Regulation of the counter flows brought about the desired oxygen levels. Routine oxygen consumption rates (mg O,/g/hr) were calculated by the equation VO, = (PO:, - PO,,) (a) (1.428) V!ww (g) (Lampert, 1984), where (Po,,) = oxygen tension of inhalent water; (Pole) = oxygen tension of exhalent water; a = solubility coefficient (from Cameron, 1986); I.428 converts ml/l to mg:l oxygen; V = flow rate (ml/hr); and ww = wet weight (g). Each experiment took a 43.5 hr period. An individual was placed within each of the respirometers at 1330 hr and held in the chamber for 18.5 hr in normoxic water (minnows = 150.1 mm Hg: mollies = 165.6 mm Hg). Between 0800 and 0900 hr, initial and final Po, readings were taken as well as a flow rate for each respirometer. A respirometer without fish was used to adjust for microbial respiration. The fish remained in the chamber for another 18.5 hr and then nitrogen was used to gradually reduce the partial pressure of oxygen (over the next 3.0 hr) flowing into the respirometers (minnows = 42.2 mm Hg; mollies = 42.8 mm Hg). Once the desired oxygen tension was obtained, the fish were allowed 2.5 hr to adjust to the lower oxygen levels. The initial and final hypoxic readings were taken between 0800 and 0900 hr. Each sheepshead minnow (n = 14) and sailfin molly (n = 14) was used only once. Surrirul
monitored at 1, 2, 4, 6, 8, 10, 12 and 24 hr intervals. Death was established when the fish did not move its operculum for 1 min. Throughout this portion of the experiments, water flow rates (121.1 k 9.0ml/min) flushed the 0.85 I tubes, on average, every 7.02min. Sheepshead minnows (n = 5) were held in the experimental conditions for 19.0 + 1.0 days and sailfin mollies (n = 5) for 31 .O k 2.1 days.
e.~prrimmrs
In order to more adequately address the effects of hypoxia on these species. survival experiments were set up at 60 and 40 mm Hg. Individual fish were placed in a 0.85 1 respirometer and were held for 18.5 hr under flowing, normoxic (162.3 + 13.6) water. Subsequently, between 0730 and 0800 hr. the oxygen tension was rapidly reduced to either 60 (59.6 f I .2) or 40 (39.6 + 1.2) mm Hg and death was
RESULTS
Normoxicjhypoxic Individual
blood constituents
sheepshead
minnow
sizes
ranged
from
24 to 40 and 31 to 45 mm SL for the normoxic and hypoxic experiments, respectively. Sailfin molly ranged from 36 to 54 and 32 to 39 mm SL. There were no significant size differences between the normoxic and hypoxic individuals (ANOVA; P > 0.05). Significantly (ANOVA; P < 0.05) elevated hematocrits were documented for both species under hypoxic conditions (Fig. 1). Only sheepshead minnows exhibited significantly (ANOVA; P < 0.01) elevated plasma osmolality, whereas there were no differences in plasma chloride ion concentration for either species (P < 0.05) (Fig. 1). Oxygen
consumption rates
For the size range examined, there was no statistically significant relationship between wet weight and oxygen consumption rates of sheepshead minnow (1.63-3.21 g ww) in either normoxic or hypoxic conditions (ANOVA; P > 0.05). However, sailfin molly (0.75-1.84 g ww) exhibited significant (P < 0.05) weight-dependent respiration in normoxic but not in hypoxic conditions (P > 0.05). This normoxic relationship, however, did not explain much of the variance (Y = 0.41) and thus the data for all sizes were pooled and analysed using a paired t-test. Significant reductions in weight-specific oxygen consumption were documented for sheepshead minnow (paired t = 10.70; df = 13: D = 0.8634; P < 0.001) and sailfin molly (paired t = 6.56; df = 13; D = 0.5391; P < 0.001) under hypoxic conditions (Fig. 2). Surcical experiments For oxygen
both sailfin molly and sheepshead tensions of 60 and 40mm Hg
minnow,
(2.6 and 1.6 ppm) were not lethal over a 24-hr period (Fig. 3). DISCUSSION
Sheepshead minnow and sailfin molly exhibit stress under hypoxic conditions, as indicated by a suite of secondary stress-response measures. Both species showed significant increases in hematocrit under hypoxic conditions. An increase in hematocrit has also been documented in other species in similar
Hypoxia-induced
physiological changes in two fishes
19
1.2, Cyprincdon variegotus -1 .o-
T
0.8-
0 6-
E’; 6 5
A c
04O.Z-
A %
‘; Q
0.0
-k
1
l
1
-II 1
T .
360
1
-
Poecdia latipinno
320
T
I4I 40 OXYGEN
TENSION
(mmHg)
Fig. 2. Comparison of oxygen consumption rates for sheepshead minnow and sailfin molly in normoxic (150 mm Hg) and hypoxic (40 mm Hg) conditions. Circle = significant difference (P < 0.05). Values are R & SD. sheepshead minnows reduced the energy associated with osmoregulation and spent more energy maintaining a higher gradient by increasing its ventilation
w
mm
-m
8
Fig. 1. Comparison of hematocrit, osmolality and chloride ion concentration in normoxic (150 mm Hg) and hypoxic (40 mm Hg) conditions for sheepshead minnow and sailfin molly. Circle = significant difference (P < 0.05) between oxygen tensions. Values are 8 & SD. hypoxic conditions and has been indicated as an initial response to hypoxia (Fievet et al., 1987). For example, Swift (1981) documented a significant increase in hematocrit for rainbow trout, Salmo gairdneri ( = Oncorhyncus mykiss: Smith and Stearley, 1989) exposed to 2.3 mg/l dissolved oxygen (or lower) for periods longer than 3 hr. Swift (1982) also suggested that increased hematocrit might pre-adapt fishes to hypoxic conditions, thus allowing them to bind more available oxygen when it is environmentally low. It is thought that elevated hematocrit is caused by an increased production of erythrocytes, fluid loss to the tissues with a subsequent decrease in plasma volume and/or swelling of the erythrocytes (Swift, 1981; Fievet et al., 1987). Although the values obtained in 30%0 salinity and normoxic conditions for both species were similar to those reported by Gustafson (1981) and Nordlie (1987), there are no osmolality and/or chloride ion data available for sailfin molly and sheepshead minnow in hypoxic conditions. The significant increase in plasma osmolality under hypoxic conditions in sheepshead minnow suggests that as the Paz gradient across the gills decreased under hypoxia,
Cyprinodon
20
variegatus
1
01
16 h
100
m
It\
m
20
6 Poecilio
0 0
.“,..‘,“.,.‘.,,.‘,‘.., 4
em W 8
12
TIME
24
,e%
16
latipinna
6OmmHq 40 mmtlg 20
24
(hrs)
Fig. 3. Comparison of survival data over a 24-hr period for sheepshead minnow and sailfin molly in two oxygen tenslons.
MARK S PETERSON
20
rate. Although I was not able to empirically measure ventilation rates in either species, rates were visibly higher under hypoxic conditions, a phenomena documented in numerous other teleosts under hypoxic conditions (Lomholt and Johansen, 1979; Boese, 1988). In contrast, there were no significant changes in either plasma osmolality or plasma chloride for sailfin molly, suggesting that this species may have taken oxygen from the relatively rich surface water surrounding the Styrofoam slabs, as has been documented in other mollies under hypoxic conditions (Poulin et al., 1987) and noticed in this study. Since this species is morphologically well adapted to using the oxygen rich layer of water near the surface (Lewis, 1970) they can survive even low oxygen tensions in nature without significant osmoregulatory disfunction. Although a significant decrease in metabolic rate with increased weight was seen for sailfin mollies in normoxia, Subrahmanyam (1980) and Gustafson (1981) documented slight but insignificant reductions in weight-specific oxygen consumption with increased weight in the same species. In contrast, there was no significant relationship between metabolic rate and weight in hypoxia. Such variations in oxygen consumption rates have been documented in a number of fish studies (Eccles, 1985; Boese, 1988). Significant weight-related differences in oxygen consumption rates in the sheepshead minnow as reported by Barton and Barton (1987) were not seen in this study. This discrepancy may have been due to differences in the size ranges examined among the studies (Table 1) and/or the techniques used. For example, Barton and Barton (1987) examined sheepshead minnows between 0.08 and 0.78 g ww, whereas minnows between 1.63 and 3.21 g ww were used in this study. This suggests that sheepshead minnows at sizes above 1.0 g ww may not have significant size-related oxygen consumption differences. The metabolic rates calculated by SubrahTable
I.
Comparison
of oxygen consumption ww
(C)
(g)
35 IO SW
? ? 25
20 20 I
SW
25
I
1.3
SW
25
I
2.4
30
30
14
2.14 k 0.44
SW
35 30
_=
N
25
25 30
0.084.78 0.09~.81 0.6
I
I .4
I
3.7
I
5.6
I8 I4
0.75-3.00 1.24kO.31
manyam (1980) for 1.45.6 g ww fish were not conclusive in terms of weight. Additionally, Barton and Barton (1987) calculated routine metabolic rates using a closed, oxygen depletion technique which introduces a series of problems not associated with flow-through respirometry (Lampert, 1984). These problems may have affected large individuals to a greater extent than small individuals. The significant reductions in oxygen consumption rates for sheepshead minnow and sailfin molly are typical for other fishes held under similar conditions (Lomholt and Johansen, 1979; Cech et al., 1985; Boese, 1988). The only previous documentation of the effects of hypoxia on oxygen consumption in these two species was a preliminary study by Subrahmanyam (1980) in which only three individuals of each species were tested. Although both species examined in this study exhibited reduced oxygen consumption rates in hypoxia, differences in activity between the two species were marked. While neither exhibited mortality in oxygen tensions as low as 40 mm Hg, sailfin molly activity was markedly increased in hypoxia while sheepshead minnow showed decreased activity. In summary, hypoxia-induced physiological changes in the sheepshead minnow and the sailfin molly were documented. The ability to tolerate hypoxic conditions depends upon physiological and behavioral mechanisms, which differ between these two species. Sailfin molly are morphologically adapted to use ASR whereas sheepshead minnow are not; however, minnows can tolerate significant fluctuations in their plasma osmolality while mollies can not. Both species are residents of mangrove swamp habitats, where major changes in dissolved oxygen occur on time scales of hours and thus these species are required to utilize both physiological and behavioral mechanisms to deal with hypoxia. Evidence from mangrove swamp habitats in east-central Florida indicates that when environmental conditions rates for sheepshead
Oxygen tension (mm Hg)
minnow
Oxygen consumption (mg 0, lgihr)
Cyprinodon cariegatus Saturation Saturation -78 -44 -85 -45 -81 -43 150. I 42.2
0.279XkO78 0.432-O. I55 - I .55 -0.23 -0.69 -0.77 -0.39 -0.18 0.88 * 0.30 0.14+0.10
Poecilia latipinna -85 -58 -84 -43 -84 -41
-0.36 -0.10 -0.33 - 0.08 -0.27 -0.08
90-155 165.6 42.8
- 0.47 0.55 * 0.31 0.13 +0.09
and sailtin molly
Authority Barton and Barton Subrahmanyam
(I 987)
(1980)
Present study ~. Subrahmanyam
(1980)
Gustafson (1981) Present study
Approximate values (from graphs); S = salinity; SW = seawater; T = temperature; ? = temperature not reported; N = sample size; ww = wet weight. Subrahmanyam (1980) used the same individual within a weight for the low and high oxygen tension experiments. Present study values are reported as X f SD.
Hypoxia-induced
physiological changes in two fishes
deteriorate, C. uariegatus undergoes severe mortality while P. fatipinna does not (Peterson, personal observation). Causal factors other than hypoxia that are common in mangrove swamp habitats and affect fishes are high hydrogen sulfide concentrations in bottom water and high temperatures. Differential mortality between these two species may be more a function of their typical habitat use pattern (epibenthic versus surface orientated) than physiological adaptations to hypoxic stress. Thus, synergistic environmental effects come into play in mangrove swamps that affect fish survival. Acknowledgements-I wish to thank K. Bird for the use of laboratory space and R. Laughlin and G. Gilmore for the use of equipment. Nancy Brown-Peterson and Drs F. G. Nordlie, T. G. Bailey and R. G. Gilmore reviewed this manuscript. Doug Scheidt, R. Brockmeyer and J. Luczkovich aided with field work. Funding was orovided bv a post-doctoral fellowship to MSP by th; Ha;bor Branch Institution. Inc. and a grant (CZM-194) to G. Gilmore from the Indian River Mosquito Control District. This is contribution No. 758 of the Harbor Branch Oceanographic Institution. Inc. REFERENCES
Barton M. and Barton A. C. (1987) Effects of salinity on oxygen consumption of Cyprinodon variegatus. Copeia 1987, 230-232. Boese B. L. (1988) Hypoxia-induced respiratory changes in english sole (Parophrys vetulus Girard). Comp. Biochem. Physiol. 89A, 257-260. Cameron J. (1986) Principles of Physiological Measurement. Academic Press, New York. Cech J. J., Massingill M. J., Vondracek B. and Linden A. L. (1985) Respiratory metabolism of mosquitofish, Gambusia a&is: effects of temperature, dissolved oxygen, and sex difference. Env. Biol. Fishes 13, 297-307. Eccles D. H. (1985) The effect of temperature and mass on routine oxygen consumption in the South African cyprinid fish Barbus aeneus Burchell. J. Fish Biol. 27, 155.-165. Fievet B., Motais R. and Thomas S. (1987) Role of adrenergic-dependent H+ release from red cells in acidosis induced by hynoxia in trout. Am. J. Phvsiol. R269-R275. Gilmore R. G.: Cooke D. W. and Donohoe C. J. (1982) A comparison of the fish populations and habitat in open and closed salt marsh impoundments in east-central Florida. Northeast Gulf Sci. 5, 25-37. Gustafson D. L. (1981) The influence of salinity on plasma osmolality and routine oxygen consumption in the sailfin molly, Poecilia latipinna (Lesueur), from a freshwater and an estuarine population. MS Thesis, University of Florida. Gainsville. FL. Harrington R. W. Jr and Harrington E. S. (1982) Effects on ^
tishes and their forage organisms of impounding a Florida
21
salt marsh to prevent breeding by salt marsh mosquitos. Bull. Mar. Sci. 32, 523-531. Lampert W. (1984) The measurement of respiration. In A Manual on Methods for the Assessment-of Secondary Productivity in Fresh Waters (Edited by Downing J. A. and Rigler F. H.), pp. 4lti68. Blackwell, Oxford. Lewis W. M. Jr (1970) Morphological adaptations of cyprinodontoids for inhabiting oxygen deficient waters. Copeia 1970, 319-326. _Lomholt J. P. and Johansen K. (1979) Hypoxia acclimation in carp-How it affects 0, uptake, ventilation. and 0, extraction from water. Physiol. ZooI. 52, 3849. Moore R. H. (1976) Seasonal oatterns in the resniratory metabolism of the mullets Mugil cephalus and Mugil curema. Contr. Mar. Sci. 20, 1333146. Nordlie F. G. (1987) Plasma osmotic, Na+ and Cl- regulation under euryhaline conditions in Cvprinodon variegatus Lacepede. Camp. Biochem. Physioi.*86A, 5761. Odum W. E., M&or C. C. and Smith T. J. III. (1982) The ecology of the mangroves of south Florida: a community profile: U.S. Fish and Wildl. Serv., Office of Biol. Services, Washinnton. D.C. FWSIOBS-81124. Peterson M. S. and Gilmore R. G. Jr (1988) Hematocrit, osmolality and ion concentration in fishes: consideration of circadian patterns in the experimental design. J. exp. Mar. Biol. Ecol. 121, 73-78. Poulin R., Wolf N. G. and Kramer D. L. (1987) The effect of hypoxia on the vulnerability of guppies (Poecilia reticulata, Poeciliidae) to an aquatic predator (Astronotus ocellatus, Cichlidae). Env. Biol. Fishes 20, 2855292. Propp M., Garber M. and Ryabuscko V. (1982) Unstable processes in the metabolic rate measurements in flowthrough systems. Mar. Biol. 67, 4749. Robertson L., Thomas P., Arnold C. R. and Trant J. (1987) Plasma cortisol and secondary stress responses of red drum to handling, transport, rearing density, and a disease outbreak. Prog. Fish-Cult. 49, I-12. Smith G. R. and Stearley R. F. (1989) The classification and scientific names of rainbow and cutthroat trouts. Fisheries 14, 410. Subrahmanyam C. B. (1980) Oxygen consumption of estuarine fish in relation to external oxygen tension. Comp. Biochem. Physiol. 67A, 1299133. Swift D. J. (1981) Changes in selected blood component concentrations of rainbow trout, Salmo gairdneri, exposed to hypoxia or sublethal concentrations of phenol or ammonia. J. Fish Biol. 19, 4561. Swift D. J. (1982) Changes in selected blood component concentrations of rainbow trout, Salmo gairdneri, following the blocking of the cortisol stress response with betamethasone and subsequent exposure to phenol or hypoxia. J. Fish Biol. 21, 2699277. Thayer G. W., Colby D. R. and Hettler W. F. Jr (1987) Utilization of the red mangrove prop root habitat by fishes in south Florida. Mar. Eeol. Prog. Ser. 35, 25-38. Whitman W. R. and Meredith W. H. (1987) Waterfowl and Wetlands Symposium. Delaware Coastal Manag. Prog., Delaware Dept. Nat. Res. Env. Control, Dover.