Study sites
Barbus neumayeri, a widely distributed cyprinid in Africa [25], were collected from two sites with very different DO levels but relatively similar in other abiotic parameters [28, 36] in the Rwembaita Swamp system of Kibale National Park in western Uganda (0°13'-0°'41'N and 30°19'-30°32'E). Rwembaita Swamp is one of the largest swamps in Kibale National Park. It is 6.5 km in length and feeds into the Njuguta River, which is a tributary of Mpanga River that drains into the Lake George basin [28]. The hypoxic site, located in the central area of the Rwembaita Swamp, displayed a monthly mean DO concentration of 1.35 ± 0.18 mg L-1 over a 2-year period [28]. The water current is extremely slow in the swamp, when compared with the numerous streams that drain into the swamp, and current is non-detectable in some areas during the dry season. One of the several streams that feed into the Rwembaita Swamp was selected as the normoxic site, the Inlet Stream West. This stream has low-flowing water current throughout much of the year, with periods of higher flows during flooding season. The monthly mean DO over 2 years was 5.58 ± 0.16 mg L-1 for Inlet Stream West [28]. Despite the DO differences in both sampling sites, almost no diel variation in temperature and DO exist at each of the sites used here [29].
Experimental design
For this study, individuals were captured at the two study sites using minnow traps, and a reciprocal transplant experiment was designed to determine the roles of collection habitat and acclimatization to alternative dissolved oxygen environments. Six cages were placed in Inlet Stream West, and six cages were placed in Rwembaita Swamp. Cylindrical cages (100 cm height × 80 cm diameter) were made from black plastic meshing (2 mm mesh size), except for the top, which was left open. The meshing allowed for free flow of water and food. Each cage was placed in water approximately 30 to 50 cm in depth and held in place with four poles implanted into the sediment or tied to adjacent trees.
Fish were haphazardly allocated into two densities, low and high, to explore the influence of density on growth rate and enzyme activity. Three low density cages and three high density cages were placed in each of the two sites. In each of the low density cages three swamp fish and three stream fish were placed; while in the high density cages, six swamp fish and six stream fish were placed. Before placing the fish into the cages, each individual was measured for standard length (L
S
± 1 mm) and total body mass (M
T
± 0.1 g), and tattooed under the skin with ink from a fine-gauge needle for individual identification [36]. The reciprocal acclimatization treatment lasted four weeks.
Tissue sampling
Fish were netted and euthanized with an overdose of buffered MS-222 (1 g MS-222 and 4 g NaHCO3 L-1), and measured again for M
T
and L
S
, after which fish were individually wrapped in tin foil and frozen in a dry shipper. Fish were maintained in the dry shipper during transport to the University of Florida and then shipped to Laurentian University on dry ice, and finally stored at - 80°C until analyzed.
Tissues were dissected on ice in the following order: liver, heart, brain, and white skeletal muscle. Tissues were weighed and homogenized in 50 mM imidazole buffer (pH 7.5), 30 mM NaF, 2 mM ethylenediamine tetraacetic acid, 5 mM β-mercaptoethanol, 0.2 mM phenylmethyl-sulphonylflouride, 50 μg ml-1 soybean trypsin inhibitor buffer, and 0.2% Triton 100X [20]. The liver, brain, and muscle tissue samples were homogenized in nine volumes of buffer; while heart tissue samples were homogenized in 49 volumes of buffer. Homogenates were made using a Polytron homogenizer (Polytron 1200C, Kinematica, Switzerland) for three 20 s periods. The samples were maintained on ice during and between the periods of homogenization. All homogenates were centrifuged at 2400 g for 15 min at 4°C, and supernatant solutions were kept on ice until enzyme activities were assayed.
Enzyme assays
Immediately prior to enzyme assays, liver, brain, and muscle supernatants were diluted in homogenization buffer to get an overall 100-fold dilution in relation to the starting tissue mass. For each enzyme in each tissue, the concentrations of substrates, cofactors, and linking enzymes were optimized to give maximal activities. Reaction conditions for the determination of PFK and LDH enzyme activities were modified from [42]. The reactions for the aerobic enzymes, CS and CCO, were modified from [43]. The optimized reaction conditions were as follows:
Phosphofructokinase (PFK; E.C. 2.7.1.11.): 50 mM imidazole (pH 7.5), 20 mM KCl, 10 mM MgCl2, 0.17 mM NADH, 1 mM ATP (liver, heart and brain) or 2 mM ATP (muscle), 2 mM AMP (liver, heart and brain) or 4 mM AMP (muscle), 10 i.u. ml-1 glycerol-3-phosphate dehydrogenase, 29 i.u. ml-1 triosephosphate isomerase and 1 i.u. ml-1 aldolase (liver, heart and muscle) or 2 i.u. ml-1 aldolase (brain). Reactions were initiated with the addition of 5 mM fructose 6-phosphate.
Lactate dehydrogenase (LDH; E.C. 1.1.1.27.): 50 mM imidazole (pH 7.0), 0.17 mM NADH. Reactions were initiated by the addition of 1 mM pyruvate.
Citrate synthase (CS; E.C. 4.1.3.7.): 50 mM Tris (pH 8.0), 0.1 mM DTNB, 0.2 mM acetyl coenzyme A. Reactions were initiated by the addition of 0.3 mM oxaloacetate.
Cytochrome c oxidase (CCO; E.C. 1.9.3.1): 61.5 mM KH2PO4 (pH 7.0), 38.5 mM K2HPO4, 0.07 mM cytochrome c reduced. Reactions were run against a 70 μmol•L-1 control of cytochrome c oxidized with 0.33% K-ferricyanide.
Maximal enzyme activities were measured in duplicate in a Varian spectrophotometer (Cary 100 Bio Varian Scientific Inc. Palo Alto CA, USA) with a circulating refrigerated water bath. Activities were measured at 25 ± 1°C. All enzyme levels were measured within 5 hours of the tissue homogenization to minimize decay of enzyme activity. LDH and PFK activities were measured at 340 nm to follow the disappearance of NADH. CCO activity was measured at 550 nm to follow the oxidation of reduced cytochrome c, and CS activity was measured at 412 nm to detect the transfer of coenzyme A to 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB). The extinction coefficients for NADH, cytochrome C, and DNTB were respectively, 6.22, 19.1 and 13.6 cm-1 μmol-1. Enzyme activities were expressed in international units (μmol substrate transformed to product min-1) g-1 tissue mass. Biochemicals and chemicals were purchased from Sigma-Aldrich (St. Louis, U.S.A.), Boehringer Mannheim Co. (Montreal, Canada) and Fisher Scientific Co. (Montreal, Canada).
Calculations and statistical analyses
Fulton's condition factor (K) was calculated as:
where M
T
is in g and L
s
is in mm.
Specific growth rate (GS) in % mass change per day was calculated as:
where G = (ln final M
T
- ln initial M
T
)(t)-1, and t is the total number of days of the acclimatization period [40, 44].
All response variables (morphology, growth, and enzyme activities) were compared against the normal distribution using one-sample Kolmogorov-Smirnov tests, and variables that were not normally distributed were log transformed. Two-way analyses of variance (ANOVA) were used to evaluate the effects of collection site (stream vs. swamp) and acclimatization treatment (site of cage; normoxic stream vs. hypoxic swamp), as well as their interaction, on response variables. Collection site and acclimatization treatment are fixed factors in this analysis because they do not represent a random sample of all possible field sites, but rather sites of known differences in dissolved oxygen level [45]. Although the density of fish was varied as part of the experimental design, all of the fish from one low density cage escaped and fish from other cages were occasionally lost due to predation, thereby altering the fish density. Therefore, density was not included as a fixed factor. Instead, cage assignment was nested within acclimatization treatment to account for differences in fish density and random factors associated with cage placement (e.g., microhabitat, food availability, competitors, and predators). Body mass was significantly correlated with brain LDH activity, and M
T
was included in the final model as a covariate for this enzyme only. Log transformation failed to normalize CS activities from heart, brain, and muscle, and consequently non-parametric Kruskall-Wallis tests were used to detect differences between collection site and acclimatization treatment. All statistical analyses were performed with Systat 10.2 and P < 0.05 indicated statistical significance.