Abstract. The responses of early life stages of Cobitis taenia to various temperatures and oxygen levels revealed the species to be warm-mesothermic and ...
Archiv für Hydrobiologie 157: 195-212, 2003
Temperature and oxygen requirements of early life stages of the endangered spined loach, Cobitis taenia L. (Teleostei, Cobitidae) and implications for the management of natural populations Jörg Bohlen Abstract The responses of early life stages of Cobitis taenia to various temperatures and oxygen levels revealed the species to be warm-mesothermic and comparatively resistant against low oxygen concentrations. Lower and upper lethal temperatures were 12.1 and 30.8° C, respectively. As estimated from the combined final size (CFS, the number of healthy survivors multiplied by their mean length), temperatures of 14.0, 15.8, 28.4 and 30.3° C were suboptimal, while temperatures of 17.8-26.1° C represented the optimum range. At 21.9 ± 1.0° C, oxygen concentrations of 0.9 to 1.8 mg l-1 were lethal; those of 2.1 and 2.2 mg l-1 suboptimal and those of 3.2 to 7.8 mg l-1 optimal. Regarding the body size the optimum range for both, temperature and oxygen concentration, was narrower than for CFS, while it was broader if reflected by the survival rate. The most critical period for temperature was prior to hatching. Under low oxygen concentration the main mortality occurred until the middle of free embryonic phase. The early life stages of spined loach express an early hatching as well as morphological (external gills, rich skin vascularisation in the dorsal fin) and behavioural (ventilation by movements of body and pectoral fins) adaptations to low oxygen supply. These adaptations are related to an evolutionary binding of spined loach to a microhabitat with frequent oxygen depletions, as it was former postulated on the base of behavioural studies. The necessity of high temperature for development binds the spined loach to warm microhabitats for reproduction. Therefore, the availability of a suited microhabitat for the early life stages with warm water becomes a key requirement for the successful maintenance of populations of C. taenia. This specialisation has to be considered in conservation measures for the Europe-wide endangered species. Introduction In fish, the sensitivity to environmental parameters changes greatly during the ontogeny. According to the critical periods hypothesis of HJORT (1914), ontogenetic periods of relatively low sensitivity are interrupted by short periods with maximal sensitivity. HJORT (1914) further states, that survival rate during these critical periods limits the number of specimens in all later periods of ontogeny. Catastrophic events during critical periods determine the strengths of year classes in natural fish populations (KAMLER 1992). In
endangered fish species with decreasing population development, the identification of the critical periods and the estimation of the tolerated range of environmental conditions is one of the most important requirements for effective conservation measures. Among the most important environmental parameters for freshwater fishes are temperature and oxygen concentration. The sensitivity to extreme temperatures is usually highest during the egg stage and decreases during ontogeny (KAMLER 1992). In contrast, the sensitivity to hypoxia shows two peaks during early ontogeny (ROMBOUGH 1988). The first peak is usually observed just before hatching and the second peak at the beginning of exogenous feeding (DOUDOROFF & SHUMWAY 1970). In juveniles and adults, the sensitivity to low oxygen concentrations and extreme temperatures remains lower than in early life stages in most species (HOLLIDAY 1969). Therefore, studies of the influence of temperature and oxygen on a fish species have to consider the early life stages. Spined loach, Cobitis taenia L., is a small (up to 12 cm), short-lived (up to 4 years) benthic fish. The species is listed as endangered in most European countries, in the Bern convention and in the directive 92/43/EEC of the European council (KOTUSZ 1996, BOHLEN & RÁB 2001). Especially the directive 92/43/EEC strongly asks for concrete conservation measures, but the reasons for the threatened status of spined loach are still unclear (LELEK 1987). The species was often assumed to be sensitive to high temperatures (LELEK 1987; BOHL 1993; VILCINSKAS & WOLTER 1993) and low oxygen concentration (BOHL 1993; VILCINSKAS & WOLTER 1993) although adults are able to compensate oxygen depletions partly by uptake of air (LUPU 1928, ROBOTHAM 1979). In the present study, the influence of temperature and oxygen concentration on survival and growth of early life stages of C. taenia was investigated. For each parameter, the range of tolerance as well as the optimum conditions were defined. The study was aimed to clarify whether or not the early life stages represent a critical stage in spined loach regarding temperature and oxygen concentration. Conclusions on habitat requirements and the potential ecological niche of this specialised fish shall provide the necessary information for further conservation measures. Materials and methods Temperature experiments Eggs of spined loach were obtained from four parental pairs that were naturally spawning in laboratory aquaria. Each aquarium was equipped with a filter, a layer of fine sand, some vegetation, one pair of spined loach and a gaze-covered plastic box with a bunch of dense moss on top. Upon spawning in the moss, the most preferred spawning substrate of spined loach (BOHLEN 1999a), the eggs fell through the gaze into the box and could be removed quantitatively without touching the eggs or removing them from the water. During the winter before spawning, the parental fish were kept at about 15 to 18° C, the temperature in the month before spawning was 23 to 25° C.
Table I. Experimental temperature regimes under which C. taenia was reared from blastula stage to exogenous feeding larvae. The temperature of the 14 treatments based on the temperatures of two or three replicates per treatment. Mean temperature of treatments (° C)
Temperature of replicates (° C) Mean
SD
Range
10.0
9.9 10.1 10.1
0.2 0.5 0.3
9.8 - 10.2 9.7 - 11.0 9.8 - 11.5
12.1
12.1 12.1 12.2
0.3 0.3 0.1
11.8 - 12.4 11.7 - 12.5 12.0 - 12.4
14.0
13.9 14.0 14.0
0.3 0.3 0.3
13.2 - 14.3 13.1 - 14.4 13.5 - 14.6
15.8
15.8 15.8 15.9
0.4 0.4 0.3
14.9 - 16.5 14.8 - 16.5 15.2 - 16.2
17.8
17.7 17.7 18.1
0.5 0.4 0.3
16.6 - 18.4 16.8 - 18.4 17.7 - 18.5
20.1
20.1 20.1
0.2 0.2
19.7 - 20.5 19.7 - 20.5
21.6
21.5 21.5 21.8
0.6 0.6 0.3
20.5 - 22.5 20.5 - 22.5 21.2 - 22.1
24.2
24.1 24.1 24.4
0.5 0.5 0.3
23.4 - 24.8 23.4 - 24.8 23.8 - 24.7
26.1
26.1 26.1 26.1
0.2 0.3 0.3
25.7 - 26.4 25.5 - 26.5 25.5 - 26.5
28.4
28.3 28.3 28.5
0.4 0.4 0.6
27.5 - 29.0 27.5 - 29.0 27.8 - 29.4
30.3
30.3 30.3 30.7
0.3 0.3 0.3
29.6 - 30.6 29.6 - 30.6 30.3 - 31.0
30.8
30.7 30.9
0.4 0.4
30.3 - 31.0 30.3 - 31.2
31.8
31.5 31.8 32.0
0.1 0.3 0.3
31.4 - 31.6 31.7 - 32.2 31.7 - 32.4
36.9
36.9 36.9
0.1 0.1
36.8 - 37.0 36.8 - 37.0
Eggs were sorted under a stereomicroscope to ensure that all eggs used in the experiments were fertilised and had not passed blastula stage. Because of the nocturnal
spawning of spined loach no eggs could be obtained at earlier stages. Artificial spawning was not applied to the loaches because it may increase the mortality rate and malformation rate of the offspring (CRAIG & HARVEY 1984). Groups of 40 eggs were incubated in 1 l of reconstituted medium in plastic boxes of 3 l volume (20×20 cm, water level 2.5 cm). The medium consisted of 0.2% CaCl2×2 H2O, 0.1% NaHCO3 and 0.1% sea salt (Instant OceanTM) in reverse-osmotic water with a conductivity of 750-800 ì S cm-1 (equalling about 0.47 ‰ salinity) and a pH of 7.6 to 7.9. Before use, the medium was intensely bubbled with air for at least 3 hours and adjusted to the temperature of the experimental treatment. Oxygen concentration in the boxes stayed above 7 mg l-1. Temperature of the incubation medium was measured twice a day. The mean of these measurements was treated as effective temperature for each box. Each day, the fish were transferred gently with a small sieve into washed boxes with fresh medium. Altogether, 39 groups of eggs were incubated at 14 experimental temperatures, ranging from 10.0 to 36.9° C (Table I). For incubation at temperatures from 24.2 to 36.9° C, the plastic boxes with the eggs were placed into water baths. Temperatures from 10.0 to 21.6° C were maintained in incubators. The number of survivors in each box was checked each day. Individuals with noticeable malformations were excluded from the experiment and counted as mortalities. Body length was measured after hatching and at the end of the experiment at 300 degree-days, when all larvae had established exogenous feeding for several days. For measurement of body length, 15 specimens per box were placed into 2.5 ml of medium in a petri dish of 34 mm diameter and their total length measured under a stereomicroscope with a measuring ocular. Larvae were fed twice per day with Artemia nauplii ad libitum. Excessive food was removed by the next water exchange. Oxygen experiments Eggs were obtained, controlled and handled as described for the temperature experiments. Eggs from the same clutch were used for all replicates. At the beginning of incubation, development of the eggs did not exceed early blastula. Groups of 40 eggs were placed in 1 l plastic bottles filled with no air bubbles with the artificial incubation medium described above. Following the results of the temperature experiment, the oxygen experiment was conducted at 21.9 ± 1.0° C in an incubator. Before filling the bottles, the medium for the three control replicates was intensely bubbled with air to reach air saturation. In order to remove the oxygen, the medium for the remaining treatments was bubbled with nitrogen (N2) until the desired concentration was roughly met. After filling the medium into the bottles the effective oxygen concentration was measured via the top opening of the bottles using an electric oxymeter (WTW Instruments, OXY 25) to consider changes during filling process. The detailed adjustment of the oxygen concentration was achieved by the exchange of small quantities of medium with higher or lower oxygen concentration. The decrease of oxygen concentration during 24 h of
Table II. Experimental oxygen concentrations under which C. taenia was reared from blastula stage to exogenous feeding larvae. The oxygen concentration of the six treatments based on the results from three replicates per treatment. Mean oxygen concentration
Oxygen concentration of replicates (mg l-1)
in treatments (mg l-1)
Mean
SD
Range
1.0
0.9 1.0 1.1
0.4 0.3 0.3
0.4 - 1.4 0.5 - 1.4 0.6 - 1.5
2.1
1.8 2.1 2.2
0.5 0.3 0.3
1.2 - 2.4 1.5 - 2.8 1.7 - 2.6
3.2
3.2 3.2 3.2
0.3 0.3 0.3
2.5 - 3.7 2.5 - 3.6 2.8 - 3.7
4.0
4.0 4.0 4.1
0.4 0.4 0.4
3.4 - 4.6 3.3 - 4.5 3.4 - 4.6
5.0
5.0 5.0 5.0
0.4 0.4 0.4
4.3 - 5.5 4.4 - 5.5 4.2 - 5.5
7.8 (control)
7.8 7.8 7.8
0.5 0.5 0.5
7.0 - 8.6 7.0 - 8.5 7.1 - 8.5
incubation was estimated by previous test runs to be 0.2 to 1.0 mg l-1 depending on developmental stage of the test animals. In order to compensate this decrease, the initial oxygen concentration was adjusted 0.1 to 0.5 mg l-1 above the intended value; therefore after 24 h it was slightly below the intended value. Oxygen concentration in each bottle was checked before and after exchange of the medium. In this way, the highest and the lowest oxygen concentrations were obtained each day for each replicate. The mean of all measurements archived from a treatment during the whole experiment represented the effective oxygen concentration during incubation. Six different incubation concentrations (including the control), each with three replicates, covered a range of tested oxygen concentration from 1.0 to 7.8 mg l-1 (Table II). Each day the test animals were gently transferred to a washed bottle with fresh medium by a fine meshed net. The number of survivors in each bottle was checked each day. Individuals with noticeable malformations were excluded from the experiment and counted as mortalities. Body length of 15 specimens per bottle was measured at the beginning of exogenous feeding and at the end of the experiment after 280 degree-days, when all larvae had established exogenous
feeding for at least two days. The protocol for body length measurements was as described for the temperature experiments. To reduce density effects and to equalise density between the treatments, the number of specimens per replicate was reduced to 30 after hatching and to 20 after beginning of feeding. Larvae were fed twice per day with Artemia nauplii ad libitum. Excessive food was removed by the next exchange of medium. Statistical analysis Combined final size (in mm) for each replicate was calculated by multiplying the number of healthy survivors by their mean length. This term integrates the response in growth and the response in survival rate. Such more general expression is evaluates the overall effect of one or more parameters, which is useful in the light of ecological questions. Relative combined final size (in %) describes the combined final size in each treatment as a percentage of the treatment with the highest value. Differences between the treatments regarding the rate of survival, the body length of the larvae and the relative combined final size were analysed using a one-way ANOVA with a multiple range test (LSD, Least Significant Difference, p
