Adult spawning grayling were intercepted during their migration and captured using fyke nets. The traps were checked three times daily and captured fish were transferred to a holding pen upstream. Once capturing was complete, adult fish were anaesthetised with benzocaine and their weight and fork length were measured (see Table 4 ) before the fish were stripped of gametes. After recovery all fish were released upstream of the capture site. Gametes were transported on ice and under oxygen by car to the fish holding facility at the Veterinary Institute of Norway, Oslo (5 hours drive). Gametes were stripped from the SW stream populations on 12th June 2007, and on 23rd June the same year from the two LC stream populations.
The eggs were pooled by deme using an equal volume (100 ml) of eggs from each female. The pooled batch of eggs from each deme was then split into a number of batches equal to the number of males from that deme. To avoid sperm competition and so maximise the number of families, each batch of pooled eggs was subsequently fertilized with sperm from one male. Following fertilization, the batches of eggs from each deme were mixed together again before they were partitioned into three treatment groups. Each treatment group was split into two replicates. Three separate experimental tanks containing water of three different temperatures (5.83 ± 0.43°C, 8.14 ± 0.29 °C, 10.02 ± 0.28 °C) were used. These temperatures were chosen to represent lower, medium and upper temperatures experienced by developing grayling larvae in nature [52 (link)], and thus we would be able to investigate norms of reaction covering the range of temperatures that grayling may be expected to tolerate. Mean summer (June - July) temperatures in the four streams investigated here differ strongly, with the two small and warm streams being approximately 1-1.5 ºC higher than the large and cold streams (Sandbekken 8.44 ± 0.52 (n = 5); Steinbekken 8.81 ± 0.60 (n = 4); Hyrjon 7.40 ± 0.94 (n = 8); Valåe 7.28 ± 0.69 (n = 0.69)). This adds to a large temperature-sum difference among streams during egg and larvae development.
Inflowing water was comprised of activated-charcoal filtered tap water. Temperatures were registered using temperature loggers (HOBO). Fertilized eggs were placed in porous containers suspended in the large treatment tanks as described in detail previously [30 (link)]. Each of the three tanks contained two replicate containers from each deme.
Animal sampling and experimentation were performed in compliance with the recommendations of National Animal Research Authority (permission ID 2008/7368.5) and under the supervision of authorized investigators.
The eggs were pooled by deme using an equal volume (100 ml) of eggs from each female. The pooled batch of eggs from each deme was then split into a number of batches equal to the number of males from that deme. To avoid sperm competition and so maximise the number of families, each batch of pooled eggs was subsequently fertilized with sperm from one male. Following fertilization, the batches of eggs from each deme were mixed together again before they were partitioned into three treatment groups. Each treatment group was split into two replicates. Three separate experimental tanks containing water of three different temperatures (5.83 ± 0.43°C, 8.14 ± 0.29 °C, 10.02 ± 0.28 °C) were used. These temperatures were chosen to represent lower, medium and upper temperatures experienced by developing grayling larvae in nature [52 (link)], and thus we would be able to investigate norms of reaction covering the range of temperatures that grayling may be expected to tolerate. Mean summer (June - July) temperatures in the four streams investigated here differ strongly, with the two small and warm streams being approximately 1-1.5 ºC higher than the large and cold streams (Sandbekken 8.44 ± 0.52 (n = 5); Steinbekken 8.81 ± 0.60 (n = 4); Hyrjon 7.40 ± 0.94 (n = 8); Valåe 7.28 ± 0.69 (n = 0.69)). This adds to a large temperature-sum difference among streams during egg and larvae development.
Inflowing water was comprised of activated-charcoal filtered tap water. Temperatures were registered using temperature loggers (HOBO). Fertilized eggs were placed in porous containers suspended in the large treatment tanks as described in detail previously [30 (link)]. Each of the three tanks contained two replicate containers from each deme.
Animal sampling and experimentation were performed in compliance with the recommendations of National Animal Research Authority (permission ID 2008/7368.5) and under the supervision of authorized investigators.
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