38 SEPTEMBER 2014 • WORLD AQUACULTURE • WWW.WAS.ORG gas bladders among teleost fishes. If swim bladder development in other teleosts is as vulnerable to genetic perturbation as it is in zebrafish, perhaps a supply of bladderless phenotypes has been readily available for selection under conditions for which the lack of a swim bladder is advantageous. Peruzzi et al. (2007) also suggested that inherited factors may contribute to the expression of swim bladder anomalies in sea bass, in addition to predominately environmental cues. The aim of this study was to clarify available information on the effect of swim bladder non-inflation on the Eurasian perch Perca fluviatilis and to assess the effect of size of hatched larvae on the level of swim bladder inflation in perch populations from different locations. Study Methods Egg collections. Fertilized egg ribbons from at least ten wild Eurasian perch were obtained from four populations in Central Europe in 2011 (populations A-D) and 18 populations in 2012 (populations A-R). Eggs were collected from natural ponds in April and May, during natural spawning periods in both years (Table 1). Eggs were transported in oxygen-saturated water in doubled plastic bags within insulated boxes for transfer to recirculating aquaculture systems in Ceske Budejovice, Czech Republic. Incubation. Approximately 100 g of eggs from each ribbon were rinsed, weighed and placed in plastic baskets, about 1 kg/basket, in 100-L tanks. Egg ribbons were placed on the substrate to avoid forming stagnant zones that promote spread of Saprolegnia. Any ribbon that was more than 20 percent infected and those with a low number of developing embryos were removed. Incubation continued for 7-10 days. Larvae hatched at an average body length of 6.5 mm and a weight of 1.8 mg. Feeding regime. For the first 10 days post-hatch (dph), larvae were fed to satiation with Artemia1 five times per day (0800, 1100, 1400, 1700 and 2000 h). Transition to exogenous feeding is a critical period in the initial stage of larval rearing. Mortality reached 99 percent, primarily because of the lack of feeding and hence starvation, with larvae from the Velke Mezirici, Czech Republic (2012), which did not consume Artemia. In further studies, this population was not used. Thereafter, feeding followed the protocol in Table 2. From 11-14 dph, defrosted and rinsed Cyclops was added to the diet. From 14 dph, Artemia was excluded from the diet and larvae were fed only Cyclops. The use of a dry starter feed (BioMar Inicioplus 0.4) was initiated at day 25-28 and continued for 5-6 weeks. From 42 dph, the starter feed was replaced with BioMar Inicioplus 1.1. Following this dietary adaptation period, larvae were fed only pellets. Water quality. Water quality was monitored with a portable meter (Hach HQ40d). The initial temperature (15 C) was increased during the culture period to 20 C; pH was 6.5-7.5. Dissolved oxygen concentration was maintained greater than 6-8 mg/L. Nitrogenous waste products (ammonia, nitrite, nitrate) were monitored three times weekly and were consistently within the normal range. Tank walls and floor were siphoned daily to remove dead larvae and waste food. All siphoned water was placed in a white container, so that any larvae inadvertently siphoned could be returned to tanks. Preservation of larvae. In most cases, average weight is established on formalin-fixed material, representing crude formalin-preserved weight, not the weight of living organisms. The ratio of the weight of preserved specimens to that of live organisms is inconsistent within species and depends on factors such as formalin concentration, fluid volume, temperature at preservation, size, and age of the organism (Borutsky 1958). The usual concentration of saturated formalin for larval fish preservation is 5 percent (Etnier and Starnes 1993). Koumoundouros (2000) evaluated the impact of fish preservation methods on the resolution of radiographic images. A concentration of 10 percent saturated formalin significantly decreases the apparent length and depth of the swim bladder. A concentration of 3-4 percent saturated formalin for fish larvae preservation is optimum (Naberezhnyj 1967); accordingly we used 4 percent saturated formalin. At the conclusion of the rearing period, larvae were examined for growth rate and for determination of the number of larvae in each population with non-inflated swim bladders. Fifty unfed larvae were removed and fixed in 4 percent saturated formalin for assessment of growth rate and swim bladder status at 14 and 28 dph, and 25 larvae were fixed at 42 and 56 dph. Fixed samples were photographed for measurement of total length (mm) with a digital camera and images were analyzed with Micro Image software. Wet weight was measured with an analytical balance after removal of excess water. Examination of larvae for swim bladder inflation. For sorting larvae with swim bladder inflation from those with non-inflated swim bladders, larvae were placed in a 3 g/L sodium chloride solution. Larvae achieving swim bladder inflation floated to the water surface (complete swim bladder inflation) or assumed a vertical position at the bottom of the container (initial stage of swim bladder inflation). Larvae without a functional swim bladder remained in a horizontal position on the substratum (Fig. 1). At least 500 larvae of each population were sorted at 56 dph following Szkudlarek and Zakęś (2000) using a 2 g/L sodium chloride solution and 25 μg/L clove oil as anesthetic. Initial swim bladder inflation is not affected by salinity in Eurasian perch (Bein and Ribi 1994). Because larvae were previously fixed in 4 percent formalin, the potential existed for changes in density, deformation of internal organs, including the swim bladder, resulting in errors in sorting. To minimize errors, larvae were examined using radiovisiography2 FIGURE 1. Sorting Eurasian perch larvae at 14 days after hatching. Larvae with inflated swim bladders (SBI) float to the water surface or assume a vertical position at the bottom of the container. Larvae without inflated swim bladders (SBN) assume a horizontal position at the bottom of the container.
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