| Abstract |
Zebrafish is a small colorful tropical fish that inhabits small ponds and slow mov-ing streams in Southeast Asia. For the past few decades, it has been a very important model-organism in research fields such as Neurobiology, Developmental Biology and Genetics. Despite that, existing data on its biology and ecology remain scarce and as a result there is an important lack of knowledge concerning its preferences towards the en-vironmental conditions, especially temperature which is considered as the most crucial. The object of the current study therefore is to examine the effect of different developmen-tal temperatures on the morphology and function of this small organism.
In order to do so, eggs from a wild type broodstock kept in the Aquaculture lab (Biology Department, University of Crete) were used. After spawning, the eggs were in-troduced in four tanks of different water temperatures (22, 25, 28 and 31 °C). All other environmental conditions were kept stable and the same between the four tanks. The eggs hatched and the larvae developed in the tanks until about metamorphosis where the sam-plings took place. Duplicate experiments were conducted, whereas the whole procedure was repeated 2 more times in order to cover the demand for samples. The characters that were chosen to be studied were the deformities, the body shape, the meristics, the sex, the swimming ability and the muscle lactate metabolism.
For the deformities study, the vertebral column and the dorsal, anal and caudal fin were studied on specimens of 13,78 ± 2,9 mm TL (Total Length). Specimens (50 per du-plicate per temperature condition) were subjected to double staining of Alizarin and Al-cian Blue and were studied under a stereoscope. Their study revealed that there is not a clear effect of temperature since in almost all groups studied, the percentage of deformed specimens was the same and total (100%, with the exception of 25 οC where the percent-age of the deformed was 99%). On the other hand, multivariate analysis showed that the type of the deformity present in each specimen was affected by the developmental tem-perature and that all different types were categorized into 2 different groups: the vertebral deformities and the fin deformities (Cluster Analysis).
For the body shape analysis, 17 landmarks were placed on the digital photographs of 50 specimens from each temperature condition sized 13,5± 1,9 mm TL. Those land-marks produced 28 distances (morphometric characters) on which the multivariate analy-
sis of the body shape was conducted. The analysis showed that developmental tempera-ture significantly affects body shape and that the characters that mostly contribute to the groups’ differentiation are located at the posterior part of the fish body.
Meristic elements were studied on the same specimens as the body shape analysis. Those specimens were subjected to double staining of Alizarin and Alcian Blue and then the vertebral column, the dorsal, anal, caudal, pectoral and pelvic fins were studied under a stereoscope. As it was shown, ontogenetic temperature has an important effect on the meristic numbers since in most cases, fish developed at the extreme temperatures (22 and/or 31 οC) were clearly differentiated from the others. Specifically, meristic counts of the vertebral column and of the dorsal fin rays were significantly higher at the 22 οC populations, whereas meristic counts of the pectoral and the pelvic fins were significantly lower at the 31 οC populations. Anal fin rays presented differentiation between the low temperature (22 and 25 οC) and the high temperature groups (28 και 31 οC), while the caudal fin rays (lepidotrichia and dermatotrichia) did not present any particular trend.
Fish rearing for the sex study was terminated at 94-107 days post hatching with the random sampling and preservation of 25 specimens per duplicate per temperature. Histological observation of the gonads performed at the Institute of Aquaculture at the Hellenic Centre of Marine Research (Dr Mylonas) revealed that lower developmental temperatures induced masculinization (22 °C, 87.1 % males) whereas higher temperatures resulted in female-biased populations (31 °C, 82.4 % females). The intermediate temperatures (25 and 28 οC) leaded to 1:1 sex ratio.
For the swimming trials, only male zebrafish from the four different developmen-tal temperatures were used. They were sized 34,4 ± 2,17 mm TL and prior to the trials, were acclimatized to the swimming temperature (26,5 οC) for about one and a half month. The swimming trials were performed on a special apparatus of the Aquaculture lab and showed that developmental temperature has an important effect on the swimming ability of zebrafish, since the populations from 31 οC proved to be the fastest swimmers while the ones from 22 οC the slowest. A number of morphometric characters such as the weight, body width, body depth, maximum body depth and body depth at the caudal peduncle were examined and no differentiation was found between the different experimen-tal groups.
For the muscle lactate metabolism study, specimens as the ones of the swimming trials were used and were also subjected to the same acclimatization procedure. Muscle lactate concentration was estimated on 35 specimens per temperature which were distrib-uted at the following sampling periods: at rest, after full exhaustion due to swimming and after recovery for half hour, one hour, three hours, six hours and nine hours. Lactate analysis was performed on the Biochemistry Lab of the Medical Department (Dr Pa-paconstanti) and revealed that in all cases, lactate metabolism follows the previously es-tablished on other species pattern, since it rises during exercise and decreases during re-covery. It was also shown that developmental temperature does not affect the original and final lactate concentration (at rest and at full exhaustion respectively) but does affect its decomposition rate since the 22 οC populations require considerably more time than the others in order to fully recover.
Based on the present results, it is clear that zebrafish is a highly sensitive fish spe-cies as far as the effect of its developmental temperature is concerned. This observed plasticity on the temperature changes is a requisite since it lives in ecosystems (ponds and streams) characterized by intense seasonal and/or daily temperature fluctuations. The suc-cessful adjustment of this species to such type of environmental changes is what finally ensures its survival.
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