Environ Biol Fish DOI 10.

1007/s10641-011-9833-z

The effect of rearing temperature on body shape and meristic characters in zebrafish (Danio rerio) juveniles
Dimitris G. Sfakianakis & Ioannis Leris & Anastasia Laggis & Maroudio Kentouri

Received: 2 December 2010 / Accepted: 18 April 2011 # Springer Science+Business Media B.V. 2011

Abstract Although zebrafish (Danio rerio) is a highly studied organism on many fields of research, many aspects of its basic biology still elude the scientific community. Its response to temperature especially developmental one - has been very scarcely studied and this is an important lack of knowledge since the species is considered quite eurythermal in nature. In the present study, zebrafish was subjected to four different developmental temperatures (22, 25, 28 and 31C) from the half-epiboly stage until after metamorphosis in order to examine whether the temperature can influence the juveniles phenotype. Morphometric and meristic characters were explored. Body shape and almost all of the meristic characters studied were significantly affected by the temperature applied during the first stages of development. Most meristic characters of the study, presented a significant differentiation in the extreme temperatures used (22 and/or 31C), whereas lower temperatures seemed to produce higher meristic counts in the majority of the characters. Zebrafish juveniles, as shown in this
D. G. Sfakianakis (*) : I. Leris : A. Laggis : M. Kentouri Biology Department, University of Crete, P.O. Box 2208, Vasilika Vouton, 71409 Heraklion, Crete, Greece e-mail: sfak@biology.uoc.gr Present Address: I. Leris Behavioural Biology, Department of Biology and Helmholtz Institute, Utrecht University, P.O. Box: 800.86, 3508 TB Utrecht, The Netherlands

study, exhibit highly variable phenotypes (phenotypic plasticity) induced by diverse thermal conditions during their early ontogenetic stages possibly in order to successfully adjust to different environments. Keywords Temperature . Zebrafish . Meristic . Body shape . Morphology . Larval rearing

Introduction Temperature is known to be one of the most important environmental factors that strongly affect all developmental processes in fishes. It influences the morphology in general (Lindsey 1988; Wimberger 1992; Tudela 1999; Koumoundouros et al. 2001; Pakkasmaa and Piironen 2001; Cabral et al. 2003; Turan 2004; Georgakopoulou et al. 2007), the muscle development (Johnston 1993, 2006; Wilkes et al. 2001; Johnston et al. 2009; Koumoundouros et al. 2009) and the appearance of morphoanatomical deformities (Wiegand et al. 1989; Polo et al. 1991; Lein et al. 1997; Vgsholm and Djupvik 1998; Wang and Tsai 2000; Koumoundouros et al. 2001; Sfakianakis et al. 2004, 2006; Abdel et al. 2005). Although the studies on the topic are numerous, very few are those that directly correlate temporal conditions (during early ontogeny) and their effect on meristic characters in any fish species (Lindsey 1988; Murray and Beacham 1989; Blaxter 1991; Georgakopoulou et al. 2007). On the other hand, only recently, it was

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reported for the first time that body shape differentiation in a fish species, sea bass (Dicentrarchus labrax, L.), was induced by the environmental temperature during the early life stages (Georgakopoulou et al. 2007). The authors used two different temperatures (15 or 20C) only in the larval rearing (from the egg stage until metamorphosis) of the species to finally conclude that developmental temperature strongly affects both body shape and meristic characters at the subsequent juvenile stage. In particular, they reported that fish body shape at 15C tended to be more slender than at 20C and that the final numbers of dorsal spines, soft rays, pectoral lepidotrichia and caudal dermatotrichia were significantly affected by temperature. Zebrafish, Danio rerio, the case study of this work, is regarded as a valuable model-organism for all kinds of research (Lawrence 2007). For the past few decades, it has been a very important modelorganism in research fields such as Genetics, Neurophysiology, Developmental Biology and Biomedicine (Amsterdam and Hopkins 2006). In spite of its popularity as a research tool, only recently did some integrated studies about its biological and ecological characteristics emerge (Engeszer et al. 2007; Spence et al. 2008) which however do not complete the puzzle of knowledge regarding its early life conditions and especially the environmental temperature. Although the temperature of the species habitat seems to be established at the range from 6C in the winter up to 38C in the summer (Spence et al. 2008) there are still some contradictions concerning the temperature range in which reproduction and early development take place (Sfakianakis et al. 2011). Our personal observations throughout many rearing trials showed that the rearing temperatures of 22C and 31C are the lower and upper limit values respectively for successful rearing in the laboratory. The purpose of the present work is to investigate whether different rearing temperature conditions result in differences on the body shape and the meristic characters of zebrafish.

University of Crete) kept in 30 l tanks at 280.5C and fed three times per day with industrial dry food (Sera Vipan, flakes for all ornamental fish, Germany). Two groups of about 80 and 100 individuals maintained at a sex ratio of 2:1 females to males were used as spawners for the purposes of the study. After the spawning, eggs were collected, separated (the live from the unfertilized ones) and submerged for 35 min in methylene blue hydrate (0.001%) (Westerfield 1995). The eggs were then randomly separated into 4 groups of 300 and each group was introduced in one of four 120 l tanks with different water temperatures (22, 25, 28 and 31C). The experiment was conducted in 2 replicates and different parental fish were used each time. The experimental temperatures (22, 25, 28 and 31C) were chosen in order to include the entire range of the suggested developmental temperatures that zebrafish encounters in nature (Engeszer et al. 2007; Spence et al. 2008; Froese and Pauly 2009) and be at the same time inside the range of the accomplishable successful rearing in laboratory facilities. The regulation of temperature was achieved with the use of electrical heaters (Aquarium Systems, VisiTherm, 100 W) and coolant device, when needed. The fish were fed ad libitum initially with Paramecium sp. (Blades Biological CO, UK) and later with newly hatched Artemia sp. nauplii (Instar I) (Westerfield 1995). Sampling and specimens handling Rearing was ended well after the ending of the larval stage, when the total length (TL) of a random sample exceeded 12 mm. Specimens were collected and anaesthetised with ethylenglycol-monophenylether (Merck, 0.20.5 ml l1), individually photographed (left-side, Olympus Camedia C-5050 Zoom) and collectively fixed in 5% buffered formalin (pH=7.2) (Taylor and van Dyke 1985). Staining for cartilage and bone was performed based on the modified technique of Park and Kim (1984). The obtained digital pictures were used to measure the TL (tpsDig, Rohlf, version 5.0.3.32) and to perform the morphometric analysis. The stained specimens were used to count the meristic characters in study. All specimens with deformities in the cephalic region, fins or vertebral column (Koumoundouros et al. 1997, 2001, 2002; Sfakianakis et al. 2003, 2004, 2006) as well as those with damaged body parts due to sampling manipulations were

Materials and methods Rearing Eggs of D. rerio were obtained from wild-type broodstock (Aquaculture lab, Biology Department,

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excluded from both the shape analysis and the meristics study. A total of 386 specimens were finally used in this study (approximately 50 per replicate). Laboratory fish were reared and handled according to the policies and guidelines of the Greek national law 2015/2001 which incorporates the convention for the protection of vertebrate animals used for experimental and other scientific purposes, of the council of Europe. Data analysis - morphometrics For the morphometric analysis, truss measurements were made on specimens by collecting XY coordinate data from morphological landmarks. Specific spots on the body, that were stable and visible at all developmental stages of the fish, were selected for landmark placement (Fig. 1), which was performed using specific image processing software (tpsDig, Rohlf, version 5.0.3.32). The distances between the landmarks corresponded to the morphometric characters used for the analysis and 28 in total were finally used (Fig. 1). The morphometric characters were significantly correlated with size, therefore each measurement was adjusted by the following allometric equation Y=a * X b, such that the standardised value of this variable in the case of an individual of size Xi would be:  b X0 Yi Yi Xi

where i is the true value of the variable Y, i* the standardised value, 0 an arbitrary reference size (in the present study, the mean TL of all the examined specimens, 14.37 mm) and b the allometric exponent. This method normalizes the individuals in a sample to a single, arbitrary size, common to all samples and, at the same time, maintains the individual variation (Tudela 1999). It has been successfully used by many researchers recently (Ibaez-Aguirre and Lleonart 1996; Tudela 1999; Salini et al. 2004; Turan 2004; Turan et al. 2006). The degree of similarity among samples and the relative importance of each measurement for group separation were assessed by stepwise discriminant function analysis (DFA) with cross-validation. Mahalanobis distances between groups and their associated probabilities were also evaluated. The significance of differences among groups was verified by multivariate analysis of variation (MANOVA) of the morphometric characters. The two replicates were pooled before the analyses which were conducted by the SPSS v.15 package. Data analysis - meristic characters Meristic characters such as the total number of vertebrae and the rays of the fins were recorded on the stained specimens. The fins used in this study were the dorsal, anal, pectoral, pelvic and caudal. The total number of rays of each fin was counted. Caudal rays were counted as upper and lower, lepidotrichia

Fig. 1 Landmarks collected on the in vivo photographed larvae and morphometric characters (distances) used in the present study. 1, anterior tip of upper jaw; 2, anterior margin of the eye, on the axis passing through the eye centre and the first landmark; 3, posterior margin of the eye, on the axis passing through the eye centre and the first landmark; 4, anterior base of the dorsal fin; 5, posterior base of the dorsal fin; 6, dorsal base of the caudal fin; 7, Ventral base of the caudal fin; 8, Distal tip of the hypural bones; 9, posterior central tip of the caudal fin; 10, posterior point of the body, on the vertical axis passing through the

posterior tips of the upper and lower caudal lobes (orientating the TL); 11, posterior base of the anal fin; 12, anterior base of the anal fin; 13, anterior base of the left pelvic fin; 14, posterior tip of the operculum; 15, ventral tip of cleithrum; 16, dorsal tip of the body on the vertical axis passing through the posterior margin of the eye; 17, dorsal tip of the body on the vertical axis passing through the 14th landmark. The 28 distances shown in the figure are those used in the morphometric analysis. The distance between landmarks 1 and 10 constitutes the TL. Landmark 9 was not used for extracting distances

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and dermatotrichia. Each fin meristic count was performed on the left side for paired fins. Skeletal terminology used is according to Harder (1975). All 386 specimens that were used for the meristics study had reached full completion of their skeletal elements (Table 1). The effect of temperature on the meristic characters of the zebrafish was studied by means of a MANOVA. The non-parametrical U test of MannWhitney for two samples was used (after the appropriate correction for tied measurements) to individually compare the meristic characters between the four thermal treatments applied (Sokal and Rohlf 1995). All tests were performed at the significance level of 0.05.

centroids of each sample in the space as defined by the first two canonical axes (Fig. 2). These two axes explain the 90.29% of the total recorded variation (Fig. 2). Meristic characters Analysis of the meristic elements showed differentiation of the total number of vertebrae and fins rays associated with rearing temperature (Wilks = 0.505, approximation F8.962, P <0.001, MANOVA). In most cases the extreme temperatures (22 and/or 31C) were significantly different from the rest of the temperatures studied (Fig. 3). Specifically, the numbers of vertebrae, dorsal and anal rays were significantly higher at the 22C group, while the numbers of pectoral and pelvic rays were significantly lower at 31C group. All the other temperature regimes on those meristic characters didnt show any differentiation (Fig. 3). In the upper caudal dermatotrichia, the two extreme temperature groups (22 and 31C) presented lower count when compared to the intermediate temperatures (25 and 28C) while in the lower caudal dermatotrichia, the number of the rays was lower at 22C. There were no significant differences between the four temperature groups with respect to the lower caudal lepidotrichia and almost no significant differences with respect to the upper lepidotrichia (Fig. 3).

Results Morphometric characters Statistical analysis showed that morphometric characters and therefore the body shape were significantly affected by rearing temperature (Wilks =0.039, approximation F15.003, P <0.001, MANOVA). The distances that contributed to the overall differentiation between temperature groups were d412, d45, d1213, d413, d78 and d56 (Table 2). The significance of the variance between the four temperature groups and the Mahalanobis distances show that each and every one of the four temperature groups is well differentiated from the others (Table 3; P <0.001). Moreover, reviewing of the Mahalanobis distances reveals that the bigger the temperature difference between two groups, the bigger the variance they exhibit (Table 3). A very high percentage (above 91%) of the examined specimens can be successfully reclassified (Table 4) between the four different thermal groups whereas the discrimination between the groups is also clearly reflected in the graphic representation of the
Table 1 Total length (mm) S.D. of all the specimens (n, total count) used for the meristics study TC 22 25 28 31 TL 13,19 13,45 13,77 13,27 SD 1,42 1,32 1,51 1,41 n 96 100 101 89

Discussion Morphometric characters The external morphology and especially the body shape of fish, has been thoroughly studied by many researchers in many different fish species. Although there are a lot of studies describing the morphological differentiation of farmed and wild populations and the factors that trigger it (Matsuoka 1987; Blaxter 1991; Wimberger 1992; Junquera and Perez-Gandaras 1993; Corti et al. 1996; Ellis et al. 1997; Mamuris et al. 1998; Tudela 1999; Hard et al. 2000; Pakkasmaa and Piironen 2001; Salini et al. 2004; Turan 2004; Turan et al. 2006), there is a lack of bibliographic data focusing on the influence of temperature on body shape of fish and especially on specimens reared in laboratory conditions. The study of Georgakopoulou

Environ Biol Fish Table 2 The distances (Fig. 1) that varied significantly between the 4 populations based on the MANOVA analysis Distances d412 d45 d1213 d413 d78 d56 Wilks' Lambda 0.053262 0.045830 0.044712 0.043969 0.043614 0.042537 F-remove (3.215) 26.09458 12.45399 10.40123 9.03674 8.38671 6.40943 p-level 0.000000 0.000000 0.000002 0.000012 0.000027 0.000354 Toler. 0.105658 0.108204 0.165672 0.026775 0.539391 0.161932

et al. (2007) is the only one that proved something similar when they recorded that sea bass body shape tended to be more slender when reared at 15C as opposed to reared at 20C. In the present study, a clear variation on the shape of the body between the four populations was observed, showing a strong influence of the rearing temperature. The results of the DFA showed that the distances that played the most important role in the discrimination were d45, d1213, d413, d78 and d56 (Table 2; Fig. 1) which are all located in the posterior part of the body. The variance in fish body shape can be explained by the general morphological plasticity that fish present in the different environments, due to alterations in muscle and bone developmental patterns (mechanical - functional approach; Wimberger 1992). In addition, physico-chemical water conditions (density, viscosity etc.), are greatly affected by environmental factors, especially temperature. So, it is possible that different conditions of the media in which fish live and swim, require different locomotive responses that can lead to the observed morphological variance, during the developmental period. Wimberger (1992) argued that if plasticity is a result of the stress regime and the various mechanical stimuli, behavioral plasticity is responsible for the morphological plasticity. Moreover, it is well known that temperature greatly affects fish metabolism, therefore the relative growth rate of some tissues

and organs of the myo-skeletal system of the fish, could be influenced by temperature during the developmental period (Lindsey 1988; Imre et al. 2002). At the same time, temperature is known to have great effects on muscle development (Johnston 1993, 2006; Nathanailides et al. 1995; Alami-Durante et al. 2000; Ayala et al. 2001; Wilkes et al. 2001; Johnston et al. 2009; Koumoundouros et al. 2009) with possible further impacts on external morphology. It is also proven that temperature affects the number and the diameter of fast and slow muscle fibres (Johnston 2006; Koumoundouros et al. 2009). Therefore, considering that zebrafishs swimming type is the subcarangiform (Plaut and Gordon 1994), achieved by the use of the posterior half of the body (1/22/3 of the overall body muscle tissue), and that the posterior part of the body was the most variant between groups, it can be stated that differences in body-shape, derive from differences in quantity and/ or size of the muscle fibres, or the relative distribution of the muscle tissue of the fish reared in different temperatures. Meristic characters Many studies have compared populations of the same species (mostly wild ones) and have found variation of the meristic characters. Most of them hypothesize
Table 4 Classification Matrix, (rows, observed; columns, predicted) Percent Correct 22 88.88889 86.76471 92.64706 96.42857 91.05691 22 48 7 0 0 55 25 6 59 2 0 67 28 0 2 63 2 67 31 0 0 3 54 57

Table 3 p-levels (***, <0.001) and squared Mahalanobis distances 22 22 25 28 31 7.94091 23.58698 34.01297 15.14899 28.72729 11.77138 25 *** 28 *** *** 31 *** *** ***

25 28 31 Total

Environ Biol Fish Fig. 2 Plot of zebrafish juveniles (N =200) reared at four different temperature conditions (22, 25, 28 and 31C) on the first 2 axes of Canonical Analysis based on their morphometric characters. Ellipses represent 95% of confidence areas

that its due to the different temperature conditions of the living area populations (Tudela 1999; O'Reilly

and Horn 2004; Turan 2004; Turan et al. 2006), whereas there are only a few studies that directly

Fig. 3 Numbers of meristic counts (MeanS.E.) on zebrafish juveniles (N =89101) of the four different temperature groups (22, 25, 28 and 31C). Same letters (e.g. a,b) indicate absence of statistical difference (MannWhitney U-test)

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correlate early developmental temperature with meristic variation (Lindsey 1988; Murray and Beacham 1989; Blaxter 1991; Georgakopoulou et al. 2007). In the present study we concluded that early life temperature influences the meristic characters of the developing fish. Variation of the meristic counts in the extreme temperatures (22 and/or 31C) was observed in most of the cases. Zebrafish seems to be a highly variable (in respect to its phenotype) fish species. Ferreri et al. (2000) studied the meristic counts in wild and reared zebrafish and reported that the variability of the meristic counts (in many of the tested anatomical areas) exhibited by the wild specimens was higher than the one exhibited by the reared ones. Reared specimens of the Ferreri et al. (2000) study developed at a constant laboratory temperature and, based on the results of the present study, it is logical to expect them to present less variable meristic counts than the wild ones which probably encountered non-stable temperature conditions in their early life. When comparing the present data with other studies of the meristic counts in zebrafish (Dentry and Lindsey 1978; Axelrod and Schultz 1990; Ferreri et al. 2000; Bird and Mabee 2003; Parichy et al. 2009) it is clear that they are in agreement as far as the total number of the characters is concerned. However, the present study is the only one that presents both the minimum and the maximum number of counts previously reported for each character and this is a result of the four different developmental temperatures used. It is therefore reasonable to argue that the temperature spectrum used in the present study covered sufficiently the observed morphological plasticity of the species. It is common knowledge that fish developing at lower water temperatures produce higher numbers of meristic counts than those developing at higher temperatures (Murray and Beacham 1989). This is in accordance with the present study, where with the exception of the caudal fin rays, all other meristic characters examined presented either higher numbers at lower temperatures (vertebrae, dorsal and anal rays) or lower numbers at higher temperatures (pectoral and pelvic rays). The caudal fin rays (upper and lower lepidotrichia and dermatotrichia) did not present any particular trend or in some cases any differentiation at all, except from the lower caudal dermatotrichia which were significantly fewer at the lower temperature used.

It has been stated before that meristic counts are continuously subjected to environmental influences from fertilization up to the final count fixation or simply during the entire larval period (Taning 1952; Fowler 1970). Georgakopoulou et al. (2007) reported recently that temperature effect from the half-epiboly stage until metamorphosis is enough to permanently alter the meristic counts of many fins in sea bass juveniles. All the above completely agree with the present findings since it was proven here that if temperature is applied only between the egg stage and metamorphosis (as opposed to during the fertilization and spawning as well), it can have a dramatic effect on a fishs final meristic count. As far as the number of the vertebral centra is concerned, which is clearly a different case than the fin rays, Lindsey and Ali (1965) suggested in an earlier period that it can be modified only during early embryogenesis. In a study conducted by Dentry and Lindsey (1978) in zebrafish, it was reported that in some cases the vertebral counts of fish cultured at the same water temperature were affected by the temperature history of the parents before and during fertilization. The present study showed clearly that vertebral numbers are modified by temperature when the latter is applied only during the embryonic and larval stages. To further specify where exactly in the developmental process does the effect of temperature act, we should try and isolate the exact time window where the meristic counts or the body shape are defined. Could it be early or later in the development (e.g. during the embryonic stage only) or are the entire embryonic and larval stages combined necessary for the effect to present itself? In order to answer these questions, a narrower stage-targeted experiment should be conducted, possibly with the same temperature range as the one used in the present study. The overall effect of temperature on zebrafish phenotype exhibited in the present study is certainly another case of phenotypic plasticity. This fishes ability of adapting successfully to all kinds of variations in their environmental conditions has long been regarded as a requisite for their survival (Fuiman and Batty 1997). Zebrafish seems to be copeing well in the different thermal treatments by adjusting successfully both its body shape and its meristic counts. The question that remains to be answered however is whether those changes are adequate for

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the specimenssuccessful living and reproduction at such extreme temperature conditions.

Acknowledgements The authors thank two anonymous reviewers for their helpful comments in producing the final manuscript. The present study was financed by the European Social Fund and National Resources (EPEAEK IIPYTHAGORAS I) to M.K.

References
Abdel I, Abelln E, Lpez-Albors O, Valds P, Nortes M, Garca-Alczar A (2005) Abnormalities in the juvenile stage of sea bass (Dicentrarchus labrax L.) reared at different temperatures: types, prevalence and effect on growth. Aquac Int 12(6):523538 Alami-Durante H, Bergot P, Rouel M, Goldspink G (2000) Effects of environmental temperature on the development of the myotomal white muscle in larval carp (Cyprinus carpio L.). J Exp Biol 203(24):3675 Amsterdam A, Hopkins N (2006) Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. Trends Genet 22:473478 Axelrod H, Schultz L (1990) Handbook of tropical aquarium fishes. McGraw-Hill, New York, p 718 Ayala M, Lpez-Albors O, Gil F, Garca-Alczar A, Abelln E, Alarcn J, lvarez M, Ramrez-Zarzosa G, Moreno F (2001) Temperature effects on muscle growth in two populations (Atlantic and Mediterranean) of sea bass, Dicentrarchus labrax L. Aquaculture 202(34):359370 Bird N, Mabee P (2003) Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae). Dev Dyn 228(3):337357 Blaxter J (1991) The effect of temperature on larval fishes. Neth J Zool 42(3):336357 Cabral HN, Marques JF, Rego AL, Catarino AI, Figueiredo J, Garcia J (2003) Genetic and morphological variation of Synaptura lusitanica Capello, 1868, along the Portuguese coast. J Sea Res 50(23):167175 Corti M, Loy A, Cataudella S (1996) Form changes in the sea bass, Dicentrarchus labrax (Moronidae: Teleostei), after acclimation to freshwater: an analysis using shape coordinates. Environ Biol Fish 47(2):165175 Dentry W, Lindsey C (1978) Vertebral variation in zebrafish (Brachydanio rerio) related to the prefertilization temperature history of their parents. Can J Zool 56(2):280 283 Ellis T, Howell B, Hayes J (1997) Morphological differences between wild and hatchery reared turbot. J Fish Biol 50 (5):11241128 Engeszer RE, Patterson LB, Rao AA, Parichy DM (2007) Zebrafish in the wild: a review of natural history and new notes from the field. Zebrafish 4(1):2140 Ferreri F, Nicolais C, Boglione C, Bmertoline B (2000) Skeletal characterization of wild and reared zebrafish: anomalies and meristic characters. J Fish Biol 56(5):11151128 Fowler J (1970) Control of vertebral number in teleosts-an embryological problem. Q Rev Biol 45(2):148167

Froese R, Pauly D (2009) FishBase website, http://www. fishbase.org/Summary/SpeciesSummary.php?ID=4653. Accessed at 7 June 2010 Fuiman L, Batty R (1997) What a drag it is getting cold: partitioning the physical and physiological effects of temperature on fish swimming. J Exp Biol 200(12):1745 Georgakopoulou E, Sfakianakis DG, Kouttouki S, Divanach P, Kentouri M, Koumoundouros G (2007) The influence of temperature during early life on phenotypic expression at later ontogenetic stages in sea bass. J Fish Biol 70(1):278291 Hard J, Berejikian B, Tezak E, Schroder S, Knudsen C, Parker L (2000) Evidence for morphometric differentiation of wild and captively reared adult coho salmon: a geometric analysis. Environ Biol Fish 58(1):6173 Harder W (1975) Anatomy of fishes. Schweizerbart, Stuttgart Ibaez-Aguirre AL, Lleonart J (1996) Relative growth and comparative morphometrics of Mugil cephalus L. and M. curema V. in the Gulf of Mexico. Sci Mar 60(23):361 368 Imre I, McLaughlin R, Noakes D (2002) Phenotypic plasticity in brook charr: changes in caudal fin induced by water flow. J Fish Biol 61(5):11711181 Johnston IA (1993) Temperature influences muscle differentiation and the relative timing of organogenesis in herring (Clupea harengus) larvae. Mar Biol 116(3):363379 Johnston IA (2006) Environment and plasticity of myogenesis in teleost fish. J Exp Biol 209(12):2249 Johnston IA, Lee HT, Macqueen DJ, Paranthaman K, Kawashima C, Anwar A, Kinghorn JR, Dalmay T (2009) Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. J Exp Biol 212(12):1781 Junquera S, Perez-Gandaras G (1993) Population diversity in Bay of Biscay anchovy (Engraulis encrasicolus L. 1758) as revealed by multivariate analysis of morphometric and meristic characters. ICES J Mar Sci 50(4):383 Koumoundouros G, Ashton C, Sfakianakis DG, Divanach P, Kentouri M, Anthwal N, Stickland N (2009) Thermally induced phenotypic plasticity of swimming performance in European sea bass Dicentrarchus labrax juveniles. J Fish Biol 74(6):13091322 Koumoundouros G, Divanach P, Kentouri M (2001) The effect of rearing conditions on development of saddleback syndrome and caudal fin deformities in Dentex dentex (L.). Aquaculture 200(34):285304 Koumoundouros G, Gagliardi F, Divanach P, Boglione C, Cataudella S, Kentouri M (1997) Normal and abnormal osteological development of caudal fin in Sparus aurata L. fry. Aquaculture 149(34):215226 Koumoundouros G, Sfakianakis DG, Divanach P, Kentouri M (2002) Effect of temperature on swimming performance of sea bass juveniles. J Fish Biol 60(4):923932 Lawrence C (2007) The husbandry of zebrafish (Danio rerio): a review. Aquaculture 269(14):120 Lein I, Holmefjord I, Rye M (1997) Effects of temperature on yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 157(12):123135 Lindsey C, Ali M (1965) The effect of alternating temperature on vertebral count in the medaka (Oryzias latipes). Can J Zool 43(1):99104

Environ Biol Fish Lindsey CC (1988) Factors controlling meristic variation. Fish Physiol 11(part B):197274 Mamuris Z, Apostolidis P, Panagiotaki P, Theodorou A, Triantaphyllidis C (1998) Morphological variation between red mullet populations in Greece. J Fish Biol 52(1):107117 Matsuoka M (1987) Development of the skeletal tissues and skeletal muscles in the red sea bream (Pagrus major). Bull Seikai Reg Fish Res Lab 65:1114 Murray C, Beacham T (1989) Responses of meristic characters in chum salmon (Oncorhynchus keta) to temperature changes during development. Can J Zool 67(3):596600 Nathanailides C, Stickland N, Lopez-Albors O (1995) Influence of prehatch temperature on the development of muscle cellularity in posthatch Atlantic salmon (Salmo salar). Can J Fish Aquat Sci 52(4):675680 O'Reilly K, Horn M (2004) Phenotypic variation among populations of Atherinops affinis (Atherinopsidae) with insights from a geometric morphometric analysis. J Fish Biol 64(4):11171135 Pakkasmaa S, Piironen J (2001) Morphological differentiation among local trout (Salmo trutta) populations. Biol J Linn Soc 72(2):231239 Parichy D, Elizondo M, Mills M, Gordon T, Engeszer R (2009) Normal table of postembryonic zebrafish development: Staging by externally visible anatomy of the living fish. Dev Dyn 238(12):29753015 Park E, Kim D (1984) A procedure for staining cartilage and bone of whole vertebrate larvae while rendering all other tissues transparent. Biotech Histochem 59(5):269272 Plaut I, Gordon M (1994) Swimming metabolism of wild-type and cloned zebrafish Brachydanio rerio. J Exp Biol 194 (1):209 Polo A, Yufera M, Pascual E (1991) Effects of temperature on egg and larval development of Sparus aurata L. Aquaculture 92:367375 Salini J, Milton D, Rahman M, Hussain M (2004) Allozyme and morphological variation throughout the geographic range of the tropical shad, hilsa Tenualosa ilisha. Fish Res 66(1):5369 Sfakianakis DG, Georgakopoulou E, Papadakis I, Divanach P, Kentouri M, Koumoundouros G (2006) Environmental determinants of haemal lordosis in European sea bass, Dicentrarchus labrax (Linnaeus, 1758). Aquaculture 254 (14):5464 Sfakianakis DG, Koumoundouros G, Anezaki L, Divanach P, Kentouri M (2003) Development of a saddleback-like syndrome in reared white seabream Diplodus sargus (Linnaeus, 1758). Aquaculture 217(14):673676 Sfakianakis DG, Koumoundouros G, Divanach P, Kentouri M (2004) Osteological development of the vertebral column and of the fins in Pagellus erythrinus (L. 1758). Temperature effect on the developmental plasticity and morphoanatomical abnormalities. Aquaculture 232(14):407424 Sfakianakis DG, Leris I, Kentouri M (2011) Effect of developmental temperature on swimming performance of zebrafish (Danio rerio) juveniles. Environ Biol Fish 90 (4):421427. doi:10.1007/s10641-010-9751-5 Sokal R, Rohlf F (1995) Biometry: the principles and practice of statistics in biological research. WH Freeman, New York Spence R, Gerlach G, Lawrence C, Smith C (2008) The behaviour and ecology of the zebrafish, Danio rerio. Biol Rev Camb Philos Soc 83(1):1334 Taning AV (1952) Experimental study of meristic characters in fishes. Biol Rev 27(2):169193 Taylor WR, van Dyke GC (1985) Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9(2):107119 Tudela S (1999) Morphological variability in a Mediterranean, genetically homogeneous population of the European anchovy, Engraulis encrasicolus. Fish Res 42(3):229243 Turan C (2004) Stock identification of Mediterranean horse mackerel (Trachurus mediterraneus) using morphometric and meristic characters. ICES J Mar Sci 61(5):774 Turan C, Oral M, ztrk B, Dzgne E (2006) Morphometric and meristic variation between stocks of Bluefish (Pomatomus saltatrix) in the Black, Marmara, Aegean and northeastern Mediterranean Seas. Fish Res 79(12):139147 Vgsholm I, Djupvik HO (1998) Risk factors for spinal deformities in Atlantic salmon, Salmo salar L. J Fish Dis 21(1):4753 Wang L, Tsai C (2000) Effects of temperature on the deformity and sex differentiation of tilapia, Oreochromis mossambicus. J Exp Zool 286(5):534537 Westerfield M (1995) The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene Wiegand M, Hataley J, Kitchen C, Buchanan L (1989) Induction of developmental abnormalities in larval goldfish, Carassius auratus L., under cool incubation conditions. J Fish Biol 35(1):8595 Wilkes D, Xie SQ, Stickland NC, Alami-Durante H, Kentouri M, Sterioti A, Koumoundouros G, Fauconneau B, Goldspink G (2001) Temperature and myogenic factor transcript levels during early development determines muscle growth potential in rainbow trout ( Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax). J Exp Biol 204(16):27632771 Wimberger PH (1992) Plasticity of fish body shape. The effects of diet, development, family and age in two species of Geophagus (Pisces; Cichlidae). Biol J Linn Soc 45 (3):197218