Effect of egg incubation temperature on the occurrence of skeletal deformities in Solea senegalensis By G. Dionı´sio 1,2 , C. Campos 1,3 , L. M. P. Valente 3 , L. E. C. Conceic¸a˜o 1 , M. L. Cancela 1,4 and P. J. Gavaia 1 1 CCMAR ⁄ CIMAR Centre of Marine Sciences, University of Algarve, Faro, Portugal; 2 Departamento de Biologia and CESAM—Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, Aveiro, Portugal; 3 CIIMAR ⁄ CIMAR, Interdisciplinary Centre of Marine and Environmental Research and ICBAS–Instituto de Cie ˆncias Biome´dicas de Abel Salazar, University of Porto, Porto, Portugal; 4 Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal Summary Temperature is a factor known to have a marked influence on metabolism and consequently on development, inducing ontogenetic plasticity in fish larvae. In this study, three different temperatures (15, 18 and 21°C) were assayed during Solea senegalensis embryonic development prior to hatching. After hatching all larvae were reared at the same temperature (21.0 ± 0.1°C) until 30 days. We observed that lowering the incubation temperature from 21 to 15°C reduced the incidence of skeletal deformities from 80 to 60%. Intermediate incuba- tion temperature (18°C), resulted in larvae that displayed a rate of abnormalities close to that observed at 15°C (64%). A similar typology of deformities was observed in fish from all rearing temperatures, with caudal vertebra being the most affected structures and with a low incidence of cranial and appendicular deformities. The effect of temperature in developmental plasticity will lead to osteological abnormalities mainly affecting the caudal vertebrae at 18°C (30%) and particularly the preural vertebra at 15°C (55%). Our results, clearly demonstrate a significant effect of water temperature during egg incubation on the skeletal development of Sene- galese sole, indicating that 18°C is the most appropriated temperature. These findings highlight the importance of tightly controlling temperature regimes during the early development of S. senegalensis, and the need for optimizing rearing proto- cols in order to avoid skeletal disorders or impaired growth. Introduction The Senegalese sole (Solea senegalensis) is a highly valued species, with particular interest for the southern European aquaculture industry. For the last two decades this species has been transposed for industrial production, but it still presents some constraints such as the high occurrence of vertebral deformities observed in fish under intensive rearing conditions (Engrola et al., 2009; Gavaia et al., 2009; Ferna´ndez and Gisbert, 2011). Water temperature is one of the most important environ- mental factors affecting survival, growth, development and influencing the appearance of early morphological abnormal- ities during early development of larval marine fish (e.g. Seikai et al., 1986; Ottesen and Bolla, 1998; Koumoundouros et al., 2001; Johnson et al., 2004; Klimogianni et al., 2004; Sfaki- anakis et al., 2004; Laurel and Blood, 2011). Previous studies have shown that inappropriate rearing temperatures will critically affect embryonic development in flatfish larvae, compromising hatching and growth as previously described for Southern flounder (Paralichthys lethostigma) (Daniels et al., 1996; Maaren and Daniels, 2001) and Northern rock sole (Lepidopsetta polyxystra) (Laurel and Blood, 2011) by altering the time of hatching, hatch success, and influencing larval growth, survival and metamorphosis. Altered tempera- ture was also reported to affect development of the skeleton by inducing metamorphosis-related morphological abnormalities in brown sole (Pseudopleuronectes herzensteini) (Aritaki and Seikai, 2004) or to affect the number of vertebrae composing the axial skeleton in halibut (Lewis-McCrea et al., 2004) and to promote an increase in the incidence of skeletal deformities in other teleosts such as gilthead seabream (Georgakopoulou et al., 2010), European seabass (Koumoundouros et al., 2001) or wolfish (Pavlov and Moksness, 1996). It was also demon- strated that temperature has a decisive effect on the size at different ontogenic events such as fin formation and metamor- phosis take place (Seikai et al., 1986; Polo et al., 1991; Koumoundouros et al., 2001). Egg incubation and larval rearing of Senegalese sole is usually conducted at temperatures ranging from 16 to 23°C (Dinis et al., 1999), although the most common range has been from 18 to 22°C (Engrola et al., 2005, 2009; Ferna´ndez et al., 2009; Blanco-Vives et al., 2010). Nev- ertheless, no data exist on the effects of temperature on the development of skeletal deformities in this species. The main objective of the present study was to evaluate the effects of different temperature regimes during egg incubation on the development of skeletal deformities in Solea senegal- ensis, up to 30 days after hatching. Materials and methods Experimental design The experiment was conducted at the CCMAR facilities, University of Algarve, Faro Portugal. Senegalese sole embryos, from a single batch, were obtained by natural spawning of a broodstock maintained in our facilities. This broodstock was captured in the wild and has been living for 3 years in captivity. Eggs were incubated at three constant temperatures (15.0 ± 0.1°C, 18.4 ± 0.1°C or 20.8 ± 0.2°C) as represented in Fig. 1. Triplicate groups of eggs were distributed among 9 fiberglass conical tanks (100 L) at a density of 100 eggs L )1 . After hatching all larvae were reared at the same constant temperature (21.0 ± 0.1°C) until 30 days after hatching (DAH). Water temperature, O 2 , salinity, pH and nitrogenous compounds were monitored regularly during J. Appl. Ichthyol. 28 (2012), 471–476 Ó 2012 Blackwell Verlag, Berlin ISSN 0175–8659 Received: October 18, 2011 Accepted: March 20, 2012 doi: 10.1111/j.1439-0426.2012.01996.x U.S. Copyright Clearance Centre Code Statement: 0175–8659/2012/2803–0471$15.00/0 Applied Ichthyology Journal of