Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Osteological Development of the Garfish (Belone belone) Larvae S. Kuz ˇir 1 *, Z. Kozaric ´ 1 , E. Gjurc ˇevic ´ 2 , B. Baz ˇdaric ´ 3 and Z. Petrinec 2 Addresses of authors: 1 Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10 000, Zagreb, Croatia; 2 Department of Biology and Pathology of Fish and Bees, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10 000, Zagreb, Croatia; 3 DALMAR D.O.O. Obala kralja Petra Kresˇ imira IV, 64, Pakosˇtane 23211, Croatia; *Correspondence author: Tel.: +00 385 1 239 02 59; fax: +00 385 1 244 13 90; e-mail: kuzir@vef.hr With 4 figures Received May 2009; accepted for publication May 2009 Summary Garfish, Belone belone (Linnaeus, 1761) is an elongate, slander fish inhabiting the Eastern Atlantic and Mediterranean Sea. These fish from the Belonidae (Actinopterygii) family have important commercial value for Croatian fisheries. Samples for research were collected from experimental hatching in the Novigrad Sea. Preparation techniques included fixation in buffered formalin, trypsin clearing and staining with alcian blue and alizarin red. As little is known of garfish osteology and bone morphology, the main goal of this study was to describe ossification process in garfish fry. At hatching, no skeletal structure is present. Newly-hatched larvae also had no osteological elements. Ossification started at 7 day post- hatching (DPH) [total length (TL) 18 mm] with head bones and vertebral neural arch. Head skeleton continued to develop mostly over the period from 7 to 10 DPH. At 21 DPH (TL 49 mm), ossification process seemed to be finished, but it was not possible to distinguish borders of all bones. The primary interest of our research was to understand the growth dynamics as well as transformation of supporting body ele- ments from cartilage to bone. At the end, developmental characteristics and functional aspects of this formation in different fish species are discussed. Introduction The garfish, Belone belone (Linnaeus, 1761) is a pelagic, carnivorous, needlefish found in the Eastern Atlantic and Mediterranean Sea. Two subspecies were found in the Adriatic Sea: Belone belone acus, Risso 1827 and Belone belone gracilis Lowe 1839 (Russel, 1976). Belone belone gracilis can reach maximal length of 80 cm and maximal mass of 1.15 kg, while the average mass is 0.6 kg (Milisˇic´, 1994). This fish lives close to the surface and as juvenile feeds on plankton, while adult needlefishes are piscivorous. For reasons of its tasty meet, the fish is used for human consumption and has an important commercial value in Croatia. In spite of that, there are very little or no studies about the development and ossification process of Atherino- morpha. Osteichthyan and chondroichthyan fish present an astonishing diversity of skeletal and dental tissues. Jean-Yves and Huysseune (2003) pointed out difficulties in classifying fish tissue into the standard textbook categories of bone, cartilage, dentin and enamel. Since the time Dingerkus and Uhler (1977) developed a new method for bone and cartilage research, information on that view of fish development has been in progress (Fritzsche and Johnson, 1980; Koumoundouros et al., 1999, 2000, 2001a,b; Vandewalle et al., 1999). Matsuoka deals with different aspects of osteological development of the red sea bream (Matsuoka, 1985) and Japanese sardine (Matsuoka, 1997). Cranial development of Dicentrarchus labrax, especially of mandibular arch elements has been investigated by Gluck- man et al. (1999) and Kuzˇir et al. (2004). However, little is known of osteological development in Belonidae family, mostly because of lack of continuous intensive hatching. The main goal of the present study was to describe ossification process in garfish fry from 1 to 21 days post- hatching (DPH) with a focus on head morphology. We believe that description of unique jaw form development as well as the whole skeleton development became important contribution to the morphology of Belonidae family. Also, we provide some additional information for future phylogeny and classification study of the order Beloniformes. Materials and Methods Garfish fry were obtained by fertilization carried out under experimental conditions in the Novigrad Sea area (Bazˇdaric´, 2007). Specimens were sampled every day from 1 to 21 DPH. Each group was represented with five specimens who were fixed in buffered formalin. Dingerkus and Uhler (1977) method was used for the staining techniques. Ontogenesis of bony elements was observed under Nikon stereoscopic microscope and Flash Bus FBG computer software was used for analyses. Specimens were measured and average total length (TL) was used for correlation with DPH. Results were given from hatching to older phases (Gluckman et al., 1999), although our research started with osteological study (Gupta, 1971) and research on the whole skeleton of adult garfish from the Fish osteological collection at the Department of Anatomy, Histology and Embryology of Veterinary Faculty University of Zagreb. Adult specimens of garfish were compared with older fry and then older fry were compared with younger fry. We wish to point out that the ossification process and the main pattern of ossification will be described only for clearly visible elements for which an affirmed bony element name can be given with great certainty. Results Day 1–5 post hatching The examined specimens (TL 11–14 mm) had well developed cartilaginous elements of neurocranium and splanchnocranium. Ó 2009 Blackwell Verlag GmbH Anat. Histol. Embryol. 38, 351–354 (2009) doi: 10.1111/j.1439-0264.2009.00953.x ISSN 0340–2096