SEXUAL MATURITY OF THE AGUJON NEEDLEFISH Tylosurus acus imperialis

SEXUAL MATURITY OF THE AGUJON NEEDLEFISH Tylosurus acus imperialis Kokokiris L.* 1, Minos G. 1, Kiriakidou Μ. 1, Alexandrou M. 1, Papadaki Μ. 2, Karidas T. 1, Economidis P.S. 3 1 Alexander Technological Educational Institute of Thessaloniki, Department of Aquaculture and Fisheries Technology, P.O.Box 157, HL-63200, Nea Moudania, Hellas, 2 Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and Aquaculture, P.O.Box 2214, 71003, Iraklion, Crete, Hellas, 3 Karakasi 79, GR- 54453, Thessaloniki, Hellas Abstract The present study was designed to identify maturity of the agujon needlefish, Tylosurus acus imperialis, using microscopic criteria. Histological analysis was applied on 110 individuals sampled between May and August in Thermaikos Gulf (N. Aegean Sea, Greece). Female gonadal development was observed in that only one functional ovary persisted in all females. This was normally on the right, while on the left side the ovary failed to differentiate. Male agujon needlefish had bilateral testes but an asymmetry between testes became evident, with the left testis being significantly less developed. All female and male individuals were in spawning capability or even in spawning active phase, providing evidence of a spawning period from May to August with a peak in June and July. The minimum size for maturity of females was 64.5 cm TL whilst minimum size for male maturity was 59.3 cm TL. The coexistence within ovary of oocytes at various maturity steps, i.e. secondary growth full grown, hydrated oocytes (eggs) and postovulatory follicles clearly indicated T. acus imperialis as a multiple spawner. Furthermore, the absence of immature individuals among specimens provided strong evidence for the existence of a spawning ground in the study area. Keywords: Tylosurus, histology, maturity, spawning period *Corresponding author: Lambros Kokokiris (lamprosk@aqua.teithe.gr) ΣΟΙΧΕΙΑ ΓΕΝΝΗΣΙΚΗ ΩΡΙΜΑΝΗ ΣΗ ΒΑΙΛΟΖΑΡΓΑΝΑ Tylosurus acus imperialis Κοκοκφρησ Λ. *1, Μίνοσ Γ. 1, Κυριακίδου Μ. 1, Αλεξάνδρου Μ. 1, Παπαδάκη Μ. 2, Καρφδασ Θ. 1, Οικονομίδησ Π.. 3 1 Σιήια Σεπκμθμβίαξ Αθζείαξ ηαζ Τδαημηαθθζενβεζχκ, Αθελάκδνεζμ Σεπκμθμβζηυ Δηπαζδεοηζηυ Ίδνοια Θεζζαθμκίηδξ, Σ.Θ. 157, 63200, Νέα Μμοδακζά, Δθθάδα, 2 Ηκζηζημφημ Θαθάζζζαξ Βζμθμβίαξ, Βζμηεπκμθμβίαξ ηαζ Τδαημηαθθζενβεζχκ, Δθθδκζηυ Κέκηνμ Θαθάζζζςκ Δνεοκχκ, Σ.Θ. 2214, 71003, Ζνάηθεζμ, Δθθάδα, 3Καναηάζδ 79, 54453, Θεζζαθμκίηδ, Δθθάδα 427

Πεξίιεςε ηδκ ενβαζία αοηή πενζβνάθεηαζ δ βεκκδηζηή ςνίιακζδ ηδξ ααζζθμγανβάκαξ Tylosurus acus imperialis, ιε αάζδ ηδκ ζζημθμβζηή ακάθοζδ ηςκ βμκάδςκ 110 αηυιςκ, ηα μπμία αθζεφεδηακ ηδκ πενίμδμ Μαΐμο-Αοβμφζημο ζημ Θενιασηυ Κυθπμ. ηα εδθοηά άημια ήηακ ακεπηοβιέκδ ιυκμ δ δελζά βμκάδα εκχ ζηα ανζεκζηά άημια παναηδνήεδηε έκημκδ αζοιιεηνία ηςκ υνπεςκ ιε ημκ ανζζηενυ υνπζ κα είκαζ ζδιακηζηά πζμ ιζηνυξ απυ ημ δελζυ. θα ηα άημια ήηακ χνζια εκχ ηα πενζζζυηενα ημοξ ιήκεξ Ημφκζμ ηαζ Ημφθζμ ήηακ ζηα ηεθζηά ζηάδζα ηδξ ςνίιακζδξ (ζηάδζμ ςμημηίαξ, ζηάδζμ ζπενιίαζδξ). Ζ πενίμδμξ ςμημηίαξ ηδξ ααζζθμγανβάκαξ ζημ Θενιασηυ Κυθπμ εηηείκεηαζ απυ ημκ Μάζμ ιέπνζ ημκ Αφβμοζημ, ιε ηδκ ςμημηία κα ημνοθχκεηαζ ημοξ ιήκεξ Ημφκζμ ηαζ Ημφθζμ. Σμ εθάπζζημ ιέβεεμξ ηςκ χνζιςκ αηυιςκ ήηακ 64,5 cm μθζημφ ιήημοξ βζα ηα εδθοηά άημια ηαζ 59,3 cm βζα ηα ανζεκζηά. Ζ ηαοηυπνμκδ ζοκφπανλδ ςμηοηηάνςκ ζε πενζζζυηενα απυ δφμ ζηάδζα ακάπηολδξ ηαζ εζδζηυηενα δ ζοκφπανλδ θεηζεζηχκ ςμηοηηάνςκ, ςμηοηηάνςκ ζημ ζηάδζμ ηδξ ηεθζηήξ ςνίιακζδξ (οαθχδδ ή εκοδαηςιέκα) ηαζ ηεκχκ ςμεοθαηίςκ, οπμδδθχκμοκ υηζ δ ααζζθμγανβάκα είκαζ πμθθαπθυξ απμεέηδξ. Ζ πακηεθήξ απμοζία ακχνζιςκ αηυιςκ ζηα δείβιαηα απυ ηδκ πενζμπή ιεθέηδξ, δείπκεζ υηζ δ ζοβηεηνζιέκδ πενζμπή ιπμνεί κα είκαζ πεδίμ ςμημηίαξ ηδξ ααζζθμγανβάκαξ. Λέξειρ κλειδιά: Tylosurus, ηζηνινγία, σξίκαλζε, πεξίνδνο σνηνθίαο *οββναθέαξ επζημζκςκίαξ: Λάιπνμξ Κμημηφνδξ (lamprosk@aqua.teithe.gr) 1. Introduction The needlefish, Tylosurus acus (Belonidae, Lacepède 1803) is an epipelagic species that is widely distributed in tropical and subtropical waters with many subspecies that have been recognized depending to their geographical distribution (Collette 2003). T. a. acus (Lacépède 1803) is found in the Western Atlantic; T. a. rafale (Collette & Parin 1970) is mainly distributed in the Gulf of Guinea, T. a. melanotus (Bleeker 1850) is found throughout the Indo-Western Pacific, extending to the Eastern Pacific and the agujon needlefish T. a. imperialis is found in the Mediterranean Sea (Collette 2003). The agujon needlefish T. a. imperialis consists part of the catches of the coastal small scale fishery in Thermaikos Gulf (N. Aegean Sea) since 2012. Agujon needlefish are usually being caught by gillnets, beach seines and night spear fishing throughout late spring and summer months reaching a relatively high price in local fish markets. Despite the existence of various information on its distribution in the Meditteranean Sea (Bello 1995, Akyol & Kara 2011, Pećarević et al. 2013, Türker Çakır & Zengin 2013, Imsiridou et al. 2014), information on its biology and particularly on its reproductive biology is still scarce. Moreover, the biological information accumulated on species of the six Tylosurus genera (Collette 2003) across their world-wide distribution is not well documented. To our knowledge, detailed information on the reproductive biology of Tylosurus genera is rather limited to T. a. melanotus in waters of southeastern Taiwan (Liao & Chang 2011). However, the reproductive biology information is essential for fisheries management schemes. The aim of this study was to identify the maturity of the agujon needlefish, T. a. imperialis, using microscopic criteria, coming to a better understanding of its reproductive biology and contributing to the development of appropriate management schemes. 2. Materials and methods A total of 110 T. a. imperialis individuals were sampled on a monthly basis between May and August, during 2013 and 2014. All fish were caught by artisanal fishery (gillnets, beach seines, permanent fish traps, night spear fishing from boat, arrowhead fixed fish trap) operating in Thermaikos Gulf (40º23 00 N; 22º47 00 E). For each specimen, total length (TL) to the nearest centimetre, as well as the total and gutted 428

weight (gw b ), and gonad and liver weights were measured (accuracy of 0.01 g). Specimens ranged from 59 to 107 cm (TL) and 0.23 to 1.7 kg (W b ). Slices of the gonads were fixed in 10% buffered formaldehyde. After 24 48 h, a segment from the central part of the left lobe was dehydrated in increasing concentrations of ethanol and clarified in xylene. Each segment was then embedded in paraffin wax and cross-sections of 4 5ιm thickness were cut and stained with Mayers haematoxylin-eosin. Sex and maturity stage (MS) were determined from microscopic observations of histological slides. Ovarian maturity was defined by the maturation stage of the most advanced oocytes and testicular maturity by the most advanced spermatic cells. For the development classification of oocytes, the classification scheme of Grier et al. (2009) was used. Ovarian and testicular maturity was divided into phases, following the conceptual models and the standardised terminology proposed by Brown-Peterson et al. (2011). The presence of postovulatory and atretic follicles was also recorded. For each individual the following indices were calculated: (i) gonadosomatic index (I g ), (I g =100 x gonads weight/gutted weight), (ii) hepatosomatic index (I h ), (I h =100 x liver weight/gutted weight, and (iii) condition index (K n =total weight/tl b ). 3. Results Specimens were caught only from May to August. Males outnumbered females. The smaller specimen collected was of 64.5 cm TL for females and 59.3 cm for males. Those sizes were also the minimum sizes for maturity of females and males, since all specimens were sexually mature with gonads capable of spawning and no immature individual was collected at lower length sizes. Female gonadal development was observed in that only one functional ovary persisted in all mature females. This was normally on the right, while on the left side the ovary failed to differentiate and underwent a degenerative fate along with its associated reproductive tract. Adult male agujon needlefish had bilateral testes but an asymmetry between left and right testes became very evident, with the left testis significantly less developed than the right testis (reaching only up to 10% of the right testis). Mature ovaries were cylindrical, swollen, ranging from 13.4 to 33.1 cm in length (Table 1), having a deep yellowish-orange in colour. Ovaries divided into those being capable of spawning ( spawning capable ) and actively spawning ( active spawning ). Spawning capable ovaries contained oocytes at all steps of oogenesis but fullgrown vitellogenic oocytes (SG fg ), were the most advanced and large oocytes. SG fg oocytes were distinguished by the formation of large regions of fluid yolk, the appearance of an ooplasm displaced into the peripheral rim surrounding the yolk mass and a zona pellucida having its maximum length. The oil droplets concentrated around the nucleus, then fused into one or a few oil drops. The germinal vesicle was of an irregular shape with an envelope became progressively folded. Numerous small and large nucleoli could also be seen. Spawning active ovaries were characterised by the presence of hydrated oocytes (final maturation step, OM); having the yolk material completely fused with a few or one oil drops formed inside. The appearance of the oocyte was homogenous, finely granular, whilst the germinal vesicle was invisible due to disintegration and dispersion of the nuclear membrane (GVBD). The ooplasm, was restricted to a narrow rim, laying next to the zona pellucida, which had became thinner. At the late portion of the spawning season (i.e. August) in some spawning-capable ovaries, empty ovarian follicles (post ovulatory follicles, POFs) were present, indicating that ovulation had occurred. Clear sings of atresia could be also identified in some ovaries. Mature testes were triangular or diamond shaped, white in colour, thick and long in size with an average length of 16.8 cm significantly less elongated than ovaries (ANOVA, P=0.04, Table 1). Testes in spawning 429

capable phase were identified by the presence of spermatozoa in the lumen of the lobules and in the sperm ducts. Some males were moved to the actively spawning subphase, releasing milt after a gentle pressure was placed on the abdomen (spermiating males). Males remained in the spawning capable phase during the majority of the period from May to August and underwent active spermatogenesis during which all stages of spermatogenesis were observed. They were classified as being in the early, middle or late portion of the spawning season based on continuous or discontinuous germinal epithelium (GE). During the early GE subphase of the spawning capable phase, they were distinguished by a continuous GE throughout testis and the presence of spermatogonia (Sg) in the spermatocyts. During the mid-ge subphase, spermatogenesis ceased in some spermatocysts in lobules near the sperm ducts, resulting in a discontinuous GE near the ducts but a continuous GE at the periphery of the lobules. By the end of July, males were in late GE subphase having a discontinuous GE throughout the testis, an increasing prevalence of anastomosing lobules and reduced spermatogenesis. During August, at the late portion of the spawning season some males with testes entering the regressing phase were seen. Table 1. Mean values (± SD) and range of values for body weight (W b ), gutted body weight (gw b ), total length (TL), gonadosomatic index (I g ), hepatosomatic index (I h ), condition index (K n ) and gonads length in female and male spawning capable needlefish, T. acus imperialis. Females (n=29) Males (n=81) Mean (±SD) Range Mean (±SD) Range W b (g) 728.3 (±359) 284-1602 480.8 (±287) 232-1690 gw b (g) 620.4 (±296) 272-1318 447.7 (±258) 219-1506 TL (cm) 81.1 (±12.5) 64.5-105.5 72.0 (±10.2) 59.3-107.4 I g (%) 9.7 (±5.5) 0.9-23.2 1.6 (±0.8) 0.4-7.1 I h (%) 2.9 (±1.5) 0.2-6.4 1.9 (±1.5) 0.2-17.4 K n (%) 1.1 (±0.08) 0.94-1.31 1.1 (±0.11) 0.92-1.48 L g (cm) 24.6 (±5.0) 13.4-33.1 16.8 (±4.8) 3.3-38.0 4. Discussion Collette (1981) and Bauchot (1987) were the first to report the existence of L-R gonads asymmetry in T. a. imperialis individuals, where only a functional ovary exists in females but they reported the presence of two asymmetric testes in male individuals. Similarly, L-R asymmetry of paired gonads has been also reported for the congeneric species, T. crocodiles (Colette 1981) but no gonads asymmetry has been reported for the subspecies T. a. melanotus or other species of Belonidae family. It seems that further research is required towards the 430

identification of factors controlling the establishment of L-R gonads asymmetry and its functional significance in this species. The histomorphological characteristics of gametogenesis of T. a. imperialis in Thermaikos Gulf are similar to those described for the needlefish T. a. melanotus in waters off Southwestern Taiwan (Liao & Chang, 2011). The capture of spawning active individuals with running milt and hydrated oocytes (eggs), additionally to the increased catches since 2012, provide evidence of a functional adaptation and successful spawning in the study area. The appearance of spawning capable individuals from May to August with a peak in June and July, provide evidence of a spawning period during the early summer months, when temperature begins to rise. Similarly, the spawning period lasts from April to August with a peak between May and July in coastal waters off the eastern coast of Tunisia (Chaari et al. 2011) or in June for needlefish T. a. melanotus in waters around Hsiao-Liu-Chiu island off southwestern Taiwan (Liao & Chang 2011). The coexistence within ovary of oocytes at various maturity steps, i.e. SG fg, oocytes, hydrated oocytes (eggs) and POFs clearly indicate T. a. imperialis as a multiple spawning species, similarly to what has been shown for the congeneric needlefish, T. a. melanotus (Liao & Chang 2011). Furthermore, the absence of immature individuals among specimens provides strong evidence for the existence of a spawning ground in the study area. Sustainable fisheries management should be complemented by other ecosystem-based measures, such as large marine protected areas where fishing is totally prohibited to ensure the survival of mature individuals. References Akyol, O. and Kara, A. (2011). Occurrence of Tylosurus acus imperialis (Rafinesque, 1810) (Osteichthyes: Belonidae) in the northern Aegean Sea. Journal of Applied Ichthyology 27, 1396. Bauchot M.L., (1987). Poissons osseux. In: W. Fischer, M.L. Bauchot and M. Schneider (eds.) Fiches FAO d'identification pour les besoins de la pêche (rev. 1). Méditerranée et mer Noire. Zone de pêche 37. Vol. II. Commission des Communautés Européennes and FAO, Rome., p. 891-1421. Bello G. (1995). Tylosurus acus imperialis (Osteichthyes: Belonidae), a fish new to the Adriatic Sea. Cahier de Biologie Marine 36, 197 199. Brown-Peterson N., Wyanski D.M., Saborido-Rey F., Macewicz B.J., Lowerre-Barbieri S.K. (2011). A standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science 3, 52 70. Collette B. (1981). Belonidae. In: FAO species identification sheets for fishery purposes. Eastern Central Atlantic. Fishing Area 34 and part of 47. Canada Funds-in-Trust. Ottawa. Fischer W., Bianchi G., Scott W.B. (eds), Department of Fisheries and Oceans Canada, by arrangement with FAO of the United Nation. Vols. 1-6: pag. var. Collette B.B. (2003). Family Belonidae (Bonaparte, 1832) needlefish. Annotated checklist of fish. California Academy of Sciences 16, 1 22. Châari M., Derbel H., Neifar L. (2013). Lecithostaphylus tylosuri sp. nov. (Digenea, Zoogonidae) from the digestive tract of the needlefish Tylosurus acus imperialis (Teleostei, Belonidae). Acta Parasitologica 58, 50 56. Grier H.J., Carmen Uribe M., Aranzabal M.C., Patino R. (2009). The ovary, folliculogenesis, and oogenesis in teleosts. In: Reproductive biology and phylogeny of fishes (Agnathans and Bony Fishes), Vol. 8, A. B.G. M. Jamieson, (Ed.) Science Publishers, Enfield, pp 25 84. Imsiridou A., Minos G., Kokokiris L. (2014). Documentary appearance of Tylosurus acus imperialis individuals in Thermaikos Gulf. Proceedings of HydroMedit 2014, 1 st International Congress on Applied Ichthyology and Aquatic Environment, Volos, Hellas (accepted). Liao Y. & Chang, Y. (2011). Reproductive Biology of the Needlefish Tylosurus acus melanotus in Waters around Hsiao-Liu-Chiu Island, Southwestern Taiwan. Zoological Studies 50, 296 308. 431

Pećarević M., Mikuš J., Bratoš Cetinić A., Dulĉić J. and M. Ĉalić (2013). Introduced marine species in Croatian waters (Eastern Adriatic Sea). Mediterranean Marine Science, 14/1,: 224-237. Türker Çakır, D. and Zengin, K. (2013). Occurrence of Tylosurus acus imperialis (Rafinesque, 1810) (Osteichthyes: Belonidae) in Edremit Bay (Northern Aegean Sea). Journal of Applied Ichthyology 29, 671 672. 432