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2 Journal of FisheriesSciences.com E-ISSN X Journal of FisheriesSciences.com E-ISSN X is published in one volume of four issues per year by Contact and Copyright All rights reserved/bütün hakları saklıdır. Aims and Scope The Journal of FisheriesSciences.com publishes peer-reviewed articles that cover all aspects of fisheries sciences, including fishing technology, fisheries management, sea foods, aquatic (both freshwater and marine) systems, aquaculture systems and health management, aquatic food resources from freshwater, brackish and marine environments and their boundaries, including the impact of human activities on these systems. As the specified areas inevitably impinge on and interrelate with each other, the approach of the journal is multidisciplinary, and authors are encouraged to emphasise the relevance of their own work to that of other disciplines. This journal published articles in English or Turkish. Chief editor: Prof. Dr. Özkan ÖZDEN (Istanbul University, Faculty of Fisheries, Turkey) Responsible editor from English paper: Prof. Dr. Fatih ÖZOĞUL (Çukurova University, Faculty of Fisheries, Turkey) Editorial assistant: Dr. Ferhat ÇAĞILTAY (Istanbul University, Faculty of Fisheries, Turkey) Dr. Deniz TOSUN (Istanbul University, Faculty of Fisheries, Turkey) Cover photo: Assoc. Prof. Dr. Abdullah KAHRAMAN (Istanbul University, Faculty of Fisheries, Turkey) I

3 Editorial board: Prof. Dr. Levent BAT (Sinop Univ., Faculty of Fisheries, Turkey) Prof. Dr. Bela H. BUCK (Alfred Wegener Institute for Polar and Marine Research, Germany) Prof. Dr. Fatih CAN (Mustafa Kemal Univ., Faculty of Fisheries, Turkey) Prof. Dr. Şükran ÇAKLI (Ege Univ., Faculty of Fisheries, Turkey) Prof. Dr. Carsten HARMS (Applied University Bremerhaven, Germany) Prof. Dr. Sedat KARAYÜCEL (Sinop Univ. Faculty of Fisheries, Turkey) Prof. Dr. G. Michael KONTOMINAS (University of Ioannina, Department of Chemistry, Greece) Prof. Dr. Sevim KÖSE (K.T. Univ., Faculty of Marine Sciences, Turkey) Prof. Dr. Robert E. LEVIN (University of Massachusetts, School of Marine Sciences, USA) Prof. Dr. Cengiz METİN (Ege Univ., Faculty of Fisheries, Turkey) Prof. Dr. Mohan J. MODAYIL (Central Marine Fisheries Research Institute Cochin, India) Prof. Dr. Sühendan MOL (Istanbul Univ., Faculty of Fisheries, Turkey) Prof. Dr. Jörg OEHLENSCHLÄGER (Max Rubner Institut and University Hohenheim, Germany) Prof. Dr. Zdzisław E. SIKORSKI (Gdańsk University of Technology, Department of Food Chemistry, Poland) Prof. Dr. Josef STOCKEMER (Applied University Bremerhaven, Germany) Prof. Dr. Krzysztof SURÓWKA (Agricultural University of Krakow, Poland) Prof. Dr. Aydın YAPAR (Pamukkale Univ., Faculty of Engineering, Turkey) Prof. Dr. Mustafa YILDIZ (Istanbul Univ., Faculty of Fisheries, Turkey) Assoc. Prof. Dr. Ahmet AKMIRZA (Istanbul Univ., Faculty of Fisheries, Turkey) Assoc. Prof. Dr. Hatice TORCU KOÇ (Balıkesir Univ., Faculty of Science and Arts, Turkey) Assoc. Prof. Dr. Hüseyin ÖZBİLGİN (Mersin Univ., Faculty of Fisheries, Turkey) Assoc. Prof. Dr. Murat YİĞİT (Çanakkale Onsekiz Mart Univ., Faculty of Fisheries, Turkey) Dr. Mark G. J. HARTL (Heriot-Watt Univ., School of Life Sciences, Scotland, UK) Dr. Muammer KAPLAN (TÜBİTAK MAM, Turkey) Abstracting/Indexing ::: ASFA ::: Oceanic Abstracts ::: Water Resources Abstract ::: FSTA ::: Fish & Fisheries Worldwide ::: CABI Abstracts ::: Chemical Abstracts ::: Ecology Abstracts ::: Academic Search Complete ::: ULAKBİM (Yaşam Bilimleri Veri Tabanı) ::: DOAJ (Direktory of Open Access Journals) ::: Zoological Record ::: AGRICOLA II

4 Journal of FisheriesSciences.com E-ISSN X All rights reserved/bütün hakları saklıdır. Vol. 7 Issue 4 Page (2013) Table of Contents/İçerik 1. Salinity Tolerance of Grey Mullet, Mugil cephalus (Linnaeus) Fry in the Laboratory Soyinka Olufemi Olukolajo, Lawal-Are Aderonke Omolara pp DOI: /jfscom Effects of String Diameter Preference Sepia officinalis During (L. 1758) Spawning in Captivity Halil Şen pp DOI: /jfscom Immersion of 17α-Methyltestosterone Dose&Duration on Tilapia Masculinization Penpun Srisakultiew, Wongpathom Kamonrat pp DOI: /jfscom Uluabat (Apolyont) Gölü'ndeki Balık Faunasının Tespiti, Tür Kompozisyonu ve Ticari Avcılığın Türlere Göre Dağılımı (Detection of Fish Fauna, Species Composition and Distribution of Commercial Fishing According to Species in Lake Uluabat) Şakir Çınar, Ramazan Küçükkara, İsmet Balık, Hıdır Çubuk, Mustafa Ceylan, Kamile Gonca Erol, Vedat Yeğen, Cafer Bulut III

5 pp DOI: /jfscom Screening for Candidate Probiotic Bacteria for The Control of Vibrio anguillarum in Rainbow Trout (Oncorhynchus mykiss, Walbaum) Behire Işıl Didinen, Seçil Metin, Özge Çayli, Ahmet Tahir Ersoy pp DOI: /jfscom Effect of Probiotic On Haematogical Paramaters of Diseased Fish (Cirrihinus mrigal) Parvati Sharma, Ram Chander Sihag, Suresh Kumar Gahlawat pp DOI: /jfscom Species Composition and Diversity of The Zooplankton Fauna of Darlık Stream (İstanbul-Turkey) and its Tributaries Özcan Gaygusuz, Zeynep Dorak pp DOI: /jfscom Serban Baraj Gölü (Afyonkarahisar) ndeki Tatlı Su Kefali (Squalius cephalus, L. 1758) nin Argulus foliaceus (Crustacea, Branchiura) Enfestasyonunun Araştırılması (Investigations on Argulus foliaceus (Crustacea, Branchiura) Infestation Fauna of Chub (Squalius cephalus, L. 1758) From Lake Dam Serban, Afyonkarahisar) Melike Açıkel, M. Oğuz Öztürk pp DOI: /jfscom Detection of Bacterial Diseases and Determination of Antibacterial Susceptibilities of Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey Necla Türk, Murat Yabanlı, Esin Baba, Canan Ontaş, Mehmet Ali Aydın pp DOI: /jfscom IV

6 10. Food and Feeding Habit of Spiny Eel Macrognathus aral (Bloch and Schneider) from Upper Assam Santoshkumar Singh Abujam, Rajesh Kumar Shah, Soram Jiten Singh, Shyama Prasad Biswas pp DOI: /jfscom V

7 7(4): (2013) DOI: /jfscom Journal of FisheriesSciences.com E-ISSN X RESEARCH ARTICLE ARAŞTIRMA MAKALESİ SALINITY TOLERANCE OF GREY MULLET, Mugil cephalus (Linnaeus) FRY IN THE LABORATORY Soyinka Olufemi Olukolajo, Lawal-Are Aderonke Omolara 1 Department of Marine Sciences, University of Lagos, Nigeria Abstract: Özet: The salinity tolerance of grey mullet, Mugil cephalus (L) fry was investigated in replicate tanks of different salinity regimes of fresh water (0 ), 5, 10, 15, 20, 25 and sea water (30 ) in the laboratory for eight weeks. The salinity tolerance experiments showed that the fish tolerated a salinity range of 5 to 25. The highest survival of 40% was recorded at salinities 10 and 15. Statistical analysis of the mortality in all the salinity regimes with ANOVA showed no significant (p>0.05) variation. The highest % gain in length and weight were and respectively in the 15 and 20 salinity regimes. The least FCR value of 2.11 and the best FCE value of were recorded in the 25 regime. The best SGR value of was recorded in both the 15 and 20 regimes. The fish has a good potential for brackish water aquaculture; its fishery and culture in the brackish environment can be sustained. Keywords: Survival, Mortality, Specimens, Fry, Tank, Regime Laboratuvar Koşullarındaki Has Kefal Mugil cephalus (Linnaeus) Yavrularının Tuzluluk Toleransı Has kefal Mugil cephalus (L.) yavrularının tuzluluk toleransı sekiz haftalık bir süre boyunca laboratuvar koşullarında tatlı su ( 0), 5, 10, 15, 20, 25 ve 30 tuzluluklarında tekrarlanmak suretiyle tanklarda araştırıldı. Tuzluluk tolerans deneyleri balıkların 5 ile 25 arasındaki tuzluluk değerlerine dayandıklarını gösterdi. Yüzde kırk gibi en yüksek yaşama oranı 10 ve 15 tuzluluk değerlerinde kaydedildi. Tüm tuzluluk değerlerindeki ölüm oranlarının ANOVA ile yapılan istatistiksel analizleri kesin bir varyasyon göstermedi (p>0.05). Uzunluk ve ağırlık bakımından en yüksek % artış 15 tuzlulukta ve 20 tuzlulukta ise olarak belirlendi. En düşük FCR değeri olan 2.11 ile en iyi FCE değeri tuzluluk değerinde kaydedildi. En iyi SGR değeri olan ise 15 ve 20 değerlerinde kaydedildi. Kefal balıkları acısu yetiştiricilik ortamları için iyi bir potansiyele sahip olup acısu ortamında balıklandırma ve yetiştiriciliği kabul edilebilir. Anahtar Kelimeler: Yaşama, Ölüm, Örnekler, Yavru, Tank, Sistem Correspondence to: Soyinka Olufemi OLUKOJO, Department of Marine Sciences, University of Lagos, Nigeria Tel: soyinka.olufemi@gmail.com 292

8 Journal of FisheriesSciences.com Olufemi and Aderonke, 7(4): (2013) Introduction Mullets (Mugilidae) are common fishes in the coastal waters of tropical and subtropical countries of the world. Sixteen species have so far been identified in West Africa (Fowler, 1936; Cadenat, 1954 and Blay, 1995), and these constitute an important proportion of the catches of commercial and subsistence fishermen in some countries in this area (Brulhet, 1975; Payne, 1976). The stripped mullet, Mugil cephalus, is perhaps the most widespread and abundant inshore teleost (Collins, 1985). Size is generally more biologically relevant than age in fishes, due to several ecological and physiological factors that are more size-dependent than age dependent. Variability in size has important implications for diverse aspects of fisheries science and population dynamics (Erzini, 1994). Adult M. cephalus are highly euryhaline, and survive in a range of salinities from 0 o / oo in fresh water to hypersaline waters. Adult M. cephalus can be found along the open coasts, but juveniles are most likely found in estuaries (Collins, 1985). Silva and De Silva (1981) reported that the percentage of grey mullet catches increased with increasing salinity. Larger fishes were found in the deeper areas. Stability of the water column and suitable food in coastal lagoons, river deltas, and estuarine mangrove areas have been identified as important factors influencing the recruitment of juvenile Mugilidae (Blaber and Blaber, 1980; Blaber, 1987; Vieira, 1991) In spite of the abundant biological data on this species from other countries, comparative data from brackish water lagoons in Africa, with Nigeria in particular is highly insufficient. Yet the fish constitutes a mainstay of the fisheries resources in the coastal communities; if the potentials are well exploited, a viable mullet fishery and culture can be maximally sustained. In this report, the salinity tolerance of grey mullet, Mugil cephalus fry in Lagos Lagoon (high brackish water), Nigeria, are investigated in the laboratory to give information for culture of the species in brackish and fresh waters. Materials and Methods Collection of specimens Collection of live fry from the Lagos Lagoon was made using hand nets at shallow portions of the lagoon near the shore for the salinity tolerance experiments. Experimental set-up The experiment was carried out in translucent rectangular white plastic tanks each of 35 litres capacity and filled with 25 litres of water. Replicate tanks for each salinity regime of fresh water (0 ), 5, 10, 15, 20, 25 and sea water (30 ) were used. The salinity in each tank was determined using a refractometer. The freshwater was obtained from tap-water that has been dechlorinated for over 24 hours, while the sea water was obtained from the Lagos bar-beach. The experimental tanks were placed in a compartment where there was a reduced effect of direct sunlight. The fries of M. cephalus used were acclimatized in lagoon water of 15 salinity for five days. Ten live specimens each were then placed in the experimental tanks of varied salinities after the acclimatization period. They were fed with formulated coppens feed at a daily ration of 5% of their total body weight. Adjustments in weight of feed fed were done fortnightly after measuring the weight of fish remaining in tanks. Growth, survival and mortality of the specimens were monitored over eight weeks. Change of water in the tanks and measurements were done forthnightly. Aerating air-pumps were used during the experimental period to aerate the tanks. Physical chemical parameter measurement The physico-chemical parameters were determined twice a week. Water temperature was measured using a simple mercury-in-glass thermometer. The salinity of the water was determined using a Refractometer (BIOMARINE, Aqua Fauna Model. Dissolved oxygen (DO) of the water samples was determined using a Jenway DO Meter (Model 4310). The ph values were determined using a Jenway Hanna ph meter (HI Model). Length weight measurement The total length (TL) of the specimens was measured on a measuring board to the nearest 0.1 centimeter. The total weight of the fish was taken on a Sartorious top loading balance (Model ) or a triple beam balance to the nearest tenth of a gram. Statistical analyses The data obtained were subjected to statistical analyses using different formulae: 293

9 Journal of FisheriesSciences.com Olufemi and Aderonke, 7(4): (2013) The Food Conversion Ratio (FCR) = dry food fed (g) live weight gained (g) The Food Conversion Efficiency = weight gggggggggggg 100% ffffffff iiiiiiiiiiii log (final body weight) log (initial body weight) x 100% The Specific Growth Rate = tttttttt (iiii dddddddd) To test if the differences in mortality observed % gain in mean weight of was recorded in in the different salinity levels were significant, the 15 and 20 salinity regimes, while the ANOVA was employed. lowest value of was in the 5 salinity regime. Results and Discussion Survival / Mortality of M. cephalus fry in varied salinities Salinity tolerance experiment carried out showed that the fries of M. cephalus could survive in varying salinity range of 5 to 25. The summary of the weekly survival / mortality record for M. cephalus fry in the tanks is given in Table 1 while Figure 1 showed the percentage survival curve for M. cephalus in varied salinity regimes. The ANOVA test indicated that there was no significant variation (p > 0.05) in the differences observed in mortality in the various salinity regimes. Physico-chemical parameters in culture tanks The weekly illustration of the physico-chemical parameters during the salinity tolerance experiment is presented in Fig. 2. The ph ranged from (mean 7.89±0.13); DO ranged from (mean 7.08±0.46) and temperature ranged from o C (mean 29.02±0.13). Effects of salinity on the growth of M. cephalus Summary of the percentage gain or loss in total length and body weight of M. cephalus in varied salinity levels is presented in Table 2. The specimens in 0 and 30 salinity regimes all died within the 1st week of the experiment. The initial mean total length of the fries ranged from cm (2.04 ±0.10); while the initial mean body weight was 0.10g (0.1 ±0.00). The final mean total length was 3.0 ±2.06, while the final mean body weight was 0.69 ±0.50. The highest % gain in mean length of was recorded in the 15 and 20 salinity regimes, while the lowest value of was in the 5 salinity regime. The highest The FCR values of the fish in all the regimes ranged from , with the highest value recorded in the 5 regime and the lowest in the 25 regime. The FCE (%) values ranged from , with the highest value recorded in the 25 regime and the lowest in the 5 regime. The SGR values of the fish in all the salinity regimes ranged from , with the highest value recorded in the 15 and 20 regimes, while the lowest was recorded in the 5 regime The differences in growth between different salinity regimes examined in the present study were not found to be significant and this agreed with the report by McDonough and Wenner (2003). Cardona (2000) revealed that the metabolic rate of young specimens was negatively affected by high salinity levels and that an improved growth performance was achieved in freshwater and oligohaline water (0.1-5 ). A stratified study on microhabitat use carried out on the Island of Minorca (Balearic archipelago), western Mediterranean sea, demonstrated that juvenile specimens, shorter than 200mm (TL), concentrated all year round in freshwater or oligohaline sites. Mesohaline areas were usually avoided, except in summer. Immature fish, with a total length between 201 and 300mm showed a similar pattern although in some seasons avoided freshwater sites. The habitat selection pattern of adults, i.e. fish longer than 301mm, changed seasonally due to their offshore migration during the spawning season (from late summer to early winter). They usually showed a greater preference for polyhaline areas and strongly avoided freshwater sites, which might also be due to their shallowness (Cardona, 2000). 294

10 Journal of FisheriesSciences.com Olufemi and Aderonke, 7(4): (2013) Table 1. Weekly survival / mortality of M. cephalus fry in varied salinity regimes. Salinity regime Initial number Mortality per week Survival at the end % Survival ( ) per tank of the 8th week Table 2. Summary of the growth performance of M. cephalus in varied salinity levels. Salinity ( ) Initial mean total length (cm) Final mean total length (cm) Gain or loss in mean total length % gain or loss in mean total length Initial mean body weight (g) Final mean body weight (g) Gain or loss in mean body weight % gain or loss in mean body weight Food Conversion Ratio Food Conversion Efficiency (%) Specific Growth Rate (% per day) The differences in growth or gain in length and weight between different salinity regimes examined in this study were not found to be significant. The high survival rate of M. cephalus fry ( cm TL) in water of different salinities (5-25 ) demonstrated in this present investigation revealed the ability of grey mullets to adapt to wide salinity fluctuations. The mortality experienced within the 1 st week in the 0 and 30 salinities was probably connected with the abrupt transfers. Ciccoti et al (1994) noticed a high survival rate and tissue osmolality regulation of M. cephalus juveniles (SL = mean 28.05, SD = 3.54mm) acclimated to freshwater gradually in 48 hours. The morphological and biochemical aspects of esophagus and gills in juveniles are similar to those of the adult, suggesting that osmotic regulatory mechanisms are precociously developed to allow the colonization of eutrophic inland waters. From their investigation, Hotos and Vlahos (1998) highlighted that M. cephalus fry acclimated in aquaria with 20 sea water acclimated gradually over days with no signs of stress. By direct transfer from 20 to salinities of 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80, mortality occurred at salinities greater than 45. The physical and chemical parameters obtained in the present study showed a similar pattern in all the varied salinity regimes. There were no wide fluctuations in those parameters and is clear that M. cephalus can thrive well under a trophic climatic condition. Bulli and Kulikova (2006) reported the response of early juveniles of the harder, Liza haematocheila (= Mugil soiuy) to changes in water salinity, and the growth and survival of larvae in water of different salinity levels. They discovered that at decreasing salinity, the growth rate, the content of defatted dry matter, and the content of lipids increased. In freshwater, the stock lipids (triacylglycerols) accumulate more intensively. From the FCR and FCE values, it could be seen that the fry in the 25 salinity regime had the lowest FCR, hence the best FCE, which meant that there was more effective utilization of less unit weight of feed to produce a unit weight of flesh than in the other regimes. However, the best SGR 295

11 Journal of FisheriesSciences.com Olufemi and Aderonke, 7(4): (2013) was in the 15 and 20 salinity regimes, which were slightly higher than those in 10 and 25 regimes. Conclusions The ability of grey mullet fry to tolerate a wide range of salinity regime demonstrated in this study further agreed to the euryhalinity of the species and its potential candidature for brackish water fish culture. Acknowledgments The authors express their profound gratitude to Professor (Emeritus) Kola Kusemiju for his invaluable contributions to the conception and design of the experiment; and the revising of the draft of the manuscript for intellectual content. We thank Mr. Anipole Biodun for the editorial assistance. References Blaber, S.J.M., (1987). Factor influencing recruitment and survival of mugilids in estuaries and coastal waters of Southeastern Africa, American Fisheries Society Symposium, 1: Blaber, S.J.M., Blaber, T.G., (1980). Factors affecting the distribution of juvenile estuarine and inshore fish, Journal of Fish Biology, 17: doi: /j tb02749.x Blay, J., (1995). Food and feeding habits of four species of juvenile mullet (Mugilidae) in a tidal lagoon in Ghana, Journal of Fish Biology, 46: doi: /j tb05952.x Brulhet, J., (1975). Observations on the biology of Mugil cephalus ashanteensis and the possibility of its aquaculture in the Mauritanean coast, Aquaculture, 5: doi: / (75) Bulli, L.I., Kulikova, N.I., (2006). Adaptive capacity of the larvae of the haarder Liza haematocheila (Mugilidae, Mugiliformes) under decreasing salinity of the environment, Journal of Ichthyology, 46(4): Cadenat, J., (1954). Note d ichthyologie oust-africain 8. Regime alimentaire sur les mullets de la cote occidental d Afrique, Bulletin de I Institute francais d Afrique noire (ser. A) 16: Cardona, L., (2000). Effects of salinity on the habitat selection and growth performance of Mediterranean flathead grey mullet, Mugil cephalus (Osteichthyes, Mugilidae), Estuarine, Coastal and Shelf Science, 50(5): doi: /ecss Ciccotti, E., Marino, G., Pucci, P., Cataldi, E., Cataudella, S., (1994). Acclimation trial of Mugil cephalus juveniles to freshwater: morphological and biochemical aspects, Environmental Biology of Fishes, 43(2): doi: /BF Collins, M.R., (1985). Species profiles: Life histories and environmental requirements of coastal fishes and invertebrates (South Florida) striped mullet. U.S. Army Corps of Engineers, TR EL pp. Erzini, K., (1994). An empirical study of variability in length and age of marine fishes, Journal of Applied Ichthyology, 10: doi: /j tb00140.x Fowler, H.W., (1936). The marine fishes of West Africa. Bulletin of the American Museum of Natural History, 70: Hotos, G.N.,Vlahos, N., (1998). Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions, Aquaculture, 167(3-4): doi: /S (98) McDonough, C.J., Wenner, C.A., (2003). Growth, recruitment and abundance of juvenile Mugil cephalus in South Carolina estuaries, Fisheries Bulletin, 101: Payne, A.L., (1976). The relative abundance and feeding habits of the grey mullet species occurring in an estuary in Sierra Leone, West Africa, Marine Biology, 35: doi: /BF Silva, E.I., De Silva, S.S., (1981). Aspects of the biology of grey mullet, Mugil cephalus L., adult populations of a coastal lagoon in Sri Lanka, Journal of Fish Biology, 19(1): doi: /j tb05806.x Vieira, J.P., (1991). Juvenile mullets (Pisces: Mugilidae) in the estuary Lagoa dos Patos, RS, Brazil, Copeia, doi: /

12 7(4): (2013) DOI: /jfscom Journal of FisheriesSciences.com E-ISSN X SHORT COMMUNICATION KISA BİLGİLENDİRME EFFECTS OF STRING DIAMETER PREFERENCE OF Sepia officinalis DURING (L. 1758) SPAWNING IN CAPTIVITY Halil Şen Ege University Fisheries Faculty Aquaculture Department, Urla, İzmir, Turkey Abstract: The aim of this study was to determine the importance of string diameter preference on spawning performance of Sepia officinalis. For this purpose, seven strings, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, and 14.0 mm in diameter were chosen in one meter length. After that two batches of strings for spawning of the cuttlefish were hanged on the two experimental tanks. Eggs were counted daily to determine which string aor stringss were preferred by the cuttlefish. The mean mantle length and body weight of the individuals used in the experiment were 12.97±1.46 cm and 231.5±48.9 g (n= 22), respectively. The string diameter preference in the cuttlefish were 6-8>10-12>3-4 mm with regards to egg numbers (χ 2 ; P<0.01). Finally, the present results showed that the 6 and/or 8 mm strings in diameter can be selected for spawning of cuttlefish in terms of aquacultural or biological studies. Keywords: Captivity, Reproduction, Sepia officinalis, Spawning, String diameter Özet: Kontrol Altında Sepia officinalis (L. 1758) in Yumurtalamasında İp Çapı Tercihi Bu çalışmanın amacı, Sepia officinalis in yumurtalama verimi üzerine ip çapı tercihinin önemi olup olmadığını saptamaktır. Bu amaçla, 1 metre uzunluğunda ve 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, ve 14.0 mm çaplarında yedi ip seçildi. Daha sonra ipler iki grup olarak sübyelerin yumurtlaması için deney tanklarına asıldı. Sübyeler tarafından kaç tane yumurta yumurtladığını ve hangi ip çapını veya çaplarını tercih ettiklerini tespit etmek için günlük kontroller yapıldı. Denemede ortalama manto boyları ve ağırlıkları sırasıyla, 12.97±1.46 cm ve 231.5±48.9 g (n= 22) olan bireyler kullanıldı. Sübyelerde ip çapı tercihi yumurta miktarı (χ 2 ; P<0.01) dikkate alındığında 6-8>10-12>3-4 mm oldu. Sonuç olarak, şimdiki sonuçlar akuakültür ve biyolojik çalışmalarda sübyenin yumurtlaması için 6 ve/veya 8 mm çapında iplerin seçilebileceğini göstermiştir. Anahtar Kelimeler: Tutsaklık, İp çapı, Üreme, Sepia officinalis, Yumurtlama Correspondence to: Halil ŞEN, Ege University Fisheries Faculty Aquaculture Department, 35440, Urla, İzmir- TURKEY Tel.: ( ) halil.sen@ege.edu.tr 297

13 Journal of FisheriesSciences.com Şen, 7(4): (2013) Introduction Sepia officinalis is one of the most easily cultured cephalopods (Richard 1971; Pascual 1978; Boletzky and Hanlon 1983; Forsythe et al. 1994; Lee et al. 1998; Domingues et al. 2001a,b, 2002, 2003a), and is a commercially important species throughout the world (Roper et al. 1984). It is highly adaptable to life in captive conditions (Forsythe et al. 1994; Domingues et al. 2001a,b, 2002, 2003a,b, 2005, 2006; Skyes et al. 2006; Şen 2009). Its life cycle in the laboratory varies from less than 6 months to 17 months with temperature, feeding regime and culture density (Richard 1971; Forsythe et al. 1994; Domingues et al. 2006). In the wild, cuttlefish live for 2 years on average, and the spawning period varies considerably with temperature (Guerra 2006), with females usually being much larger and older when they start spawning compared to laboratory-cultured cuttlefish. Moreover, it s known that cuttlefish lay their eggs on various things especially floating things such as wood or branches of some macroalga and corals, fish net and/or other manmade objects (Choue, 1966; ; Arnold et al., 1972; Okutani, 1978; Boletzky and Boletzky, 1973; Şen, 2009). Nevertheless, there is only one information can be found from the Boletzky s (1983) study in the literature. So far, the data related to appropriate diameter of strings or other materials for laying eggs of S. officinalis hasn t been indicated in terms of rearing or culture studies. Therefore, preference to string diameter may be important while spawning of the cuttlefish in captivity. The aim of this study was to determine the importance of string diameter preference on spawning performance of S. officinalis. Materials and Methods The experimental animals were captured off the Izmir Bay by trammel nets on April 3, Totally 22 S. officinalis (11 males and 11 females) were selected and placed in an open flowthrough filtered sea water system with two cylindrical polyester tanks (450 L volume) in indoor facilities of the Fisheries Faculty of Ege University (Urla, Izmir, TURKEY) at the end of the spawning (April 3-17, 2012). Avoiding congestion the cuttlefish were divided in two groups (5 males and 5 females, and 6 males and 6 females). Natural photoperiodicity was adjusted. The oxygen and ph meter YSI 5750 and salinity hand refractometer 508-IIW were used for measurements. The specimens were fed ad-libitium with low market price fish species (i.e. Sardine pilchardus, Engrualis encrasicolus) by hand. The following day, uneaten part or remains were removed by siphoning. The mean mantle length and body weight of the individuals using the experiment were 12.97±1.46 cm and 231.5±48.9 g (n= 22), respectively. The seven strings, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, and 14.0 mm in diameter were chosen in one meter length. After that two batches of strings for spawning of the cuttlefish were hanged on the experimental tanks (Figure 1.). Daily control was carried out for how many eggs were laid on the strings and which string diameter or diameters were preferred for spawning by the cuttlefish. The obtaining data were given as mean ± SD values in the text. The differences among means of the spawned egg numbers and the spawning numbers on chose string diameters were tested using chi-square test to determine any significance (P<0.01). Results and Discussion The salinity, ph, O 2 saturation and temperature in tanks were measured as 37±0.2, 8.1±0.1, 65±5%, and 16.5±1 C, respectively. The experiment lasted 14 days. During this period the cuttlefish mated multiple times in the tanks. Additionally, it s observed that after each mating the males generally allowed females to feed first. Also, agonistic behaviour occurred frequently in the tanks. Furthermore, total 53 spawning occurred and total 1519 eggs were laid on the strings. There were significant differences among string diameters (P<0.01) based on the egg numbers. On the other hand, there wasn t any significance between string diameters according to the spawning numbers (P>0.01). The string diameter preference in the cuttlefish were 6-8>10-12>3-4 mm in diameters with regards to egg numbers (P<0.01) (Table 1.). However, the 14 mm in string diameter was excluded from the statistical analysis because it was hardly chosen. Present findings related to mating behaviour in the cuttlefish is parallel to that of Hanlon and Messenger (1996). Furthermore, the males allowed to first food intake of the mated females after mating which is observed firstly in the current research. Also, the agonistic behaviour is a known phenomenon in the cuttlefish (Hanlon and Messenger, 1996). In conclusion, the current re- 298

14 Journal of FisheriesSciences.com Şen, 7(4): (2013) sults indicated that the cuttlefish prominently preferred the 6 and 8 mm in string diameters (P<0.01). The 14 mm in string diameter was chosen hardly ever. Therefore using this diameter is inadvisable according to the present study. Also, Boletzky (1983) said that S. officinalis lays its eggs on the oblong object, not more than 1 cm in diameter. The current results generally in agreement with that of the Boletzky (1983) s findings. Conclusions Finally, the present results showed that the 6 and/or 8 mm in diameter of strings can be selected for spawning of cuttlefish in terms of aquaculture or biological studies. Unfortunately, the actual evidences couldn t be compared with any studies related to this subject. However, more detailed behavioural and/or physiological studies on why cuttlefish prefer the definite diameter for spawning is needed. Figure 1. A general view of laying eggs on the strings and the cuttlefish from the one of the experimental tank. Table 1. Diameter (mm) Total egg numbers Total spawning number The total numbers of chosen string diameters and eggs * 120 a 111 a 360 b 358 b 292 c 236 c 42* * 299

15 Journal of FisheriesSciences.com Şen, 7(4): (2013) References Arnold, J.M., Singley, C.T., Williams-Arnold, L.D., (1972). Embriyonic development and post hatchling survival of the Sepiolid squid Euprymna scolopes under laboratory conditions, The Veliger, 14: Boletzky, S.V., (1983). Sepia officinalis. Boyle P.R. (ed) Cephalopod life cycles. Vol.1. Academic Press, London, pp Boletzky, S.V., Boletzky, M.V., (1973). Observation embriyonic and early postembryonic development of Rossia macrosoma (Mollusca: Cephalopoda), Helgolender wiss Meeresunters, 25: Boletzky S.v., Hanlon R.T. (1983) A Review of the Laboratory Maintenance, Rearing and Culture of Cephalopod Molluscs, Memoirs of the National Museum Victoria, 44: Choe, S., (1966). On the eggs, rearing, habits of the fry and growth of some Cephalopoda, Bulletin of Marine Science, 16: Domingues P., Kingston T., Sykes A., Andrade J., (2001a). Growth of young cuttlefish, Sepia officinalis (Linnaeus, 1758) at the upper end of the biological distribution temperature range, Aquaculture Research, 32: doi: /j x Domingues P., Sykes A., Andrade J., (2001b). The use of Artemia or mysids as food for hatchlings of the cuttlefish Sepia officinalis Linnaeus, 1758; effects on growth and survival throughout the life cycle, Aquaculture International, 9: doi: /A: Domingues P., Sykes A., Andrade J., (2002). The effects of temperature in the life cycle of two consecutive generations of the cuttlefish Sepia officinalis (Linnaeus, 1758), cultured in the Algarve (South Portugal), Aquaculture International, 10: doi: /A: Domingues P., Poirier R., Dickel L., Almansa E., Sykes A., Andrade P., (2003a). Effects of culture density and live prey on growth and survival of juvenile cuttlefish, Sepia officinalis, Aquaculture International, 11: doi: /A: Domingues P., Sykes A., Sommerfield A., Almansa E., Lorenzo A., Andrade J. (2003b). Effects on feeding live or frozen prey on growth, survival and the life cycle of the cuttlefish Sepia officinalis (Linnaeus, 1758), Aquaculture International, 11: doi: /B:AQUI a Domingues P., DiMarco F., Andrade J., Lee P. (2005). The effects of diets with amino acid supplementation on the survival, growth and body composition of the cuttlefish Sepia officinalis, Aquaculture International, 13(5): doi: /s Domingues P.M., Bettencourt V., Guerra A. (2006) Growth of Sepia officinalis in captivity and in nature, Vie et Milieu, 56: Forsythe J., DeRusha R., Hanlon R., (1994) Growth, reproduction and life span of Sepia officinalis (Cephalopoda: Mollusca) cultured through seven consecutive generations, Journal of Zoolology London, 233: doi: /j tb08582.x Guerra A., (2006). Ecology of Sepia officinalis, Vie et Milieu, 56: Hanlon, R.T., Messenger, J.B., (1996). Cephalopod behaviour. Cambridge University Press; 230 pp. Lee P., Turk P., Forsythe J., DiMarco F., (1998). Cephalopod culture: physiological, behavioural and environmental requirements. Suisanzoshoku, 46(3): Okutani, T., (1978). Studies on early life history of decaodan Mollusca. VII Eggs and newly hatched larvae of Sepia latimanus Quoi ans Gaimard, Venus (Japanese Journal of Malacalogy), 37: Richard A. (1971) Contribution a` l e tude expe rimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (Mollusque, Ce phalopode). The`se 248, Univ. Lille, France. Roper C.F.E., Sweeney M.J., Nauen C.E., (1984) F.A.O. Species Catalogue, Cephalopods on the World. An Annotated and Illustrated Catalogue of Species of Interest to Fisheries, in F.A.O. Fisheries Synopsis,

16 Journal of FisheriesSciences.com Şen, 7(4): (2013) Sykes A., Domingues P.M., Correia M., Andrade J.P., (2006). Cuttlefish culture State of the art and future trends, Vie et Milieu, 56: Şen H., (2009). Kontrollü Koşullarda Sübye (Sepia officinalis L.) nin Yumurtlaması, Yumurtaların Gelişimi ve İnkübasyonu, Journal of FisheriesSciences.com, 3(3): doi: /jfscom

17 7(4): (2013) DOI: /jfscom Journal of FisheriesSciences.com E-ISSN X RESEARCH ARTICLE ARAŞTIRMA MAKALESİ IMMERSION OF 17α-METHYLTESTOSTERONE DOSE&DURATION ON TILAPIA MASCULINIZATION Penpun Srisakultiew 1, Wongpathom Kamonrat 2 1 Department of Fisheries, Faculty of Agriculture, KhonKaen University Khon Kaen, Thailand 2 Department of Fisheries, Ministry of Agriculture and Cooperatives in Kaset Campus, Jatujak, Bangkok Abstract: Thirty five Nile Tilapia (Oreochromis niloticus L. 1758) eggs or fry at the ages of 2 and 14 days post fertilization (dpf) were randomly selected and put into a 1L plastic bottle incubator. These were then immersed either in 0, 250 or 500µg/L of 17α-methyltestosterone (MT) for 6, 12, 24, 48, 72 and 96h separately. Controls were established using eggs or fry at the same age groups immersed only in ethanol (250µL/L) instead of MT at the same amount and duration. At the end of the treatment, they were washed and nursed in 10L plastic containers and 100L glass aquaria which were replenished with water according to their growth. They were fed with 40% protein pellet until 62-65dpf then dissected for gonadal sex determination using acetocarmine squash method. The results showed that both the 250 and 500µg/L MT induced (P<0.05) males ( and %) higher than the controls ( and % when immersed at 2 and 14dpf, respectively). While the immersing duration between 6-96h of both ages however, showed no differences to the male percentages. Keywords: Sex reversal, Monosex, Male, Inversion, Methyltestosterone Correspondence to: Penpun SRISAKULTIEW, Department of Fisheries, Faculty of Agriculture, KhonKaen University, Khon Kaen, THAILAND Tel/Fax: penpunsri@gmail.com 302

18 Journal of FisheriesSciences.com Srisakultiew and Kamonrat, 7(4): (2013) Özet: Tilapia Maskülinizasyonu Üzerine 17α-metiltestosteron Daldırmanın Doz ve Süre Etkisi İki yaşında yumurtalar ve 14 günlük dölleme sonrası zigotları içeren 35 adet Nil Tilapia (Oreochromis niloticus L. 1758) sı rastgele seçilerek 1 L lik plastik küvez şişe içine konulmuştur. Bunlar daha sonra hem hem 500 mg/l 17 a-methyltestosterone (MT) içine ayrı ayrı 6, 12, 24, 48, 72 ve 96 saatlik sürelerle daldırılmıştır. Kontrol grubu olarak aynı yaş grubu yumurtalar kullanılarak aynı miktar ve sürede MT Yerine 250 ml/l etanole daldırılarak değerlendirilmiştir. Uygulamanın sonunda bunlar 10 L plastik konteynırda yıkandı ve büyümelerine göre suyla tazelenen 100 L lik cam akvaryumda bakıldılar dpf boyutuna gelene kadar % 40 lık protein peletiyle beslendiler ve sonra Aceto-carmine squash yöntemiyle gonadal cinsiyet tayini için parçalara ayrıldılar. 250 ve 500 mg/l MT her ikisine daldırılan (P<0.05) erkek bireylerdeki (% ve % ) sonuçların kontrol grupdakilerden (2 ve 14 dpf daldırılanların sırayla % ve % ) daha yüksek çıktığını göstermiştir. Her iki yaştan 6-96 saatlik daldırma sürelerinde erkek yüzdeleri arasında önemli bir fark bulunmamıştır. Anahtar Kelimeler: Cinsiyet dönüşümü, Monoseks, Erkek, İnversiyon (değişme), Metiltestosteron Introduction Tilapia (Oreochromis niloticus) is one of the most important freshwater species cultured in many parts of the world. They consume low cost feed but grow fast, show tolerance to low water quality and tilapia meal is a good protein source for human consumption throughout the world as well as Thailand. Nile Tilapia was introduced from Japan to Thailand in 1965 as the King s souvenir. The fish were propagated at the King s palace and then distributed to Thai people the following year. In 2008, tilapia production of 217,200 tons equaled 41.6% of the total aquaculture production at the value of US$ million (Anonymous, 2010). Tilapia is the most economic freshwater cultured species for domestic consumption and export. As the male tilapias grow faster than females, culture of a single sex stopped the fish breeding. In addition, Beardmore et al. (2001) reported benefits of monosex male culture on high growth, preventing large energy diversion into ovary as well as reproductive behavior, reducing aggressive interaction and uniform size at harvest. Thus, culture of male tilapia is the most favored way for commercial scale farming (Mires, 1977; Guerrero, 1982). However, the male tilapia can be cultured from fry, produced by either manually sexing, hybridization or hormonal sex reversal (Mires, 1983). The hormonal sex reversal is the most reliable and widely used method in many countries. 17α-methyltestosterone (MT) is the most commercial synthetic androgen used for effective masculinization of more than 42 fish species (Devlin & Nagahama, 2002). In addition, the MT is also commercially used in monosex-male tilapia hatcheries worldwide including Thailand. In the commercial scales tilapia hatcheries of Thailand, fish farmers are able to produce male tilapias with a percentage of % (Bhujel, 1998). The method however, had to be applied and conducted in hapas suspended in an earthen pond in order to reduce labor costs on cleaning the cement tanks otherwise used. In addition, the sex reversal process used in earthen pond might cause MT contamination to workers and the surrounding environment. The sex reversal by immersion technique had been developed and conducted in salmonids (Baker et.al., 1988; Piferrer & Donaldson, 1989). The use of MT at µg/L immersed new hatched larvae for 2h and then repeated with another 2h immersion the following week resulting in % males (Baker et.al., 1988). Similarly, Piferrer & Donaldson (1989) reported on a single immersion of 6 days post hatching Oncorhynchus kisutch in 400µg/L MT for 2h resulted in 73% males. In tilapia, Yang Yi (1992) immersed day post-hatched tilapia fry in 5mg/L MT for 3 days and produced 90% males. However Fitzpatrick et al., (1999) got 79.3% males by immersing the 13 days post-fertilization (dpf) tilapia larvae in 200µg/L methyldihydrotestosterone for 2h. Doses and duration of hormone immersing tilapia are vastly different between 200µg/L up to 303

19 Journal of FisheriesSciences.com Srisakultiew and Kamonrat, 7(4): (2013) 5mg/L and 2h up to 3 days. Nevertheless, aging tilapia on a different basis using either on dph or dpf, may affect labile or sensitivity period, thereby affecting the efficacy of the technique. Thus, we decided to use the dpf aging basis throughout the study, and the tilapia at either egg (2dpf) and late fry (14dpf) stages were selected for examination of different doses and durations of sex reversal by immersion technique. Materials and Methods Experimental fish A total of 32,500 tilapia samples used in this study were supplied from the KhonKaen Inland Fisheries Research and Development Center. Twenty five concrete spawning tanks (5x10x0.6m) containing 50 females and 25 males in the center were used and the fish were fed with 25% protein pellet at 1-2% daily and egg were taken weekly. Each batch of egg or larvae was kept individually in a metal bowl. They were classified into age (dpf) according to their embryonic development. Pigmented eggs (2dpf) were selected from 13 females. The eggs were cleaned with 100ppm formalin for 2 min in order to remove any external parasites and washed with fresh water 2-3 times before being placed into a plastic tray for the experiment. Thirty five eggs were randomly selected and put into a plastic bottle incubator containing 1L fresh water with an air stone to circulate the eggs during incubation. The experiment was carried out in 27.5 ±1.2 o C water temperature. Hormonal preparation Hormonal stock solutions were prepared by dissolving 15 and 30mg 17α-methyltestosterone (MT; Fluka Chemie, Buchs, Switzerland) in 15mL absolute ethanol. Then, the MT stock solution concentrations were established at 1,000 and 2,000µg/mL, respectively. Experiments Factorial of 2 factors consisting 3 dose levels (MT: 0, 250 and 500 µg/l) and immersing durations (either 6, 12, 24, 48, 72 and 96h or 6, 12, 24 and 48h) were designed in 2 age groups (2 and 14 dpf) with 4 replicates each. At the beginning of the experiment, 250µL of each MT stock was randomly added into the bottle incubators (1L) at 2dpf age. Thus, the MT concentrations in the bottles were 250 and 500 µg/l, respectively. For the control, only 250µL absolute ethanol was added to replace the MT stocks. The eggs (2dpf) were immersed for 6, 12, 24, 48, 72 and 96h. Later, the hormone solutions were drained off and thoroughly washed with fresh water. They were further incubated in the same bottles until 8dpf (start feeding fry). They were counted (70 fry) and transferred into plastic containers containing 2L of water. The fry were fed with fish meal 5 times a day. The containers were cleaned and water was changed daily. At 14dpf, the untreated fry from the same egg batch of 2dpf immersion were treated with either 250 or 500 µg/l MT stocks in the plastic containers (70 fry/2l) while controls were added with absolute ethanol in the same amount as MT stocks. The fry were statically immersed in MT solution for 6, 12, 24 and 48h before being drained off, thoroughly washed and nursed in the same manner as the 2dpf immersion. They were nursed in the plastic containers for 3 weeks then transferred into aquaria supplied with 30L of bio-filtered and recycled water. The water volume was then slowly increased according to fish sizes and densities. They were nursed in the aquaria until at least 60dpf. Sexing fish and data analysis Numbers of the remaining fry at the end of the experiment were recorded to assess survival rate. Fifty fry (62-65 dpf ages) from each aquarium were sampled, dissected for gonads and individually phenotypic sexed using Aceto-carmine Squash Method (Guerrero and Shelton, 1974) under a binocular microscope (x100). Arch sine transformation of the male proportion was used prior to Analysis of Variance to analyze the effects of doses, immersing durations and interaction using SPSS version Chi-square test was also used to detect sex ratio compared to the control. The differences were considered statistically when the p-value was equal or less than Results and Discussion MT was used in the present study in order to find out efficacy of MT immersion ( µg/l) at different durations (6-96h). Both dosages of MT immersion significantly increased the percentage of male (P<0.05) while the immersion duration and interaction showed no effect. There were no significantly differences on survival rates of the fry when immersed at either 2 or 14 dpf ages (79.0 ± ±0.4 % and 71.8 ± ±3.2%, respectively; Table 1 and 2). These indicated the fry dead were random and had no effect on particular sexes. 304

20 Journal of FisheriesSciences.com Srisakultiew and Kamonrat, 7(4): (2013) At egg stage (2dpf), the MT immersion (250 and 500µg/L) induced significantly (P<0.05) males ( MT immersions showed higher males ( %) than those of the controls ( % males; Table 2). The results showed the possibility of the tilapia MT immersion stages at either eggs (2dpf) or late fry (14 dpf). In another words, tilapia have a long labile period between 2 14 dpf before occurring gonadal differentiation and presenting some special cells, receptors or enzymes involved in steroid production (Devlin & Nagahama, 2002). The 14dpf age was correlated to the on- set of sex differentiation of Nile tilapia, which Srisakultiew & Rana (1991) indicated at the early age as dpf at 27 ±2 C. This finding is related to Baroiller et al. (1996) who suggested the critical period of the fish to steroid sex reversal by oral application, must begin between 9-13dpf and last for 21 days (27 C). Thus, the 14dpf was selected as the late fry immersing ages while the egg stage at 2dpf was used for comparing efficacy of MT sex reversal in the present study, which resulted in significantly (P<0.05) higher males. The results indicated that both 2 and 14 dpf were sensitive ages (labile period or sex lability) for tilapia sex reversal. Similar to Haffray et al. (2009) who reported earlier and longer period of sex lability in brook trout, which needed to initiate several immersions during the week before hatching to 2 weeks after hatching and combined with oral MT treatment. Table 1. The effects of MT immersion on Nile tilapia egg (2 dpf) at different durations and dosages on percentage (mean±sd) of sex and survival rate. Duration (h) Dosage (µg/l) N Male (%) Inter-sex (%) Survival rate (%) ± ± ± ± ± ± ± ± ± b ± a ± a ± ± ± ± b ± a ± a ±2.1 Different super scripts in the same immersing duration showed significantly different from the control (P<0.05, χ 2 ) 305