Turkish Journal of Zoology Turk J Zool (2013) 37: 576-581 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1209-22

Reproductive biology of pink orbignyana in the (eastern Mediterranean)

Derya DURSUN, Elizabeth Grace Tunka ERONAT*, Meryem AKALİN, Mehmet Alp SALMAN Faculty of Fisheries, Ege University, Bornova, İzmir, Turkey

Received: 19.09.2012 Accepted: 17.04.2013 Published Online: 12.08.2013 Printed: 06.09.2013

Abstract: Some aspects of the reproductive biology of , such as fecundity, spawning strategies, and gonad histology, have been studied in 1048 specimens (538 males and 510 females) caught during trawl surveys from May 2008 to April 2009 in the Aegean Sea. The length at 50% maturity was estimated as 50 mm for males and 70 mm for females. The smallest mature female had a length (ML) of 36 mm; the smallest mature male had a 32-mm ML. The total egg counts (potential fecundity) in females of 60–84 mm in ML varied between 201 and 1532. The spermatophore counts in mature males (ML = 42–67 mm) were between 68–1055, with a mean of 485.

Key words: Reproductive biology, pink cuttlefish, S. orbignyana, Aegean Sea

1. Introduction 2. Materials and methods Sepia orbignyana Ferussac, 1826 inhabits the eastern Samples were collected onboard commercial vessels Atlantic Ocean from 55°N to 17°S and occurs throughout during daytime by bottom trawl (44-mm mesh size in the between depths of 15 to 570 m, cod-end) on sandy and muddy bottoms at depths of 150, and mostly between 50 and 250 m. S. orbignyana, which is 350, and 550 m in the Aegean Sea (eastern Mediterranean often mistaken for S. elegans, occurs mainly on sandy and Sea). Samplings were carried out monthly from May 2008 sandy–muddy bottoms (Jereb and Roper, 2005). to April 2009 (Figure 1). The duration of each haul was 1 h, Research on Mediterranean had shown and towing speed varied between 2.2 and 2.5 knots. that S. orbignyana is one of the most common species in The entire catch was preserved in 4% the area (D’Onghia et al., 1991; Belcari and Sartor, 1993; formalin solution onboard. A total of 1048 S. orbignyana D’Onghia et al., 1996; Salman et al., 1997). As part of their individuals (538 males and 510 females) were collected. The dorsal mantle lengths (MLs) were measured within 1 work regarding cephalopod fishery in both the Aegean Sea mm, total body weights (TWs) within 0.01 g, and gonad and the eastern Mediterranean Sea, Salman and Katağan weights (GWs) within 0.0001 g of precision. Specimens (2004) reported that S. orbignyana is the most abundant were examined under microscope after dissection. A cephalopod bycatch species from depths of 100–200 m. In maturity stage was assigned visually using a universal European countries it is consumed fresh or frozen (Jereb maturity scale adapted for cuttlefish by Arkhipkin and Roper, 2005). (1992). Gonadosomatic indices (GSIs) of both sexes were Unlike Sepia officinalis, data on the calculated for each sampling period (GSI = (GW/TW) × reproductive biology of S. orbignyana are scarce (Hastie 100) (Gabr et al., 1998). Subadult and adult large female et al., 2009). The species has been studied in the western were then dissected and the ovary and oviducts Mediterranean Sea (Mangold-Wirz, 1963), but not in the were excised; out of 26, 4 were stage II, 5 were stage III, 5 eastern part of this water body, which is different in many were stage IV, and 12 were stage V. Potential fecundity (PF) oceanographic and biological aspects (Sonin et al., 2007). of females was calculated as the sum of the oocytes in the The aim of this study is to describe different aspects ovary plus the eggs in the oviduct. Since the fixated stage V of the species’ reproductive biology such as fecundity, oviduct eggs were torn during counting in the laboratory, spawning strategies, and gonad histology, which knowledge true potential fecundity could not be estimated. To get is necessary for sustainable management of its stocks. true totals of oviduct eggs, the unfixated mature stage V * Correspondence: [email protected] 576 DURSUN et al. / Turk J Zool

38.2°N

38.0°N

37.8°N

37.6°N 26.6°E 27.0°E 27.4°E Figure 1. Sampling stations (A: 150 m; B: 350 m; C: 550 m). female specimen oviducts were dissected and oviductal 3.2. Bathymetric changes in cuttlefish eggs were fixated separately during trawling. All oocytes The minimum monthly average mantle length of females from the ovaries and the oviducts were counted separately was recorded in May (38.66 ± 14.39 mm), and the maximum and measured along the major axis to the nearest 0.1 was recorded in November (67.54 ± 7.55 mm). In males, mm. Spermatophores were counted in 16 mature males, the minimum monthly average mantle length occurred in and a total of 30 spermatophores were measured in each January (45.01 ± 8.86 mm) and the maximum occurred in specimen. November (60.45 ± 9.59 mm), though no monthly trend To validate our conclusions on oocyte development, was obvious (Figure 3). histological studies were used. Samples from ovaries were When the combined data of the bathymetric distribution first rinsed with water. After that, they were dehydrated in of the MLs were examined, 73.8% of the MLs at 150-m a graded ethanol series (dehydration processes started with depths were between 22 and 85 mm, and the average ML 70% alcohol and increased to a series of 96% and 100% alcohol) and then stained with Mallory’s hematoxylin and 100 eosin to reveal the start of yolk accumulation. Mallory’s hematoxylin stains the membrane and proteinaceous Fema le N = 538 90 TW = pow (ML,2.55267) * 0.000802773 structures like yolk droplets blue, and eosin stains R-squared = 0.956003 cytoplasmic structures pink (Laptikhovsky and Arkhipkin, 80 2001). The slides were examined using a CX-31 phase 70 Male N = 510 contrast microscope and photographed with an Olympus TW = pow (ML,2.30968) * 0.0019176 ) R-squared = 0.863104

DP-20 digital camera. (g 60 t gh Histological study of mature gonads with ripe eggs was 50 we impossible because of the very large size of the oocytes. l ta 40 To 3. Results 30 3.1. Cuttlefish size and weight Females were the larger sex (Figure 2); their size in 20 monthly samples varied between 22 and 90 mm ML versus 21 and 78 mm ML in males. The length–weight relations 10 2309 were estimated as TW♂ = 0.0019 ML and TW♀ = 0 2552 0.0008 ML for males and females, respectively. When 0102030405060708090100 the length–weight relationships were compared by t-test Mantle length (mm)

(P > 0.05), it showed that there was a difference (ttab < tcalc) Figure 2. Length–weight relationship between the 2 sexes of S. between the sexes. orbignyana from the Aegean Sea.

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80 Male Female + 70 m) (m

60 th

ng 50 le

le

nt 40

Ma 30 20

08 08 08 08 8 08 08 08 08 09 09 09 09 r- - - l- -0 - t- - - - - r- r- p ay un u g ep c ov ec an eb a p A M J J Au S O N D J F M A Figure 3. Monthly average length and standard deviation distribution of S. orbignyana in the Aegean Sea. and standard deviation was 53.47 ± 13.92. At a depth of 350 diameter (Figure 3). Oocyte growth and maturation was m, 20.1% of MLs of the specimens were between 21 and 90 asynchronous, and oocytes of 0.1–2.0 mm predominated at mm, and the average ML and standard deviation was 56.12 all maturity stages (Figure 8). There was no evidence of egg ± 13.52 mm. At a depth of 550 m, 6.1% of the specimens’ size increase with an increase in female ML (r = 0.0407), but MLs were between 25 and 82 mm (Figure 4). the sample size was too small to judge this. 3.3. Maturity Mature males had between 68 and 1055 (mean: 485) The smallest mature female was of 36 mm in ML, and the spermatophores. Spermatophore length (SpL) increased smallest mature male was of 32 mm in ML. The size at with male size (Figure 9). The calculated SpL/ML index varied between 7.7% and 12.7% (mean 10.2%). maturity (ML50) was 70 mm for MLs in females and 50 mm for MLs in males (Figure 5). Maturity increased with ML 3.5. Ovary development (Figure 6). Asynchronous oocyte development was obvious after The mean monthly female GSIs demonstrated 2 annual maturity stage I (Figures 10A and 10B). The connective peaks, in spring (May) and in autumn (November). Male tissue in the center of the gonad holds the oocytes together GSIs were similar throughout the year (Figure 7). and provides nourishment via the net of blood vessels. 3.4. Fecundity Growing oocytes lie more sparsely in the ovary. Potential fecundity of females (60–84 mm ML) varied between 201 and 1532 eggs. Mature specimens had from 4 to 4. Discussion 82 ripe eggs of 6.7–8.3 mm (average: 7.9 mm) at the largest The earlier maturation and thus the smaller size of males are in agreement with data from the by 25 Ragonese and Jereb (1991) and from the Adriatic by Bello Fema le; N= 375 20 (2001). 15 Male; N=395 10 150 m 5 100 0 25 ) Female; N= 130 20 (% Ma le; N= 84 75 y 15 nc 350 m ue 10 eq

5 ty (%) Fr

r 50 0

25 Female; N=33 Matu 20 Male; N=31 25 Male 15 Female 10 550 m 5 0 0 10 20 30 40 50 60 70 80 90 100 0510 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Mantle length (mm) Mantle le ngth (mm) Figure 5. Male and female distribution of 50% of the population

Figure 4. Bathymetric distribution of the different-sized S. (ML50) at the size of maturity of S. orbignyana obtained from the orbignyana in the Aegean Sea. Aegean Sea.

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80 N= 196 the eastern and western Mediterranean: productivity, N= 55 Female temperature, and salinity (Sonin et al., 2007). This study, 70 N= 107 Male combined with previous results (Salman, 1998; Önsoy and 60

(mm) N= 160 Salman, 2005; Salman and Önsoy, 2010; Salman, 2011), th 50 N= 20

ng testifies that Mediterranean nanism can also be seen in N= 342 le 40 cephalopods. le

nt 30 N= 39 N= 76 Histological analysis of pink cuttlefish ovaries revealed Ma 20 N= 9 N= 44 that oogenesis is asynchronous (sensu Rocha et al., 2001) and spawning likely is intermittent, as reported by 10 0III III IV V Laptikhovsky et al. (2003) for S. officinalis. Gonad stage Because both mature females and recruitment of MLs Figure 6. Average mantle length distribution by gonad stages of of 20–30 mm could be found in the Aegean Sea year- S. orbignyana. round, we might conclude that spawning happens in any season. Dynamics of GSI values indicate 2 spawning peaks (spring and fall). Similar results were reported from the Vertical distribution of the ML showed that S. western Mediterranean Sea by Mangold-Wirz (1963) and orbignyana was widely distributed in waters from less than Jereb and Roper (2005). 100 m to deeper than 500 m. There is no vertical migration The species’ fecundity (ca. 200–1500, generally 600– for reproduction, as Jereb and Roper (2005) reported. 700) was higher than some reports of 400 in the West The minimum size of mature , both males and Mediterranean (Mangold-Wirz, 1963). females, was lower in the Aegean Sea than in other parts Mangold-Wirz (1963) reported that there was no of the Mediterranean system: 32 mm versus 35–38 mm in strong correlation between ML and spermatophore length males, and 36 mm versus 40–65 mm in females (Jereb and of male S. orbignyana specimens from the Catalan Sea, Roper, 2005; Ciavaglia and Manfredi, 2009). and this was also observed in the Aegean S. orbignyana Boyle et al. (1988) reported that cephalopods’ first specimens in this study (R = 0.548). maturation length decreases from the Atlantic to the Mature male S. orbignyana specimens from the Aegean Mediterranean, and other results on other Mediterranean Sea generally have many more (mean: 485) spermatophores cephalopods such as Sepietta oweniana (Salman, 1998), S. than their counterparts from the western Mediterranean officinalis (Önsoy and Salman, 2005), Rossia macrosoma (ca. 100) (Mangold-Wirz, 1963). We suppose that the higher (Salman and Önsoy, 2010), and Neorossia caroli (Salman, number of spermatophores is necessary to fertilize due to 2011) also demonstrated a decrease in the minimum size the higher fecundity of females. The ripe egg size found of mature animals from west to east that is in agreement in this study (6.7–8.3 mm) was similar or slightly smaller with our results on S. orbignyana. than that in the western Mediterranean (7.0–9.0 mm) A phenomenon of so-called Mediterranean nanism (Mangold-Wirz, 1963; Jereb and Roper, 2005). All of this had been suggested for various taxonomic groups of shows that S. orbignyana is more r-strategist in the Aegean fishes and invertebrates such as sponges, crustaceans, Sea than in the rest of the species’ range. This difference is and (Sonin et al., 2007). The reason for probably caused by a higher level of competition for scarce this phenomenon is the unidirectional impact of 3 food sources, so there is higher natural mortality in the main environmental factors that are different between nutrient-poor eastern Mediterranean area.

15 30 14 Female 13 Male 12 25 T Temp em 11

10 20 pe ) 9 r a (% 8 t I 7 15 u r e

GS 6

(

5 10 C) 4 3 2 5 1 0 0 8 8 8 8 8 8 8 8 8 9 9 9 9 -0 -0 -0 -0 -0 -0 -0 -0 0 0 0 0 0 r y n l g p t v c- n- b- r- r- p a u J u u e c o e a e a p A M J A S O N D J F M A Figure 7. Monthly gonadosomatic index values of S. orbignyana from the Aegean Sea.

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100 Immature 9

80 (mm) ML= 31 mm; N= 796 Y = 0.125863 * X -1.2115 60 th 8

ng R= 0.548 40 le 7 20 e 0 A or 6 ph

100 to 5

80 Immature ma er 4 60 ML= 50 mm; N= 1438 sp ) 40 e 3 % ag ( 20 er cy B 2 0 Av en 20 30 40 50 60 70 80 90

qu 100 Mantle length (mm) re 80 F Matur ng 60 ML= 77 mm; N= 829 Figure 9. Average spermatophore length–mantle length 40 relationship. 20 0 C 100 Mature spawn ng Acknowledgments 80 ML= 82 mm This study was financially supported by EBİLTEM (Science 60 Gonad; N= 1258 and Technology Center of Ege University) (Project No: 40 Ov duct; N= 51 20 2008/SUF/004) and the Scientific and Technological 0 D Research Council of Turkey (TÜBİTAK, Project No: 012345678910 108Y102). We are indebted to Prof Dr Stephen Astley from Oocytes’ d ameter (mm) the Ege University Science Faculty for editing the text. We Figure 8. S. orbignyana egg diameter distribution percentages for would also like to thank both referees for their valuable different gonad stages. and constructive comments.

A B

200µ

Figure 10. Histological section of the gonad stages; A) stage II and B) stage III.

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