Reproductive biology of largescale tonguesole Cynoglossus arel in coastal waters of Bandar Abbas, Persian Gulf, Iran

Animal Reproduction Science 154 (2015) 142–157

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Reproductive biology of largescale tonguesole Cynoglossus arel in coastal waters of Bandar Abbas, Persian Gulf, Iran H. Ghaffari a,∗ , H. Hosseinzadeh Sahafi b , G.H. Engelhard c , M. Mekhanik Babaei d a b c d

Iranian National Institute for Oceanography and Atmospheric Science (INIOAS), Tehran, Islamic Republic of Iran Iranian Fisheries Research Organization (IFRO), Tehran, Islamic Republic of Iran Centre for Environment, Fisheries & Aquaculture Science (Cefas), Lowestoft, UK Faculty of Marine Science and Technology, North Tehran Branch, Islamic Azad University, Tehran, Islamic Republic of Iran

a r t i c l e

i n f o

Article history: Received 6 March 2014 Received in revised form 29 November 2014 Accepted 3 December 2014 Available online 24 December 2014 Keywords: Cynoglossus arel Reproductive cycle Maturity Spawning Flatfish Fisheries

a b s t r a c t The objectives of this study were to determine the reproductive cycle of largescale tonguesole Cynoglossus arel, a commercially valuable flatfish species, in coastal waters of Bandar Abbas, along the south coast of Iran in the Persian Gulf. From October 2009 to September 2010, 905 fish were collected in monthly samples, and their length, weight, sex, gonad weight, and maturity status recorded. These data revealed that ovary weight in females is low from July to September, then increases to a peak in February followed by a decrease, indicating that the peak spawning season is from February to March with some spawning lasting until June. Males showed a corresponding seasonal pattern in testis weight, although with much less pronounced seasonal differences than gonad weight in females. Five maturity classes were described based on ovarian and testicular histology, corresponding with macroscopic analysis of gonads. The spawning season in C. arel is prolonged, similar to several other tropical flatfish species, and larger adult females tended to have an even more prolonged spawning period than smaller, presumably younger adult females. Combined, our results indicated that C. arel is a winter-to-spring batch spawner with an asynchronous type of ovarian development. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Worldwide, flatfishes (Pleuronectiformes) are of great commercial and ecological significance. However, the overwhelming majority of research on their reproductive biology, exploitation and management has focussed on species living in temperate seas, especially the North Atlantic and North Pacific; by comparison, knowledge on the reproductive biology of tropical flatfish species remains

∗ Corresponding author at: No. 3, Etemadzadeh St., Fatemi Ave., Tehran 1411813389, Islamic Republic of Iran. Tel.: +98 912 212 45 20. E-mail address: [email protected] (H. Ghaffari). http://dx.doi.org/10.1016/j.anireprosci.2014.12.004 0378-4320/© 2014 Elsevier B.V. All rights reserved.

limited (Rijnsdorp and Witthames, 2005). Yet tropical flatfishes provide an important food source to the inhabitants in many coastal communities, such as along the shores of the Indian Ocean (Gibson, 2005). In the Persian Gulf, five species in the family of tonguesoles (Cynoglossidae) are caught and marketed fresh, frozen, dried, or as salted fish products (Gibson, 2005). These include largescale tonguesole Cynoglossus arel, fourlined tonguesole Cynoglossus bilineatus, hooked tonguesole Cynoglossus carpenteri, shortheaded tonguesole Cynoglossus kopsii, and speckled tonguesole Cynoglossus puncticeps. All are valued for their delicious meat, and largescale tonguesole C. arel in particular is an important source of protein in the diet of local communities in southern Iran (personal communication).

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This species is distributed throughout the Persian Gulf and Oman Sea (Yasemi et al., 2007) and can be found at the continental shelf down to 125 m depth (Fischer and Bianchi, 1983). Although there is not a directed fishery for largescale tonguesole, it is taken in considerable quantities as bycatch in the local shrimp trawl fisheries (Topp and Hoff, 1972). Nominal catches of tonguesoles from the western Indian Ocean (FAO Fishing Area 51) have previously been reported as about 1000 t in 1981, all taken by Pakistan (Fischer and Bianchi, 1983); as this only includes the reported catch, it is likely that this does not represent the full commercial significance of tonguesoles which may currently be considerably higher. So far, research on tonguefishes in the Persian Gulf included the identification of closely related species by means of morphometric and meristic characteristics (Yasemi et al., 2007) and the recent description of a new species, Cynoglossus persicus, from Iranian coasts (Kousha et al., 2008). Elsewhere, the reproductive biology of C. arel and Cynoglossus lida has been studied in southern India (Rajaguru, 1992), where the length–weight relationships in Cynoglossus macrostomus and C. arel have also been described (Jayaprakash, 2001), as well as growth, mortality (Kutty, 1967), reproductive biology, and fisheries in related tonguesole species (Seshappa, 1973, 1974, 1980; Ramanathan et al., 1977). In the Persian Gulf, knowledge on reproductive biology of tonguesoles is limited to macroscopic studies on C. arel (summarised in Ghaffari et al., 2011) which might partly reflect less scientific attention due to the species’ relatively small size (total length range 11–33 cm in females, 10–31 cm in males; Ghaffari et al., 2011). The general seasonality of reproductive and body condition indices showed that C. arel is a winter–spring spawner (Ghaffari et al., 2011). This study complements earlier work on large-scale tonguesoles by including

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macroscopic analyses of maturation and reproductive condition, but also focuses on the gonad histology using microscopic techniques (Narimatsu et al., 2007). Gonad histology, although more costly and time-consuming than macroscopic analyses, can provide an independent assessment of maturity and a more detailed insight into the process of oogenesis (McBride et al., 2013). The purpose of this study was to determine the reproductive biology of C. arel in coastal waters of the Persian Gulf near Bandar Abbas, Iran. To do so, this paper (1) characterises the phases of ovarian follicle and testicular development and the stages of ovarian and testicular maturation; (2) determines the spawning season, spawning periodicity, type of spawning and size at first maturity; and (3) assesses demographic trends in spawning periods. 2. Materials and methods 2.1. Collection and processing In this study a total of 905 specimens of C. arel (493 females, 412 males) were collected monthly during day time, from October 2009 to September 2010, from the coastal waters of Bandar Abbas, south coast of Iran (27◦ 17 N, 56◦ 26 E) (Fig. 1). Commercial trawling nets were used during the shrimp fishery season, and trammel and gill nets during other seasons. Total length (TL) of each individual was measured to the nearest 1 mm, and body weight (BW) recorded to the nearest 0.1 g. The gonads of each specimen were also weighed to the accuracy of 0.01 g (GW), while the sex was determined by examination of the gonads either with the naked eye or with the aid of a binocular microscope, if necessary, and the gonads in females were macroscopically classified. Gonad length (GL) was measured to the nearest 1 mm. For both

Fig. 1. Location of sampling in coastal waters of Bandar Abbas, Persian Gulf, Iran.

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Table 1 Classification of maturity for female largescale tonguesole, Cynoglossus arel. Macroscopic character traits (e.g., gonad size, shape, and volume) are listed on the left as they were estimated using histological characters on the right side. The origins of this scheme are reviewed by Afonso-Dias et al. (2005). Macroscopic class and criteria

Gonad histology class and criteria

I–Immature or resting (I) No female smaller than 222 mm was mature, and no female larger than 300 mm was immature.The ovary wall was thin and tore easily, smallest in length, and not extending more than 4 cm down the side of the body. In fish <200 mm, the ovary was clear, transparent. Color was evident in larger immature fish, pinkish. Small translucent organ located at posterior curve of gut cavity. There were no visible oocytes II–Developing (D) Ovaries grew rapidly in this class; increase in length and in width, they become thicker and opaque with blood vessels becoming prominent, small opaque specks are visible as ovary develops to occupy 2/3 of ventral cavity. Ovary extending posteriorly fromgut cavity; bright yellow and firm texture. The fully vitellogenic oocytes were large enough to be visible to the unaided eye III–Maturing (M) The ovaries increase considerably in volume and usually distend the body. Many opaque and a few hydrated oocytes are visible

I–Immature or resting (Im) Oogonias and small oocytes in chromatin nucleolus stage (CNS); no oocytes beyond the perinucleolus stage (PNS) and Early perinucleolus (EPN) were found. Observed in spring to autumn

IV–Ripe (R) Ovary may fill body cavity. Large, turgid, and full ovary with hydrated oocytes visible. Hydrated oocytes were darker than the background of yellow to orange yolked eggs. In the running stage the oocytes were extruded copiously under light pressure of the ovary V–Spent and recovering (S) Ovaries were empty or partially empty and flaccid. If the ovary was cut open to confirm, the sac-like nature of the ovary was apparent. There may be residual hydrated or larger vitellogenic oocytes scattered in a state of reabsorption with a lot of slime. The ovary was usually discolored, red-purple, and shrank in size during this class. Gonads were highly vascularized with some ruptured capillaries, bloodshot in appearance. Gonad continued to shrink in length during the resting and recovering period

II–Developing (De) Some oocytes in CNS but most of the small oocytes were in PNS with one or more nucleoli in the periphery of the nucleus. The larger oocytes were in the cortical alveoli stage (CAS). Observed in spring to autumn

III–Maturing (Ma) Oocytes continue to increase in size, most in CAS. Vitlongenic (Vit) and small numbers of mature (Mat) oocytes with a migrating nucleus are present. Small PNS oocytes are still present. Could be observed in all season, but mostly in autumn to winter IV–Ripe (Ri) Most advanced oocyte (MAOS) was a hydrated oocyte still within the follicle and lamellae. Many mature and hydrated oocytes were present. Numerous oocytes in CAS and in an advanced stage of vitellogensis; pre-vitellogenic oocytes were still present. No post-ovulatory follicles (POF) were evident. The gonad wall was stretched. Observed in autumn to winter with the further in winter V–Spent (Sp) Numerous post-ovulatory follicles (POF) were present. Many oocytes were in different stages of perinucleolus and cortical alveoli. Many empty spaces in the ovaries. Mature oocytes that were not released in different stages of atresia; observed in spring to summer

Table 2 Classification of maturity for male largescale tonguesole, Cynoglossus arel. Macroscopic character traits (e.g., gonad size, color, and volume) are listed on the left as they were estimated using histological characters on the right side. The origins of this scheme are reviewed by Rajaguru (1992) and Kume et al. (2006). Macroscopic class and criteria

Gonad histology class and criteria

I–Immature No male smaller than 123 mm was mature, testis minute, pale white

I–Immature or resting (Im) Spermatogonia present in testes. Immature class characterized by SG in the germinal epithelium and by the early formation of testis lobules that contain only SG. The lobules do not have a lumen and spermatogonial proliferation in the form of mitotic division is the only type of spermatogenic activity occurring II–Developing (De) Primary spermatocytes, secondary spermatocytes, and spermatids present in testes. Seminiferous lobules develop moderately. As males move into the gonadotropin-stimulated developing class, SG within spermatocysts that line the lobules divide to form SSC, which then enter meiosis, and active spermatogenesis occurs III–Maturing (Ma) Lobules and sperm ducts packed by sperms. Many spermatids observable in lobules with sperms. This class containing all stages of spermatogenesis, including ST IV–Ripe (Ri) Lobules and sperm ducts more densely packed by sperms. Few cysts of spermatids observable in lobules. This class identified by the presence of SP in the lumen of the lobules and in the sperm ducts V–Spent (Sp) Large empty lobular spaces are present in testes, except for some residual sperm

II–Maturing Testis slightly enlarged, sac-like, creamy white; no milt oozes out on pressure

III–Mature Testis enlarged, sac-like, creamy white; whitish milt running from vent on slight pressure IV–Spent Not found during the present study

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The timing and duration of the spawning season were derived from the monthly data on the incidence of mature individuals, proportions of each maturity stage and changes in standardized gonad weight. Monthly estimates of standardized gonad weight were also calculated for two size groups (202–266 and 266–330 mm, both larger than the minimum size at maturity) to observe whether there is an effect of fish size on the timing and duration of the spawning season. As far as we know, spawning duration was first estimated by Qasim (1956) (and see also Iles (1964) in herring Clupea harengus and Rijnsdorp (1989) in plaice Pleuronectes platessa). The maturation rate was examined at every millimeter increment in TL. Definition of maturation is described below. The relationships between the maturation rate and TL was fitted to the logistic formulae. Using the formulae, changes in the maturation rate dependent on TL was analyzed. Length at 50% maturity (L50% ) for females was estimated as the length at which the probability of being mature (stages III and IV) was 50%: Maturity (M) = 1/{1 + exp[−A(TL − B)]}. In order to establish the size-frequency distribution of oocytes in C. arel microscopic histology of females (n = 149) was carried out with ovaries in different stages of maturity. The sizes of the oocytes were measured to the nearest 0.01 mm in diameter under a light microscope (Nikon ECLIPSE 80i) with a micrometric scale on the ocular (Romagosa et al., 2001; Honji et al., 2009). For each section, at least 30 oocytes were measured.

sexes, we calculated indices of standardized gonad weight and standardized gonad length, following definitions of McBride et al. (2013). Standardized gonad weight was defined as gonad weight/(ovary-free body weight) × 100, or GW/(BW − GW) × 100 (n = 905). Standardized gonad length was defined as (GL/TL) × 100 (n = 456). The gonads were then preserved for 12 h in Bouin’s solution for subsequent histological analysis (Kume et al., 2006). 2.2. Histological examination Following a standard histological manual (Hinton, 1990), a histological study on gonads was conducted on 149 females and 96 males taken from each monthly sample. Briefly, fragments were removed from previously fixed gonads, and dehydrated with a series of alcohol that varied from 70% aqueous alcohol to 100% ethanol. After being embedded in ascending solutions of resin, the tissue was sliced into 8–10 mm sections, and stained with hematoxylin and eosin (HE staining). We designated the most advanced oocyte stage (MAOS) observed in each female, in increasing order, as the perinucleolar, cortical alveoli formation, vitellogenic or migration of the germinal vesicle stage (McBride et al., 2002). The effect of gonad sampling location on interpretation of histology was also examined with another set of samples taken from the gonad anterior, middle, or posterior of some fishes in different maturity classes, but no differences were noted so these results are not discussed further. Histology slides (n = 245) were examined using a microscope (10–100×), independently by one of the authors (HG) and a single, experienced reader.

3. Results 3.1. Morphometrics

2.3. Maturity index and reproductive mode Length distribution of C. arel is shown in Fig. 2. Females reached slightly larger sizes than males. Females ranged from 113 to 330 mm TL (mean ± SE, 226 ± 2 mm) and from 4.1 to 201 g total weight (mean ± SE, 54 ± 1.4 g), males ranged from 99 to 308 mm TL (mean ± SE, 211 ± 2 mm) and from 3.5 to 137.9 g total weight (mean ± SE, 43 ± 1.2 g). The gonads of C. arel are quite distinct between sexes and easy to identify. They show a well-marked size

For female C. arel, five macroscopic and histological classes and criteria were used, in line with the maturity stages in Afonso-Dias et al. (2005) (Table 1). For males, four macroscopic classes of maturity were determined based on the colour and size of the testes, following Rajaguru (1992), and five histological classes and criteria were adapted from Kume et al. (2006) (Table 2). 60

Male Female

% Frequency

50 40 30 20

330-321

310-301

290-281

270-261

250 241 250-241

230-221

210-201

190-181

170-161

150-141

130-121

110-101

0

90-80

10

Length h class (mm) Fig. 2. Length distribution of 412 male and 493 female largescale tonguesole, sampled in 2009–2010 in coastal waters of Bandar Abbas, Persian Gulf, Iran.

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Fig. 3. Macroscopic images of largescale tonguesole, Cynoglossus arel ovaries: (A) immature or resting (I) female (gonad length [GL] 21 mm in eye side and 17 mm in blind side), (B) developing (II) female (GL 73 mm in eye side and 64 mm in blind side), (C) maturing (III) female (GL 87 mm in eye side and 76 mm in blind side), (D) ripe (IV) female (GL 117 mm in eye side and 102 mm in blind side), (E) spent or recovering female (V) (GL 122 mm in eye side and 112 mm in blind side).

asymmetry between the eyed and blind side. The ovaries exhibit a flattened tubular structure much wider in the anterior region that runs along the ventral part of the animal’s body. 3.2. Maturity classification scheme in females The ovaries of immature or resting (I) females were smallest in length and not extending more than 4 cm down the side of the body, with a wall that was thin and tore easily. They were clear, transparent. Colour was evident in larger immature fish, and pinkish. There were no visible oocytes with unaided eye (Fig. 3A). In primary growth, many pre-vitellogenic oocytes were in chromatin nucleolus stage (CNS); no oocytes beyond the perinucleolus stage (PNS) or early perinucleolus stage (EPNS) were found (Fig. 4A). Secondary growth is typically categorized into the developmental stages: cortical alveolar stage, vitellogenesis, and oocyte maturation (Fig. 4B–D). A developing class, developing females (De), was also identifiable with gonad histology. Class De females were intermediate in body length and gonad size relative to

class I females and all mature classes (Fig. 5). In this class, ovaries grew rapidly; increase in length and in width, they coloured yellow-orange, become thicker and opaque. Small opaque specks are visible (Fig. 3B). In continue of developmental stages, some oocytes were in CNS stage but most of the small oocytes have found perinucleolus stage (PNS) with one or more nucleoli in the periphery of the nucleus. The larger oocytes were in the cortical alveoli stage (CAS) (Fig. 4B). The developing female was generally unambiguous macroscopically, because the gonad increased rapidly in size, became opaque, colourful, sometimes even bright orange or yellow, and the vitellogenic oocytes were large enough to be visible to the unaided eye. Class De females were gonadotropin dependent, as evident by the presence of cortical alveolar or vitellogenic inclusions in the cytoplasm; however, class De females were not ready to spawn in the season they were collected (Table 1). Instead they were preparing to spawn for the first time at the end of winter and in the following spring. Microscopic classes corresponded to the macroscopic classes (Table 1). When using macroscopic characters at the laboratory, misclassification of immature females (I)

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Fig. 4. Photomicrographs of cross-sections of Cynoglossus arel ovaries at different developmental stages. (A) Immature or resting (I) female, (B) developing (II) female, (C) maturing (III) female, (D and E) ripe (IV) female, (F) spent female (V). OO, oogonia; CNS, chromatin nucleolar stage; EPNS, early perinucleolus stage; LPNS, late perinucleolus stage; CAS, cortical alveoli stage; PNS, perinucleolus stage; EVit, early vitellogenic stage; LVit, late vitellogenic stage; Mat, mature; POF, post-ovulatory follicle; N, nucleus; Nu, nucleolus. Magnifications: (A) 400× and (B–F) 100×.

as resting females occurred, indeed in the resting class, gonads reduced in length and gonad wall was thick and opaque. Gonad length overlapped among these classes, but generally differed enough that this character should help classify maturity correctly (Fig. 5). The gonad colour and transparency was notably different among these classes (Fig. 3), presumably as a result of the differences in the most advanced oocyte stage (MAOS): immature (transparent), resting (opaque). This demonstration of the difficulty of distinguishing these classes was the primary motivation to estimate maturity parameters using gonad histology.

The presence of fully vitellogenic oocytes in the ovary was diagnostic at the end of the developing stage and the beginning of the maturing stage (Fig. 4). Based on the macroscopic and microscopic observations, there were also associations between these in maturing class (Figs. 3 and 4Figs. 3C and 4C). Using histology it was possible to recognize class Ma, maturing female, which had oocytes in an early stage of maturation; however the appearance of cellular nuclei migrating to the animal pole was not distinguishable macroscopically, so most maturing females were assigned at laboratory to class M rather than R. In this class, oocytes continue to increase in size, most in

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CAS, vitellogenic (Vit) and small numbers of mature (Mat) oocytes were present, small PNS oocytes are still present (Fig. 4C). Maturing gonads increase considerably in volume and usually distend the body. Many opaque and a few hydrated oocytes are visible (Fig. 3C), a maturing class could be observed in all season, but mostly in autumn to winter. Using histology, one could also recognize initiating a new group of oocytes into vitellogenesis. Initiating a new group of oocytes into vitellogenesis was distinguished by a thicker gonad wall and an increased amount of connective tissue throughout the lamellae; however, this class was not typically assigned at sea to the developing class, presumably because the primary criterion for recognizing it was not apparent macroscopically. Ripe female as a class IV is to be used when female is ready to spawn, is spawning, or has not yet completed spawning; large, turgid and full ovary with hydrated oocytes was visible. The ovary appeared speckled with hydrated (i.e., clear) oocytes that were darker than the background of yellow to orange yolked eggs. In the running stage the oocytes are extruded copiously under light pressure of the ovary (Fig. 3D). Microscopic traits expressed that most advanced oocyte (MAOS) was a hydrated oocyte still within the follicle and lamellae and the gonad wall was stretched, numerous oocytes in CAS and in an advanced stage of vitellogenesis; pre-vitellogenic oocytes were still present. No post-ovulatory follicles (POF) were evident (Fig. 4D,E), these results portray that the ovary reached its largest size in this class. Female largescale tonguesole ovulated in a single wave, so spent and recovering (Sp) females may have had remnant (few) hydrated eggs but they were recognizable with histology by numerous POF. The ovaries were empty or partially empty and flaccid, if the ovary was cut open to confirm, the sac-like nature of the ovary was apparent. There may be residual hydrated or larger vitellogenic oocytes scattered in a state of reabsorption with a lot of slime. The ovary was usually discoloured, red-purple and shrank in size during this class. They were highly vascularized with some ruptured capillaries, bloodshot in appearance (Fig. 3E). According to the histological observation, numerous post-ovulatory follicles (POF) were present throughout the section, most were collapsed. The MAOS may be hydrated oocytes, but if so, these were uncommon or rare, often representing residual eggs, usually atretic. Otherwise, the MAOS were no more advanced than cortical alveolar. Many oocytes were in different stages of perinucleolus and cortical alveoli. Mature oocytes that were not released in different stages of atresia and many empty spaces in the ovaries have been observed (Fig. 4F). The gonad continued to shrink in length during the resting and recovering period, this class and its length overlapped with that of Sp and repeat developing classes (Fig. 5), which was a likely source of confusion at the

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macroscopic level among these classes. These classes were not difficult to distinguish from each other with histology, but the primary microscopic characters – POF and partially vitellogenic oocytes – could not be observed with the unaided eye. The size of the resting and recovering gonad was also very similar between larger immature and first-time developing females. This may contribute to misclassification of larger immature fish as adult, resting or recovering fish. The histological observations revealed that the development of oocytes was asynchronous with oocytes of all stages of development present in the population without any clearly predominating stages. The ovary appeared to be random mixture of oocytes, at each conceivable stage and gonads contained individual oocytes in four or more development stages (Fig. 4B–F). 3.3. Maturity classification schemes in males Male gonads were bean-shaped and extremely small, occupying less than 1% of the abdominal cavity (Fig. 6). Each testis was divided by a septum of connective tissue into numerous testicular lobules. Within each testicular lobule, cysts containing germ cells at different stages of development were found and germ cells within each cyst developed synchronously. According to the macroscopic and histological characters, spermatogenesis could be divided into four and five classes, respectively (Table 2). Spermatogenesis was initiated throughout the entire length of the lobule. Unreleased sperms were observed as residual sperms in a number of testes between June and September. Maturity classes are defined above and thinsections for each class are shown in Fig. 7. 3.4. Reproductive seasonality 3.4.1. Seasonal changes of standardized gonad weight The standardized gonad weight of female C. arel was higher between January and April than during the rest of the year, with highest values in February (mean ± SD, 6.01 ± 0.52), and declined between May and July (Fig. 8A). Standardized gonad weight was lowest from July to October, suggestive of a resting period; after this period the gonad increased in weight continuously in relation to body weight, with some acceleration around February. In males, the standardized gonad weight was higher between January and June, started to increase from September with the highest mean in May (0.10 ± 0.01), and decreased from June to September (Fig. 8). Studies of standardized gonad weight in females in two size groups (Fig. 8B) demonstrated that there was an effect of fish size on spawning season, such that the duration of the spawning period and reproductive activities was longer in larger than in smaller fish.

Fig. 5. Morphometrics of immature and mature female largescale tonguesole, Cynoglossus arel, with respect to histology maturity class. (A) Body length, (B) standardized gonad weight, and (C) standardized gonad length. Maturity classes are defined in Table 1. Each panel is a box–whisker plot, where the dark horizontal line is the median value, the outer box represents the first and third quartiles, extended bars indicate the approximate 95% confidence limits, and the individual points are outliers. Total sample size (n = 456) is for gonad length/total length only and total sample size (n = 493) is for other metrics.

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Fig. 6. Macroscopic images of largescale tonguesole, Cynoglossus arel testis. (A) Immature (stage I); (B and C) maturing (II); (D) mature (III).

3.4.2. Monthly variation of maturity stages in females According to the monthly percentage of ovarian maturity stages, ripe females (class IV) were high in February with 67% and 33% in class III. Maturing females were found mainly in January (50%), March (58%) and April (77%). Spent females were observed more frequently from May to September and were absent from January to April. Immature fish were found only from May to November (Fig. 9A).

(stages IV and V). The distance between the three lines at the points where 50% of the population had reached at least maturity stage III and V indicates the duration of spawning of an average individual tonguesole. Thus estimated, the average female was in a spawning stage (maturity stages III–V) for about eight mouths from October to June.

3.5. Timing and duration of spawning

Since spawning occurred most frequently in March, this month was defined as the birth-month of all largescale tonguesole and could be used as the start-month for age determination. Estimated total length ranged from 113 to 330 mm in 905 specimens. Size at first maturity was estimated at 202.7 mm TL (Fig. 11). The relationship between TL and maturation rate (M) is given by the following formula:

The resting (stage V) and ripening and developing stages (I and II) dominated during the growing season: from June to January in females and from August to January in males (Figs. 8 and 9). In females visual inspection and microscopic evidence of ovaries indicated that vitellogenesis started in June and continued until January when the spawning period commences (Figs. 8 and 9). Spawning females (stage IV and V) were observed from February to June (Fig. 8) and spawning males (stage III and IV) from July to December, so male tonguesole thus started their activity at an earlier date and continued for a long time. The few male and female tonguesole that were recorded as being in spawning condition outside the main spawning period were probably due to errors in the maturity staging. In Fig. 10 the cumulative proportion of maturity stages (maturity stages II–V) within the adult population (length group = 202–330) are shown. The descending lines, which connect the cumulative proportions of the successive maturity stages (II, II + III, II + III + IV, etc.), demonstrate the transition of adult fish through successive maturity stages. The shaded area in Fig. 10 indicates the proportion of fish within the adult population that were in a spawning stage

3.6. Length at first maturity in females

M = 1/{1 + exp[−0.09(TL − 202.7)]} 3.7. Size-frequency distribution of oocytes The size frequency distribution of oocyte diameter (Fig. 12) showed that oocytes in different stages of development were found at each maturity stage. During class I, oocytes in primary growth (chromatin nuclear and perinucleolar stage) with a narrow diameter range distribution were present (15–65 ␮m). In class II, oocytes in primary growth and cortical alveoli with noteably larger diameters were present. A wide oocyte diameter range distribution was observed (35–175 ␮m). In class III, yolk granule stages were present along with the previous oocytes types (110–375 ␮m). In class IV, many mature and hydrated oocytes were present, numerous oocytes in CAS and in an

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Fig. 7. Photomicrographs of cross-sections of Cynoglossus arel testes at different developmental stages. (A) Stage I, immature or resting; (B) stage II, developing; (C) stage III, maturing; (D) stage IV, ripe; (E) stage V, spent. SG, spermatogonia; SSC, secondary spermatocyte; ST, spermatid; SP, sperm; RS, residual sperm, L, lobule. Magnifications: (A–E) 400×.

advanced stage of vitellogenesis; pre-vitellogenic oocytes were still present (260–400 ␮m). In class V, post-ovulatory follicles (POF) with many oocytes in different stages of perinucleolus and cortical alveoli were present, including oocytes that had not been not released in different stages of atresia. Combined these results suggest that C. arel are batch spawners, exhibiting asynchronous oocyte development and spawning multiple batches of oocytes over the course of the reproductive season. 4. Discussion The present study is the first to present detailed information on the reproductive biology on a member of

the flatfish family Cynoglossidae. The cytological events associated with oogenesis observed here were similar to other studies on flatfishes (e.g., Rajaguru, 1992; AfonsoDias et al., 2005; Kume et al., 2006). In this study, ovaries increased in volume in association with increase in weight, during different stages of gonadal maturation and development of oocytes was observed (Fig. 4A), whereas C. arel has batch spawning during the spawning and the ovaries have an asynchrony pattern, it seems that increase in weight and ovarian volume was related to the vitellogenic stages in first oocyte development. This increase was also indicated by Afonso-Dias et al. (2005) and Kume et al. (2006). In the immature or resting stages, all germ cell stages (chromatin nucleolus, early perinucleolus, late perinucleolus stage) in

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A

Female

Male

0.64

6

5

0.32

4 0.16

3 0.08

2 0.04

1

0

B 8

Standardized gonad weight (male)

Standardized gonad weight (female)

7

0.02

202 - 265

266 - 330

Standardized gonad weight

7 6 5 4 3 2 1 0

Fig. 8. (A) Monthly changes in standardized gonad weight (mean ± SE) of female and male Cynoglossus arel. (B) Monthly changes in standardized gonad weight (mean ± SE) of females in two length groups (202–265 and 266–330 mm).

different sizes in ovary were visible and the ovary was in the primary stages (Fig. 4B). In class II ovaries, oocytes in perinucleolus and cortical alveoli (CAS) stages were seen. As the ovarian maturation proceeded, the oocytes began to absorb yolk and vitellogens, the oocytes’ membranes became thicker, and follicular layer and zona radiata were visible. In continuation sexual maturation stages and vitellogenic stages, yolk granules accumulation is initiated in cytoplasm (Fig. 4C). With the final development stages of maturity, the oocytes are ready to spawn (Fig. 4D–F). The events were especially visible in histological observations of class III and IV in February and March. Based on the above-mentioned information, it may be concluded that C. arel in coastal waters of Bandar Abbas spawns from February to June, with a spawning peak in February or March. Ovaries containing both early vitellogenic and late vitellogenic oocytes occurred from February to June, implying that some well-developed oocytes will have also been recruited for ovulating during the spawning season. Thus, C. arel appears to release eggs more than once in a single spawning season and have a protracted spawning season in coastal waters of Bandar Abbas. Studies on the reproductive biology of marbled sole Pleuronectes yokohamae in Tokyo Bay Japan indicated that gonadal maturation of females

started in September and females at stage IV appeared in January. Specimens with spent ovaries began to appear in December, suggesting that the spawning season lasted from November to March and with especially high activity during December and January (Kume et al., 2006). Spermatogenesis is the process during which spermatogonia undergo meiosis, produce primary spermatocytes then each primary spermatocyte divides into two secondary spermatocytes and finally spermatids, this process has been observed in most of mature C. arel males and it increased during the late winter and early spring (Fig. 7A–E). C. arel male displayed an asynchronous pattern of spermatogenesis, where all germ cell stages were present at the same time. Within the lobules spermatogenesis occurred in cysts and germ cell development occurred synchronously in each cyst. In marbled sole P. yokohamae in Tokyo Bay gonadal maturation of males, stage 2 started in November and males at stages 3 and 4 appeared between November and March. Specimens with spent testes began to appear in January. Although the present study reveals the reproductive characteristics of C. arel in coastal waters of Bandar Abbas, we need further investigation of the reproduction of this species and other flatfishes, emphasizing methods to assess within population variability and

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Fig. 9. Monthly percentage of ovarian maturity stages of Cynoglossus arel. (A) All length group, (B) 202–265 mm length group, (C) 266–330 mm length group.

demographic effects, also further studies needs to be done at the Gulf of Oman (Makran area) to establish effective conservation and management strategies for other flatfish species in the near future. The spawning season is commonly described in terms of the months at which spawning starts and ends (Russell, 1976; Whitehead et al., 1986; Minami and Tanaka, 1992). This allows a crude estimate of the duration of spawning. The study of standardized gonad weight in females over the years showed that the main spawning peak was in winter (February and March) with less spawning activity in

spring (April and May) and that the fish have a protracted spawning season. At low latitudes spawning periods of C. arel and C. lide may extend over up to 10 months (Rajaguru, 1992), in line with Qasim (1973) who indicated that in Indian waters many fish species may be prolonged breeders, although shorter spawning periods are reported as well (Ramanathan et al., 1990; Garcia Abad et al., 1992). In tropical waters, productivity is often low throughout the year, to which a protracted spawning period may be an adaptation (Garcia Abad et al., 1992; Rajaguru, 1992), although locally, spawning may be linked to the seasonal

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V 75

%

IV 50 III II 25

0 Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Jun

Jul

Fig. 10. Cumulative percentage of the adult maturity stages II–V of female tonguesole. The spawning stages IV and V are shaded. The estimated spawning duration is indicated by the horizontal line at 50% encompassing the spawning stages.

patterns of rainfall or wind (Ramanathan et al., 1977). The protracted spawning period was confirmed by our results on asynchronous oocyte development, where oocytes in a variety of developmental stages were present in the ripening ovaries. Entry into the spawning capable phase is characterized by the appearance of late vitellogenic (LVit) oocytes (Fig. 4C); fish in this phase are capable of spawning due to the development of receptors for maturation-inducing hormone on the LVit oocytes. Any fish with LVit oocytes is assigned to the spawning capable phase, yet histological differences between batch spawners and total spawners and between synchronous and asynchronous species are most pronounced in this phase. In total spawners, LVit or early oocyte maturation and primary growth oocytes are the only oocyte stages present. Total spawners complete the sequestration of yolk into all growing oocytes during the spawning capable phase, and the time required for this process is species specific (Brown-Peterson et al., 2011). In contrast, asynchronous oocyte development, characteristic of batch spawners, allows production of successive batches of oocytes multiple times during the spawning season (Lubzens et al., 2010). Oocytes are recruited continuously into the cortical alveolar stage (CAS) and then

into vitellogenesis throughout the spawning capable phase. Thus, ovaries of these species may have CAS oocytes as well as a variety of vitellogenic oocyte stages in the spawning capable phase (Fig. 4E). In our study the standardized gonad weight in males represented the cyclical fluctuations in testicular activity during the year. The standardized gonad weight in males was relatively high from the January to June with a peak in May and relatively low from July to December, suggesting that release of spermatozoa was over a long period, in line with prolonged spawning in females. To reveal the reproductive features of C. arel, we compared data with previous studies on seasonality of spawning in Indian Ocean flatfishes, which showed that not all species have a prolonged spawning season. Some, including Cynoglossus macrolepidotus and Psettodes erumei, have a short spawning window which in the case of the latter is linked to the monsoon season (Hickling and Rutenberg, 1936; Pradhan, 1964; Ramanathan et al., 1977; Seshappa, 1980; Das and Mishra, 1990). Others have a long spawning season (Qasim, 1973; Seshappa, 1974; Rajaguru, 1992; Seshappa and Bhimachar, 1955; AfonsoDias et al., 2005; Kume et al., 2006; García-López et al., 2007; Narimatsu et al., 2007), as we also found for C. arel.

Maturation rate (%)

100%

50%

L 50% = 203 mm 330

310

290

270

250

230

210

190

170

150

130

110

50

0%

Total length (mm) Fig. 11. Relationship between total length (TL) and maturation rate of female largescale tonguesole. The maturation rate was fitted to a logistic curve.

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Fig. 12. Distribution of oocyte diameter in largescale tonguesole at each maturity classes.

Wright and Trippel (2009) indicated that spawning periods of most species are not discrete, extending over weeks and in many cases months due to individual differences in the onset and duration of spawning within the breeding. The duration of the spawning season tends to become more extended from the polar regions to mid-latitudes with the population of some tropical fish spawning all year. Age and size related differences may be linked to spawning experience with large, old repeat spawners usually spawning before young, first-time spawners (Leino and McCormick, 1997; Wright and Gibb, 2005) although not in all species (Morgan, 2003) and stocks (Hutchings and Myers, 1993). In addition to variation in the onset and peak of spawning, the duration of the spawning season in many fish populations is protracted due to multiple spawning by individuals. In multiple-batch spawners individuals may, as they grow larger and older, produce more batches over a longer period (Trippel et al., 1997; Secor, 2000; Claramunt et al., 2007; Wright and Trippel, 2009). In temperate multiple-batch spawners, the peak and duration of spawning can vary by weeks to months. Spawning duration of young age-classes within a stock may only be as little as half that of older adults (Parrish et al., 1986; Wright and Gibb, 2005; Wright and Trippel, 2009). Lastly, many species demonstrate demographic differences in spawning periods: older, larger fish spawn sooner and often for longer durations than younger fish (Kjesbu et al.,

1996; Wright and Trippel, 2009), presumably increasing the reproductive success of these individuals and the population as a whole. Such differences also appear to exist in largescale tonguesole, as suggested by the comparison in spawning duration between larger (TL 266–330 mm) and smaller adult females (TL 202–266 mm), which revealed that the larger, presumably older fish had a more extended spawning period (Fig. 9). In “asynchronous ovulators”, eggs are recruited and ovulated from the population of yolked oocytes in several batches over a protracted period during each spawning season (Murua and Saborido-Rey, 2003), and our study demonstrated the protracted spawning in different sizeclasses (Fig. 9). The timing of sexual maturity in fishes is a critical component of population dynamics and life history theory (Lowerre-Barbieri et al., 2011). A fish becomes mature after it has passed some fixed size or age threshold (Roff, 1982). However, in a more general representation, fish mature according to a trajectory in the length–age space (Stearns and Koella, 1986; Rijnsdorp, 1993a). Fitting a logistic curve to sex-specific maturity data distributed by size or age is the traditional method of estimating sexual maturity (Hunter and Macewicz, 2003; Lowerre-Barbieri et al., 2011). The percentage of mature female was adequately described by a logistic curve and sexual maturity of C. arel (L50% ) on the coastal waters of Bandar Abbas in Persian Gulf

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was 203 mm. However, in Indian waters Rajaguru (1992) reported that both sexes of C. arel began to mature after the 140–154 size-group and from the 155 to 169 mm sizegroup onwards, percentage occurrence of mature males and females increased steadily. Lm (L50% ) for C. arel was 217 mm for males and 225 mm for females, which was slightly above the value reported here (202.7 mm) for females. In another tonguefish, C. lida, Lm was demonstrated 167 mm for male and 179 mm for females that both species, male mature at a smaller size than females. Ramanathan et al. (1977) have studied the biology of the largescaled tonguesole C. macrolepidotus along the coast of south-east India and indicated the females below 131 mm size group were all immature and the size at first maturity in female may be fixed at about 191 mm. For blackcheek tonguefish, Symphurus plagiusa length at sexual maturity observed in females and males ranged between 80–130 mm TL and 70–110 mm TL, respectively, and length at 50% maturity was 101 mm TL for females and 91 mm TL for males (Terwilliger and Munroe, 1999). The frequency distribution of oocyte diameter and the percentage of different developmental oocyte stages within oocyte cluster corroborates that the C. arel is a batch spawner with asynchronous oocyte development. Analyses in different maturity stages taken from the gonad anterior, middle, and posterior regions indicated no variation in their mean diameter. It is therefore concluded that the development of ovarian eggs proceeds uniformly throughout the ovary. Such an observation has been reported in C. arel and C. lida (Rajaguru, 1992) and also for Indian halibut P. erumei and flounder Pseudorhombus arsius (Ramanathan and Natarajan, 1979). Although previous studies have been done on flatfish biology in the Persian Gulf, these have largely been restricted to systematic surveys without reporting on reproductive characteristics. This study may provide a basis for the future studies on life history parameters, such as growth, size and age at maturity, and histological studies on the reproductive biology of flatfishes in the Persian Gulf. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments We would like to acknowledge the Iranian National Institute for Oceanography and Atmospheric Science (INIOAS) and also we express our gratitude to a significant number of commercial fishermen from Bandar Abbas for their support in collecting and preparing the samples. We would like to express our thanks to Professor Adriaan Rijnsdorp for his helpful comments. References Afonso-Dias, I., Reis, C., Andrade, P., 2005. Reproductive aspects of Microchirus azevia (Risso, 1810) (Pisces: Soleidae) from the south coast of Portugal. Sci. Mar. 69, 275–283. Brown-Peterson, N.J., Wyanski, D.M., Saborido-Rey, F., Macewicz, B.J., Lowerre-Barbieri, S.K., 2011. A standardized terminology for describing reproductive development in fishes. Mar. Coast. Fish. 3, 52–70.

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