By R. Sa nchez-ca rdenas 1, M. Arellano-Martı nez 1, M. C. Valdez-Pineda 2, R. E. Mora n-angulo 2 and B. P. Ceballos-Va zquez 1

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1 Journal of Applied Ichthyology J. Appl. Ichthyol. (2011), 1 7 Ó 2011 Blackwell Verlag, Berlin ISSN Received: July 8, 2010 Accepted: November 11, 2010 doi: /j x Reproductive cycle and sexual maturity of Sphoeroides annulatus (Jenyns, 1842) (Tetraodontiformes, Tetraodontidae) from the coast of Mazatlan, Sinaloa, Mexico By R. Sa nchez-ca rdenas 1, M. Arellano-Martı nez 1, M. C. Valdez-Pineda 2, R. E. Mora n-angulo 2 and B. P. Ceballos-Va zquez 1 1 Centro Interdisciplinario de Ciencias Marinas, Instituto Polite cnico Nacional, Playa Palo de Santa Rita, La Paz, B.C.S., Me xico; 2 Facultad de Ciencias del Mar, Universidad Auto noma de Sinaloa, Mazatla n, Sinaloa, Me xico Summary The reproduction of any fish species may be influenced by environmental factors, knowledge of which is required for an adequate control of the reproductive process to improve culture practices. Thus, the reproduction of a wild population of bullseye puffer, Sphoeroides annulatus (Jenyns, 1842), and the influence of temperature, photoperiod, lunar cycle and tide level were analyzed. Ovarian ripeness is asynchronous, and the ovary may ripen again at least once following spawning. Testes also display an asynchronous ripeness, but once sexual maturity is attained, spermatozoa are continually produced and released. The reproduction is highly seasonal, with an intense spawning period during the spring-summer, when the sea surface temperature is C and a h photoperiod. The observations suggest that the timing of spawning is synchronized by a semi-lunar cycle together with the rise of the average tide level. Size at first maturation was similar for females (28.2 cm TL) and males (28.6 cm TL). However, some specimens may start their gonad maturation when are as small as 19 cm TL. Introduction The bullseye puffer Sphoeroides annulatus (Jenyns, 1842) is a rising fishery resource with high commercial value (Sánchez- Cárdenas et al., 2007) and is a potential marine species for aquaculture on the Pacific coast of Mexico. Significant advances have been made on its reproduction in captivity (Duncan et al., 2003) and the rearing of larvae with live food organisms (Garcı a-ortega et al., 2003). However, studies on the reproductive biology of wild populations of S. annulatus are rather limited. In Mexico, Castellanos-Rodrı guez et al. (1982) conducted a macroscopic monitoring of gonad ripeness of S. annulatus and, more recently, Sa nchez-ca rdenas et al. (2007) determined a morphochromatic scale with five ovarian stages (resting, early developing, late developing, ripe and spent) and four testicular stages (resting, developing, ripe and spent). Some investigations have been conducted on gonad ripening in wild populations of the genus Sphoeroides from the coast of Parana, Brazil (Rocha et al., 2002; Schultz et al., 2002). Available information suggests a population sex composition determined as one male per female in Sphoeroides and other tetraodontid species (Table 1). Moreover, the temporality of reproduction is highly variable, ranging from seasonal (with one or two annual reproductive periods) to year-round spawning (Table 1). Accurate identification of the reproductive season and a thorough knowledge of spawning strategy are required for adequate control of the reproductive process to improve culture practices for any fish species (Carrillo et al., 1995; Cerqueira, 2002), together with knowledge of the influence of environmental factors on them. Due to the growing importance of S. annulatus as a fishery resource and as a culture species, this study was conducted to characterize the basic reproductive biology of wild bullseye puffer using histological approaches and analyzing the influence of some environmental factors. It is expected to be useful as a reference in the implementation of its fishery management, in the culture protocols and in other biological research. Materials and methods Fishermen from the Playa Norte fishing camp working at Mazatlan, Sinaloa, Mexico (23.17 N W to N W) (Fig. 1) provided weekly samples from January 2004 to August Capture was diurnal using a hand line or fishing rods with hook. Total length (L t, cm) and total mass (M t, g) were recorded for each specimen. The gonad was removed, its mass (g) recorded, and then fixed in 10% formaldehyde. Samples of gonad tissue were sectioned for histological processing, which consisted of dehydration, inclusion, sectioning (5-lm thick), and staining with hematoxylin eosin (Humason, 1979). The observations by Wallace and Selman (1981) and Tyler and Sumpter (1996) were considered for the description of the ovarian ripeness process. The testicular ripeness process was based on the work by Grier and Taylor (1998). Changes in the internal arrangement of the gonad associated with gametogenesis were categorized in development phases. Seasonal presence of these development phases was described through the relative monthly frequency. Additionally, oocyte diameter was measured for different growth phases using digitalized images and SCAN PRO software (Version 5.0). As an attempt to determine which environmental factors influence the reproductive season, specifically as a spawning trigger, the frequencies of ripe, spawning and spent stages by sampling date were analyzed. Sea surface temperature was obtained from monthly average images of the Aqua-Modis satellite, through the website Photoperiod, percentage of illumination of the moon and average tide level were calculated with MAR V0.7 software. Lastly, the size at first maturity L 50 (length at which 50% of the specimens have reached sexual maturity) was calculated,

2 2 R. Sánchez-Cárdenas et al. Table 1 Size at maturity and reproductive period of Tetraodontidae species around the world Species Sex ratio Size at maturity (cm) Reproductive period Locality References Canthigaster valentini 1 : SL Year-round Off Lizard Island, Australia Gladstone and Westoby (1988) Takifugu niphobles May August Tomioka bay, Japan Yamahira (1994) Sphoeroides greeleyi 1 : TL August January Paranaguá bay, Brazil Schultz et al. (2002) Sphoeroides testudineus 1 : TL September January Paranaguá bay, Brazil Rocha et al. (2002) Sphoeroides annulatus 1 : 1 April May, October November Off Mazatla n, México Sa nchez-cárdenas et al. (2007) Sphoeroides annulatus 28.2 TL April July Off Mazatla n, México Present study SL, standard length; TL, total length. Table 2 Morphometric variables (mean ± error standard) by sex of Sphoeroides annulatus. Data analyzed by one-way ANOVA at 0.05 significance level Female Male ANOVA, P Total weight (g) ± ± Total length (cm) 29.5 ± ± Standard length (cm) 23.7 ± ± Fig. 1. Sites of Sphoeroides annulatus caught by coastal fishery landings at Playa Norte fishing camp (marked with O), at Mazatlan, Sinaloa, Mexico. Weekly sampling January 2004 August 2005 Sexuality Sphoeroides annulatus appears to be a species with no sexual dimorphism in body shape or colour. Morphologically, both ovaries and testes consist of two lobes located in the peritoneal cavity and suspended by a mesentery, mesovaria and mesorchia, respectively, connected to the urogenital pore through a duct (oviduct or spermiduct, respectively). In the ovarian stroma, ovigerous lamellae are arranged concentrically towards the centre. In testes, seminiferous ducts are arranged longitudinally and become compacted when not storing and or releasing spermatozoa. The collecting duct or ejaculatory canal runs dorsally along each lobe and opens at the union between both lobes. considering organisms undergoing vitellogenesis, vitelline granule and final maturation. Specimens in the resting phase were excluded, as there was no certainty whether they were sexually mature. The punctual value of the size at first maturity by sex was calculated by fitting the accumulated relative frequency per 3-cm length interval to a logistic function. Once the model for each sex was obtained, it was optimized through an iterative technique using the Excel Solver tool. The size at first maturity (punctual length for 50%) was calculated using the optimized parameters. Results Sex ratio A total of 715 specimens were sampled. Overall, 318 (44.5%) were females measuring between 19.5 and 44 cm TL and g in total mass; 397 (55.5%) were males between 20 and 42 cm TL and g in total mass. The overall sex ratio was 1M : 0.8F, with no significant differences relative to the hypothetical 1M : 1F ratio (v 2 = 0.05, P = 0.05).When morphometric variables were compared between females and males no significant differences were obtained (Table 2). Gonad ripeness Histologically, seven phases of oogenesis (chromatin nucleolus, perinucleolus, cortical alveoli, lipid vesicle, yolk-granules, migrating-nucleus, and yolk-fusion) were identified (Table 3). On the other hand, six ovary ripeness phases [resting, early development, late development, ripening, spawning (Fig. 2) and spent] were characterized, whereas testis ripeness involved four phases (resting, development, spawning, and spent) (Table 4). In the resting stage, only oocytes undergoing the primary growth phase are present. However, their frequencies decrease whereas the oocytes undergoing the secondary growth phase appear sequentially accordingly with the increment in ovary ripeness. For instance, ripe ovaries mainly show oocytes in the migrating-nucleus stage and or yolk-fusion stage. In a similar manner, in resting males spermatogonia and scarce spermatocytes prevail, and the number of spermatocytes and the accumulation of spermatozoa increases with the advance in testis ripeness. Additionally, one ovary undergoing generalized reabsorption was observed in August Microscopical analysis revealed that the reproductive activity was arrested in the ripening phase, with generalized atresia of oocytes in late

3 Reproduction & maturity of S. annulatus 3 Table 3 Oocyte growth phases of Sphoeroides annulatus by histological analysis of ovary sections stained with haematoxilin eosin Stage Histological description Previtellogenesis Primary growth Chromatin Oocytes measuring ± 1.53 lm (mean ± standard error) in diameter, wrapped in a thin layer of follicular cells and nucleolus with a smooth dense ooplasm. The nucleus is located towards the center and includes a larger nucleolus relative to the other phases Perinucleolus Oocytes measure 67.5 ± 1.43 lm, wrapped in a thin layer of follicular cells. Ooplasm density decreases as size increase and acquires a lumpy appearance; the nucleus is located to the oocyte center, displaying multiple nucleoli (n > 10) arranged towards the periphery Cortical alveoli Oocytes with a diameter of ± 1.54 lm, wrapped in thin layers: follicular cells (theca and granulose cells) and the radiata zone. These oocytes contain cortical alveolus of an increasing size. The nucleus remains at the center, with aggregated chromatin and nucleoli arranged towards the periphery Vitellogenesis Secondary growth Lipid vesicle Yolk-granules Migrating-nucleus Yolk-fusion Oocytes with a diameter of 162 ± 2.29 lm, wrapped in follicular cells and the radiate zone which displays an increasing thickness. Lipid droplets fuse forming larger vesicles. Small yolk droplets are observed in the ooplasm. The nucleus contains a lower number of nucleoli compared with previous phases Oocyte diameter is ± 2.03 lm, wrapped in a layer of follicle cells and the radiate zone reaching its maximum thickness. Ooplasm is completely filled with yolk granules of different sizes Oocyte diameter is 481 ± 4.41 lm. The nucleus leaves its central location and starts migrating towards the animal pole. The yolk granules of various sizes appear clustered around the nucleus, seemingly merging together The yolk fusion is apparent in the ooplasm. The nucleus and its components are not evident (SST, C; Php, h) In October November 2004 (SST, C; Php, h) a slight reproductive activity was found with females in late development phase (27.3%) and males in development and spawning phases (8.8 and 8.4%, respectively). Factors influencing reproductive season Spawning organisms were observed at intervals of approximately 30 days (Fig. 6). In general, spawning coincided with an increase in the average tide level. Spawning occurred between 1 and 4 days before the full moon or new moon. However, it was not a clear relationship to spawning, either with percentage of illumination of the moon or the photoperiod. On 22 July 2005, 1 day after a full moon, the spawning phase coincided with a decrease in the average tide level. Fig. 2. Photomicrograph illustrating spawning phase in ovary of Sphoeroides annulatus. Note presence of oocytes in vitellogenesis phase (VO) along with post-ovulatory follicles (POF). Staining technique haematoxylin eosin. Scale bar = 100 lm vitellogenesis and no evidence of previous spawning observed (Fig. 3). According to the described ovarian ripeness phases and its temporal variation, the ovarian cycle of S. annulatus follows the sequence schematized in Fig. 4. Reproductive cycle Figure 5 illustrates the reproductive cycle of females and males in relation to sea surface temperature (SST) and photoperiod (Php). Phases with no gametogenic activity (resting and spent) were recorded in practically all months; however it was possible to identify a resting season from July to November 2004 and August 2005, where both phases represented a relative frequency between 72 and 100%. Phases with advanced gametogenic activity display a clear seasonality. Organisms (either females or males) in late development, ripe and spawning stages were observed mainly from April to July (SST, C; Php, h), and from March to July Size at first maturity (L 50 ) Mature females were observed at cm TL and mature males at cm TL. It was estimated that 50% of females display sexual activity at 28.2 cm TL, and that 50% of males are sexually active upon reaching 28.6 cm TL (Fig. 7). Adults were thus defined as those individuals at or exceeding the L 50 size and at least undergoing vitellogenesis. Estimated L 50 sizes for females and males respectively corresponded to 64 and 65% maximum size (44 cm) observed in the present study. Discussion Sphoeroides annulatus displays a bigeneric sexuality, both genera with the same morphology and coloration, each producing either oocytes (females) or spermatozoa (males), thus evidencing that it is a monomorphic and monochromatic species. Gonad characterization in S. annulatus matches the description of cystovarian ovaries (Jameson, 1988; Helfman et al., 1997; Grier, 2000) and lobular testes (Billard, 1986; Nagahama, 1994; Grier and Taylor, 1998), without any features of hermaphroditic gonads (see: Sadovy and Shapiro, 1987). The general pattern of oogenesis in S. annulatus follows the cell differentiation according to the oocyte growth phases:

4 4 R. Sánchez-Cárdenas et al. Table 4 Gonad ripeness phases of Sphoeroides annulatus by sex via histological analysis of ovary and testis sections stained with haematoxilin eosin Histological description Ripeness phase Resting Development Ripe Spawning Spent Female Oocytes undergoing the primary growth phase prevail. Oocytes in the nucleolar chromatine stage are found closer to the lamellar wall whereas perinucleolar stage oocytes are found in a central position Early development Oocytes in the cortical alveolus stage are predominant although oocytes in vitellogenesis phase are being increasing in quantity as the development advance. Primary growth oocytes are still present, arranged closer to the lamellar wall Late development Oocytes in the yolk-granule stage fill up a great portion of the ovarian stroma, although oocytes in previous phases are also apparent Ripe ovaries show oocytes in migrating-nucleus stage and or yolk-fusion stage depending on the advance in ripening grade. Primary growth oocytes are still present in low quantity and they are less evident The distinctive characteristic of this stage was the presence of postovulatory follicles together with oocytes in the ripening phase. In some ovaries, the presence of oocytes in the cortical alveolus and or vitellogenesis phases in addition with the post-ovulatory follicles (undergoing reabsorption) was evident (Fig. 2) Ovarian reabsorption and reorganization takes place in this phase. Early, after spawning, atretic oocytes are evident along with resorbing post-ovulatory follicles. Late, after spawning, primary growth oocytes in addition to evident connective tissue and only atresias of ripening oocytes are present Male Spermatogonia and scarce spermatocytes are attached to spermatogenic tubules wall. Spermatogenic tubules appear semi-compact, with a narrow lumen and in general are inactive. The collecting tubules may contain residual spermatozoa Active spermatogenic activity into the spermatogenic tubules. Substantial spermatocyte clusters are present in addition to spermatids. Increasing quantity of spermatozoa in the lumen of spermatogenic tubules according with the advance of ripeness A traditional ripe phase was not characterized since spawning start before a total fullness of testis occurs Spermatozoa release is evidenced by the gradual emptying of spermatogenic tubules. The collecting tubules are full of spermatozoa. Spermatocyte and spermatid proliferation persists, and spermatogenic activity is observed to the periphery even when stored spermatozoa are observed in the collecting tubules, ready to be released Spermatogenic activity is scarce. Spermatogenic tubules are not compact as in the resting phase and contain residual spermatozoa Fig. 4. Ovarian cycle of Sphoeroides annulatus. Sequence of ovarian ripeness phases described as affected by environmental conditions at Mazatlan, Sinaloa, Mexico Fig. 3. Photomicrograph illustrating ripe ovary of Sphoeroides annulatus undergoing generalized reabsorption. AT: atresia of oocytes in late vitellogenesis. Staining technique haematoxylin eosin. Scale bar = 100 lm early growth, cortical alveoli, vitellogenesis and final maturation, as in S. testudineus and S. greeleyi (Wallace and Selman, 1981; Tyler and Sumpter, 1996; Rocha et al., 2002; Schultz et al., 2002). Unlike the morphochromatic classification of Sa nchez-ca rdenas et al. (2007), our histological classification of ovarian and testicular ripeness of S. annulatus allows differentiation of the spawning phase. On the other hand, the presence of postovulatory follicles along with vitellogenic oocytes confirms that this species is a partial spawner, coinciding with the reports for S. testudineus (Rocha et al., 2002) and S. greeleyi (Schultz et al., 2002). During the ovarian ripeness of S. annulatus, the presence of oocyte cohorts undergoing different growth stages was characteristic; this is known as asynchronous ripeness (Scott, 1987; Tyler and Sumpter, 1996), which characterizes species with multiple spawnings and an extended reproductive period (Mun oz-cueto et al., 1997). Furthermore, the presence of

5 Reproduction & maturity of S. annulatus 5 Fig. 5. Monthly changes in relative abundance of ripeness phases of Sphoeroides annulatus by sex, reproductive cycle. Temporal variation in water temperature (broken line) and photoperiod (solid line) throughout the study at Mazatlan, Sinaloa, Mexico also shown Fig. 6. Temporal relation between environmental factors (photoperiod, percentage of moon illumination and average tide level) and reproductive season (represented as ripe, spawning and spent phases), in 2004 (upper graph) and 2005 (lower graph) Fig. 7. Length at first maturity of Sphoeroides annulatus from coasts of Mazatlan, Sinaloa, Mexico. Logistic models (optimized through an iterative technique using the Excel Solver tool) by sex and their corresponding correlation coefficients are shown oocytes in the cortical alveolus and or vitellogenesis phases, in addition to postovulatory follicles undergoing reabsorption (Fig. 2), may indicate that a new ripening is possible, enabling a S. annulatus female to spawning at least twice in a single reproductive season. In the case of males, an active spermatogenic development was observed even in spawning testis. This feature makes us think that males actually have a continuous sperm production during the reproductive season, enabling them to fertilize oocytes more than once. This same pattern has also been described for Centropomus undecimalis (Grier and Taylor, 1998). Follicular atresia or reabsorption is a natural event, and it was reported that from 75 to 99% of the follicles affected by atresia, over all stages of ovary ripeness, are a product of programmed cell death (Tilly et al., 1991). However, when the reabsorption is generalized in a ripe ovary such as that observed in S. annulatus, it is associated with adverse environmental conditions that affect reproductive success (Luksˇ iene et al., 2000). Reabsorption was observed in August 2004, the month following the reproductive period; this may be the reaction of mature individuals to unsuitable environmental conditions for reproduction of the species at the begin of the non-reproductive period.

6 6 R. Sánchez-Cárdenas et al. The reproduction of S. annulatus is seasonal and its pattern of occurrence shows a clear reproductive period, which coincides with sea surface temperature values from 24.7 to 30.4 C and with photoperiod (light hours) from to h. In contrast, two reproductive periods in an annual cycle were described for S. annulatus but using less precise methods such as visual maturity scale and monthly variation of mean gonadosomatic index values (Sa nchez-ca rdenas et al., 2007). In 2005 the reproductive period began a month earlier (in March) than in 2004 (April). The onset of the reproductive period was not explained by sea surface temperature because their values were not consistent in both periods. In this case, the begin of the breeding season, marked by the accumulation of yolk in oocytes, could be explained by other factors such as food availability, or any other biological factor or activity that compromises the nutritional status of the fish, which seems most directly related to the accumulation of yolk. Nevertheless, the timing of spawning was synchronized with the semi-lunar cycle, around each new moon and full moon, and with a rise in the average tide level. Although on 22 July 2005 the spawning phase coincided with a drop in the average tide level, it is believed that spawning occurred during the rise of the average tide level, because the postovulatory follicles were in an advanced state of reabsorption, so that the spawning could have occurred days earlier. The semi-lunar spawning cycle seems to be adaptive from the viewpoint of larval hatching success (Yamahira, 1997) and occurs commonly among fish species that produce benthic eggs (Thresher, 1984) such as S. annulatus (Komar et al., 2004). The same spawning timing was reported for Takifugu nipholes (Yamahira, 1994). Finally, thus far no data is available concerning size at first maturity of S. annulatus. Our results indicated that individuals of this species attain their first maturity at 28.2 cm total length for females and 28.6 cm TL for males, when they had already attained 64 65% of the maximum observed total length. S. testudineus and S. greeleyi specimens reach size at first maturation when they attain only 28 and 39% of their respective maximum total lengths (Rocha et al., 2002; Schultz et al., 2002), thus it is probable that the size at first maturation of S. annulatus was overestimated as a result of the narrow size range analyzed. However, intra- and inter-specific variations in size at first maturation derived from genetic, environmental, and ethological factors, among others are common (Rodríguez et al., 2001). Acknowledgements This research was funded by projects SIP , , y of Instituto Polite cnico Nacional. The authors deeply thank the fishermen from Playa Norte for their committed efforts to attain the objective of this investigation. We are also thankful to A. Inzunza-Rojas, J. M. Sa nchez- Caniza lez, L. A. Salcido-Guevara, A. Sa nchez and V. Rios for their support during sampling. R. Sa nchez-ca rdenas is a fellow student of PIFI (IPN) and CONACyT, and the results presented here are part of her M Sc thesis. B.P. Ceballos- Vázquez and M. Arellano-Martı nez received grants from SIBE (COFAA), EDI (IPN), and SNI-CONACYT. Thanks to Marı a Elena Sa nchez-salazar, M. Sc., for her editorial contribution to the English manuscript. 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7 Reproduction & maturity of S. annulatus 7 Thresher, R., 1984: Reproduction in reef fishes. T. F. H. Publications, New Jersey. 399 pp. Tilly, J. L.; Kowalski, K. I.; Johnson, A. L.; Hsueh, A. J. W., 1991: Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129, Tyler, C. R.; Sumpter, J. P., 1996: Oocyte growth and development in teleost. Rev. Fish Biol. Fish. 6, Wallace, R. A.; Selman, K., 1981: Cellular and dynamic aspects of oocyte growth in teleost. Am. Zool. 21, Yamahira, K., 1994: Combined effects of tidal and diurnal cycles on spawning of the puffer, Takifugu nipholes (Tetraodontidae). Environ. Biol. Fishes 40, Yamahira, K., 1997: Hatching success affects the timing of spawning by the intertidally spawning puffer Takifugu nipholes. Mar. Ecol. Prog. Ser. 155, AuthorÕs address: Bertha P. Ceballos-Vázquez, Centro Interdisciplinario de Ciencias Marinas, Instituto Polite cnico Nacional. Av. Instituto Polite cnico Nacional s n, Playa Palo de Santa Rita, Apartado Postal 592, La Paz, B.C.S., 23096, México. bceballo@ipn.mx