Reproductive period of the swimming crab Callinectes danae at the Santa Cruz Channel , a highly productive tropical estuary in Brazil

The aim of this study was to analyze reproductive aspects of Callinectes danae in one of the most productive estuarine systems in the Northeast Brazil, Santa Cruz Channel. A total of 1.573 individuals, being 756 females (23 ovigerous) and 817 males were examined from January to December 2009. Mature males and females, as well as couples of C. danae, occurred in all months of the year. Ovigerous females occurred in nearly all months. The reproductive activity, based on the macroscopic observation of the gonads, occurs continuously, with distinguishable peaks in February, March and September. Ovigerous females were only recorded nearest to the sea, while couples were only in the inner estuary. Thus, a migration pattern is proposed, with couples mating inside the estuary, followed by a migration of females to the area of greater marine influence for egg laying and, probably, larval release. After this, probably the females of do not return to the estuary, characterizing habitat partitioning. The present study is the first contribution on the reproductive period of C. danae in an estuarine ecosystem of the Brazilian northeast coast, and elucidates some aspects of its reproductive behavior.


Introduction
Reproduction is the most important biological phenomenon to ensure the continuity of a species (Cobo and Fransozo, 2000).The determination of the reproductive pattern is of great importance especially for commercially exploited species.It allows the elaboration of conservation mechanisms that Brazilian coast, the genus Callinectes Stimpson 1860 is represented by species confined to shallow waters (Melo, 1996).Besides being a bycatch in many fisheries, especially shrimp trawls, these swimming crabs are also targeted by poor coastal communities that use them for their subsistence and informal commerce of the pre-treated meat (Santos, 1990;Branco and Fracasso, 2004).In southern Brazil, C. sapidus Rathbun, 1896 is the most abundant species of the genus, followed by C. danae Smith, 1869 (Weber and Levy, 2000).However, from Santa Catarina State to the North, C. danae becomes more abundant (Sawaya and Pereira, 1946;Farias, 1980;Medeiros, 1982;Pita et al., 1985a, b).In northeastern Brazil, the species of preference for regional cuisine is C. danae (Calado and Souza, 2003).
The reproduction of C. danae has been studied in some coastal environments of southern and southeastern Brazil (Pita et al., 1985a;Costa and Negreiros-Fransozo, 1998;Chacur et al., 2000;Baptista-Metri et al., 2005).Meanwhile, no study has determined the reproductive period of this species on the largest segment of the Brazilian coast, North/ Northeast.The aim of this study was to analyze some reproductive aspects of Callinectes danae, including the determination of the reproductive period, in one estuarine system of Northeast Brazil.
At the laboratory, the specimens were dissected for the observation of the gonads.Three stages of testes maturation (immature -IM, developing -DG and developed -DD) and four stages of ovarian maturation (immature -IM, developing -DG, ripe -RP, spent -SP) were considered (adapted from Costa andNegreiros-Fransozo, 1998 andKeunecke et al., 2009a).
The percentage of each gonad stage in the total sample was determined.The monthly frequencies of mature individuals (♂ -DG and DD; ♀ -DG, RP, SP) were obtained.The determination of the reproductive period was based on the declining frequency of females with gonads at the RP stage and the increasing frequency of females with gonads at the SP stage (Vazzoler, 1996).
Based on the months in which ovigerous females (DP stage) were found, reproduction was characterized as: seasonal (presence of ovigerous females in only certain months or seasons), continuous (when ovigerous females occur during all months of the year with similar intensity) or seasonal-continuous (with the presence of these females in all months of the year, but with distinguishable peak of high reproductive activity in some months or  (Pinheiro and Fransozo, 2002).The spatial distribution of ovigerous females and couples was analyzed to investigate a possible reproductive migration.

Results
A total of 1.573 individuals of Callinectes danae, being 756 females and 817 males, were examined.The four stages of ovarian maturation of C. danae were observed.The majority of females had IM gonads (72.2%), followed by the DG (23.7%).Females with RP and SP gonads had little representation in the samples (1.9% each one).
Only the RP and SP stages did not occur in all months.Females with SP gonads occurred in February, March, from June to September, November and December, while the presence of females with RP gonads was restricted to January and from June to October (Fig. 2).In February and March, there was a peak of females with SP gonads and no females with RP gonads, indicating the posture of the ovigerous mass.There was a peak of females with RP gonads in July, which decreased in August.In September, there was the second peak of females with SP gonads (Fig. 2), indicating the posture of the ovigerous mass again.However, mature females (DG, RP and SP) occurred in all months of the year.
Considering each season, it can be observed that the higher frequency of mature females occurs in the second half of the summer and winter.
The three stages of testes maturation of C. danae were observed in all months of the year.The majority of males had IM gonads (83%), followed by the DD (11.4%) and DG (5.6%) gonads.Males with DD gonads were in greater numbers from August to December (Fig. 3).
In all sampling months, C. danae couples were observed, with most being captured in guarding position.The male was always visibly larger than the female, which had triangular sealed abdomen and IM gonads.Few couples were caught copulating, when the male gonopods were introduced into the female gonopores.On these occasions, the female had a very soft carapace, semicircular unsealed abdomen, decalcified gonopore and IM gonads.While couples were registered in the upper estuary, the ovigerous females (SP gonads) were collected in lower estuary (Tab.1).

Discussion
The presence of mature males and females in all months of the year, and of ovigerous females in almost all of them, indicates that the reproductive activity occurs continuously.However, the higher frequency of mature and

Callinectes danae Upper estuary Lower estuary
Couples 13 0 Ovigerous females 0 21  (Batoy et al., 1987) and Ovalipes punctatus (De Haan, 1833) at South Africa (Du Preez and McLachlan, 1984) also follow the seasonal-continuous pattern.Continuous or seasonal-continuous reproduction is typically found in species of tropical and subtropical environments (Pinheiro and Fransozo, 2002), as the northeastern coast of Brazil and the SCC, where the temperature remains more or less constant throughout the year.It also ensures a year-round larvae supply, and thus, a continuous recruitment of juveniles to the population (Díaz and Conde, 1989;Negreiros-Fransozo, 2002).
At the Santa Cruz Channel, the recruitment of juveniles into the population occurs year-round, but it is enhanced from March to June and from October to December (Araújo et al., 2012).These pulses of recruitment are probably a result of the reproduction seasonality observed herein.The larvae coming from the ovigerous females of February contribute to the juvenile population from March to June, and the larvae coming from ovigerous females of September to the juveniles from October to December, considering a mean period of 12-15 months from hatching to attaining the mean size of juveniles (Branco and Masunari, 2000).Besides, since the majority of portunids accomplish multiple spawning (Pinheiro and Fransozo, 1999;2002), and like in C. ornatus Ordway, 1863, the recruitment can exhibit several pulses throughout the year (Baptista et al., 2003).
All couples of C. danae were found in the upper estuary and it is probable that mating occurs in this area.While males remain copulating with other females inside the estuary, it is possible that the fertilized females migrate to the sea for spawning.Such pattern indicates a reproductive migration, phenomenon observed by Keunecke et al. (2009b) and Keunecke et al. (2011).Literature has showed that C. danae stores energy in habitats of low salinity, but the female incubate the eggs and releases them in environments with higher salinity (Norse, 1978).These saltier waters facilitate the flotation and dispersal of the larvae.Besides, the osmotic pressure is important for the development of the embryos in C. danae and C. ornatus (Pita et al., 1985b;Mantelatto, 2000;Fernandes et al., 2006), since the larval stages are much less tolerant to salinity variations (Paul, 1982;De Vries et al., 1983).Keunecke et al. (2011) suggested that the females of C. danae return to the estuary after spawning.However, Araújo et al. (2012) observed that males outnumbered females in the upper estuary, while females outnumbered males in the lower estuary at the Santa Cruz Channel.Thus, probably the females of this population do not return to the estuary, characterizing habitat partitioning.
According to Gleeson (1991), the female of C. sapidus Rathbun, 1896 normally copulates only once during their lifetime.This mating occurs immediately after its puberty molt (Hartnoll, 1969;Barreto et al., 2006), which is also its final molt (Van Engel, 1958).The male of C. sapidus is observed guarding a female that is about to molt, holding the female beneath him in a position called "cradle-carry" (Carr, 1988), also observed in the couples of C. danae at SCC. Copulation is usually initiated by the female, when the carapace is soft, the abdomen is semicircular and unsealed, and the gonopore is decalcified, as observed in C. danae in the present study.The female stands beneath the male, with the abdomen extended upward, allowing the insertion of gonopods.After the spermatophores are deposited in the spermathecae, the "cradle-carry" position is restored and maintained until the carapace is hardened.Females store the spermatophores until the full development of the gonads (Subramonian, 1991), since they may copulate while their gonads are still immature, as observed in the present study and by Barreto et al. (2006) in a nearby population.The sperm remains viable for at least a year and is used to fertilize two or more egg laying that the female performs during her lifetime in C. sapidus (Hard, 1942;Van Engel, 1958).
The present study is the first contribution on the reproductive period of C. danae in an estuarine ecosystem of the Brazilian northeast coast, and elucidates some aspects of its reproductive behavior.However, a study on its larval dynamics is needed for a better understanding of the biological cycle of this crab in the region.

Figure 2 .
Figure 2. Callinectes danae.Frequency of females with gonads in the stages of development: DG = developing; INT = intermediary; AD = advanced; DP = depleted, at the Santa Cruz Channel.

Figure 3 .
Figure 3. Callinectes danae.Frequency of males with gonads in the stages of development: DG = developing; DD = developed, at the Santa Cruz Channel.

Table 1 .
Callinectes danae.Number of couples and ovigerous females by sampling area, at the Santa Cruz Channel.