Reproductive cycle of Macrobrachium amazonicum females ( Crustacea , Palaemonidae )

Macrobrachium amazonicum is considered a favorite Brazilian species of freshwater prawn for cultivation as a result of its quick development and because it is easy to maintain in captivity. The aim of this work is to describe the sexual cycle stages and determine maturation age of the female M. amazonicum, which was collected monthly from June, 2002 to May, 2003 in the Jaguaribe River, Itaiçaba, Ceará. A monthly sample of water was also collected to determine the following parameters: temperature, dissolved oxygen, pH and salinity. A monthly sample of females was selected among the individuals caught, to determine the total weight (W T ), carapace length (L C ) and abdomen+telson length (L A+T ) and to register the number of non-ovigerous females (NOF) and ovigerous females (OF). Determining ovarian maturation stages of M. amazonicum was done in a laboratory by observing macroscopic characters such as coloring, size, location and appearance of ovarians examined by transparent carapace. The first maturation age was determined from the relative frequency of the total length (L T ) of young and adult females. The environmental parameters of the Jaguaribe River did not hold any influence in the number of individuals collected. A total of 1,337 prawns were sampled, 513 males (38.4%) and 824 females (61.6%). The proportion between males and females in the studied population was of 1:1.6. Among the collected females, 492 (50.7%) did not carry eggs in their abdomens (NOF) and 332 (40.3%) carried eggs in their abdomens (OF). There was no record of intact females. Non-ovigerous females with mature ovaries were recorded throughout all the months of collection. The female ovaries were classified as immature (IM), rudimentary (RU), intermediary (IN) and mature (M). M. amazonicum females reach their first sexual maturity between 4.5 and 5.5 cm of total length.


Introduction
According to data from the FAO, mentioned by Valenti (2002), worldwide prawn production went from 21,000 to 118,500 t yearly between 1990 and 2000, which corresponds approximately to a 500% growth, and to this we should add roughly 27,000 t of "other prawns and crustaceans" registered by Vietnam, bearing in mind this production refers to M. rosenbergii; as well as M. nipponense, which in China was of the order of 100,000 t in 2000.Therefore, the worldwide production of prawns surpassed 240,000 t in 2000, an amount considered historical since this represents approximately 20% of the total amount manufactured, when taking into account that for a long time it was only 5%.
The term prawn has a very broad meaning.It is used to characterize species whose entire life cycle is restricted to freshwater, as well as to those that need brackish water at the beginning of their development and later freshwater, after metamorphosis (New, 2002).
Prawns are decapod crustaceans belonging to the Suborder Pleocyemata, Infraorder Caridea, Superfamily Palaemonoidea and Family Palaemonidae (Martin and Davis, 2001).Although they are also called prawns, like salt water shrimps, they are considered closer to lobsters, since they share many similarities, mainly regarding their reproductive habits because the prawn females, similar to lobsters, keep their eggs in the abdomen until spawning time (Valenti, 1990).
Most of the prawn species of commercial interest belong to the Macrobrachium Bate, 1868 genus, distributed in the tropical and subtropical regions of the world (Jalihal et al., 1993).According to (Holthuis, 2000) this genus is cincumtropical and indigenous to all continents, except Europe.Currently, there are nearly 210 species of the Macrobrachium genus known in the world (Short, 2004), of which 45 are registered in the Americas and 18 in Brazil (Melo, 2003).
In the early 1970s, researchers from the Oceanography Department of the Federal University of Pernambuco (DOUFPe) began to study three of the most important Brazilian native species: M. acanthurus, M. amazonicum and M. carcinus.Their objective was to develop adequate technology for their cultivation (Coelho and Ramos-Porto, 1985).However, due to importation of post-larvae of M. rosenbergii from Hawaii by DOUFPe in 1977, and the development of its cultivation worldwide, studies related to our species underwent discontinuity (Valenti, 1992;1993).Notwithstanding and despite advances achieved in the cultivation of M. rosenbergii all over the world, it is necessary to study the life cycle, reproduction, growth and habitat of these species once again.
Among the native Brazilian species, M. amazonicum is the selected choice for cultivation, owing to its rapid growth and easy maintenance in captivity.Despite its growth being smaller than other species of the same genus, they do not seem to have the aggressive characteristic of M. acanthurus and M. carcinus, apart from the fact that they are more resistant to illness and predators (Lobão and Rojas, 1991).
M. amazonicum is an indigenous species and vastly distributed in many basins of South America, of the Amazon River, of Northeastern Brazil, of the São Francisco River and of the Paraguay River (Bialetzki et. al., 1997).This species was brought over from the Amazon Bay to the Brazilian Northeastern region in the 1940s and was introduced by the Fishery Services of the National Department of Services Against Droughts (DNOCS) in several dams of the region as forager species for carnivorous fish.Owing to its immediate acclimatization and quick reproduction, it spread to several dams and rivers of the area, thus becoming among the most important fishing resources (Gurgel and Matos, 1984;Paiva and Campos, 1995).
There is little information concerning the population structure and reproduction biology of M. amazonicum collected in state rivers of Ceará.In their papers, Rebouças (2002), Santos (2003) and Silva et al. (2002) determined the morphometric relationships of male and female, female fertilization and fertility, and the morphotypic distinctiveness of the M. amazonicum male collected in the River Jaguaribe, Itaiçaba, Ceará, respectively.
Therefore, it has become necessary to continue studies of this species in order to develop cultivation technology appropriate for the reality of the Brazilian Northeast and thus enable farmers to have alternative species for cultivation.
The objective of this work is to establish and categorize phases of the sexual cycle of M. amazonicum females collected in the Jaguaribe River, Itaiçaba, Ceará Brazil, its annual distribution and its relationship with the abiotic factors and also to determine the first maturation size of M. amazonicum female.

Location of collection
The collection was carried out in the River Jaguaribe, Itaiçaba, Ceará, located above Work Channel dam, at 200 m latitude and 04° 40' 28" S and 37° 49' 21" W.
The Jaguaribe River is 633 km long and is divided into three basins: Jaguaribe High, Medium and Low.In the lower part, where the dam is located, the river runs along 137 km and flows into the Atlantic Ocean in the city of Aracati (Iplance, 1995).

Environmental parameters
The temperature, pH, salinity and dissolved oxygen were determined monthly.The water was collected at 6 m deep with a Van Dorn bottle.
The temperature was determined with a regular thermometer (Incoterm), pH with a pocket pH-meter (Orion, Quikchek) and salinity with a portable refractometer (Alfakit, 211 model).The oxygen dissolved was determined in laboratory using the Winkler method.
The monthly data of pluviometer precipitation were supplied by the Foundation for Meteorology and Water Resources from the State of Ceará (FUNCEME).

Collected material
Specimens of M. amazonicum were collected monthly during the months of June, 2002 andMay, 2003.The prawns were captured with casting nets of 5.0 m diameter and mesh with openings of 20.0 mm between knots.The trap was thrown from a raft into a sandy bottom, always at the beginning of the morning for one hour.After collecting, the prawns were transported alive to the Biological Science Laboratory in the State University of Ceará (LABIO/UECE).
A monthly sample of up to 150 individuals of M. amazonicum of both sexes was selected randomly, regardless of the number of prawns collected.In this paper, only females were used, the males were separated for posterior studies.
The females were divided into three groups, according to their reproductive stage.The first group was composed of non-ovigerous females (NOF), the ones that had an abdominal incubator cavity, showing traces of previous spawns; the second group was of ovigerous females (OF), those with pleopods carrying eggs and the third group was of intact females (IF), those that did not have an abdominal cavity.

Morphometric data
For each of the three female groups, the total weight (W T ); carapace length (L C ) and abdomen+telson length (L A+T ) was determined.The total length (L T ) is the sum of the last ones added.
The length measures were obtained using a steel caliper ruler with precision of 0.05 mm (Mitutoyo, caliber 150 mm x 6").The total weight was determined by a semi-analytic scale with a precision of 0.1 g (Gehaka Model BG 2000).

Ovarian maturation stages
Determining the ovarian maturation stages of M. amazonicum was done by observing macroscopic characters such as coloring, size, location and appearance of the ovary observed by transparent carapace according to criteria established by Porto (1998) for M. amazonicum and adapted by Carvalho and Pereira (1981) and Carvalho-Pinheiro (1983) for M. acanthurus.

Size of first maturation
According to Vazzoler (1982), the first sexual maturation is reached at the time when 50% of the individuals of a population go into gonadic activity at maturation stage.To determine the first maturation size, the total length monthly data of females were used, considering two groups: 1) young females: those with immature gonads and 2) adult females: those with gonads in maturation, mature and empty, meaning, all that are not immature.Afterwards, the relative frequency of immature and mature females was obtained for each length class.
Data relating to the total length of each of the two categories referred to above were plotted in the graph and X was the total length and Y the relative frequency of females.The intersection point of the two curves indicated in which total length class the average size of the females in their first maturation is located.

Environmental parameters
During monthly collection, the water temperature varied between 29.0 ± 1.13 °C; dissolved oxygen between 5.9 ± 0.66 mg.L -1 and pH between 7.9 ± 0.37.Monthly salinity throughout the entire period was equal to 0 ppt.It was impossible to collect water in July; therefore the environmental parameters were not determined in this month (Table 1).5.9 ± 0.66 7.9 ± 0.37
Absolute frequency of M. amazonicum females was higher than males in nine of the twelve months of collection, with marked decrease of their number in the period between November, 2002 and January, 2003 (Figure 1).

Reproductive cycle
Among the collected females, 332 (40.3%) carried eggs in their abdomen and 492 (50.7%) did not.No intact females were recorded.
Ovigerous M. amazonicum females were found during the entire year.However, recurrence of non-ovigerous females was higher than ovigerous in ten of the twelve months of collection, at which time they markedly reduced in number in the period between November/2002 and January/2003 (Figure 2).

Ovarian maturation stages
It was possible to detect four ovarian development stages: Immature (IM), Rudimentary (RU), Intermediary (IN) and Mature (M) for M. amazonicum females captured in the Jaguaribe River, Itaiçaba, Ceará.
Females belonging to this species were found in all stages of ovarian development, ovigerous as well as non-ovigerous.The relative frequency of non-ovigerous females in mature stages has always been higher than ovigerous females in the same stage.On the other hand, the relative frequency of ovigerous females in an immature stage was higher than non-ovigerous females in the same stage, although mature females were found throughout the entire year, even at low frequencies (Figures 3 and 4, respectively).

Size of first maturation
To determine the size of first maturation, the total lengths of 768 M. amazonicum females were used, 271 were young and 479 were adult.
Considering that 50% of the non-ovigerous and ovigerous M. amazonicum females showed mature gonads in the total length classes situated between 4.5-5.0 and 5.0 and 5.5 cm, it could be suggested that the first gonad maturation of this species occurs between these classes (Figure 5).

Environmental parameters
According to Zimmermann (1998), the shrimps show physiological response to physical and chemical factors that are most important in their habitat.In a cultivation  nursery, the water temperature and the dissolved oxygen dominate the behavior and the physiology of most of the aquatic organisms.While the temperature controls the molecular dynamic and most of the chemical reactions, the dissolved oxygen acts as the main limitation factor in the biochemical reactions controlled by the temperature.
According to Sipaúba-Tavares (1998), the temperature, dissolved oxygen, pH and water salinity often interfere in the reproduction, distribution and periodicity of organisms.Valenti (1996) and Pinheiro and Hebling (1998) reported that in natural environments, the reproduction of M. rosenbergii is usually continuous throughout the whole year.However, this presents higher density in the months when the environmental factors are more favorable to the gonadal development and to offspring survival.Mossolin and Bueno (2002) report in a study also carried out with M. olfersi from São Sebastião, São Paulo, Brazil, in which the periods of more reproductive activity coincide with the months when the temperature was higher, on the other hand, with opposite occurrences in the colder months.
According to Bond and Buckup (1982), M. potiuna and M. borelli reproductive cycles are more strongly stimulated by seasonal light variation than by thermal water fluctuations.These authors also inferred that in equatorial regions and even in low latitudes, a light influence is less significant due to the small variation of the length of the day.
The physical-chemical parameters that were analyzed did not have an influence in the quantity of collected organisms in the natural population of M. amazonicum in the Jagaribe River, Itaiçaba, Ceará, because, although the number of males, non-ovigerous and ovigerous females have varied quite considerably throughout the collection period, the physical-chemical parameters present little variation since they were in the zone suggested by New (2002) for the tropical species of this genus.
Owing to the fact that the location is in an equatorial region, the water temperature was always high, perhaps favoring the occurrence of ovigerous females in all months.It is worth mentioning that the values observed are found within the ideal zone referred by Sampaio and Valenti (1996) for M. rosenbergii, which are cultivated in ponds.
Ours data is compatible with work by Biswas and Qureshi (1994), who observed that the natural population of M. dayanum shows seasonal variations of density.However, these authors did not find any straight connection of the population density variation to the temperature, pH, dissolved oxygen or free CO 2 .
Another exogenous factor that can have an effect in the reproductive cycle of M. amazonicum is the pluviometric configuration.Bragagnoli and Grotta (1995) observed that the highest percentage of ovigerous M. amazonicum females occurred in the months with the highest pluviometric precipitation, while Gamba (1984) studying M. amazonicum and M. jelskii reproduction also observed that the highest number of ovigerous females of this species was collected at the beginning of the rain season.On the other hand, Lobão et al. (1978) demonstrated an inversed relation between mature M. holthuisi females in pluviometric precipitation.
The non existence of any relationship between pluviometric precipitation in the region of the Jaguaribe River and the occurrence of ovigerous females is reinforced by the observations made by Sagi and Ra'anan (1988), who in their studies affirm there is no relationship between the reproductive stages of M. rosenbergii and the seasonal changes.

Population structure
The proportion between males and females in the natural population of M. amazonicum collected in the Jaguaribe River, Ceará was quite similar to that found by Relative frequency (%) Young females Adult females Porto (1998) for the same species collected in the Meia Ponte River, Goiás, with the females predominating over the males.However, Silva et al. (2002) studying the same species collected in the State of Pará found the contrary, that is, males predominating over females.
The sexual proportion changed between the Macrobrachium species, so Barros (1995) and Ammar et al. (2001) studying the M. olfersii population of the coast of Santa Catarina and Mossolin and Bueno (2002) studying samples of the same species collected in the coastal waters of the State of São Paulo found a predominance of males over females at a proportion of 1: 0.53; 1: 0.71 and 1: 0.23, respectively.Antunes and Oshiro (2004) found for M. potiuna, collected in the coast of the State of Rio de Janeiro, Brazil, a ratio of 1: 0.94 between males and females, higher than M. olfersii.On the other hand, Mantelatto and Barbosa (2005) discovered the opposite for M. brasiliense collected in Serra Azul, São Paulo, that is, females predominated over males in a proportion of 1:2.6.

Reproductive cycle
Pinheiro and Hebling (1998) assert that the reproductive period of freshwater decapods crustacean is closely associated to the rainy period, to thermal variations and to the photoperiod of the region where it happens.
The occurrence of ovigerous M. amazonicum females of the Jaguaribe River during the collection period indicates that this species is capable of reproducing the entire year, having a continuous reproduction.This observation is similar to the one by Chaves and Magalhães (1993); Bragagnoli and Grotta (1995); Bialetzki et al. (1997); Porto (1998) and Silva et al. (2002) for samples of the same species collected in various regions of Brazil.
The predominance of non-ovigerous females during most of the year of collection may be related to a difficulty in catching ovigerous females, since during the reproductive phase they migrate to estuary regions where egg incubation occurs or they look for shelter in protected areas, that is, in marginal plants to avoid predation pressure (Ismael and New, 2000).
Chaves and Magalhães (1993); Porto (1998) and Silva et al. (2002) observed that ovigerous M. amazonicum females are more abundant in the rainy season than at other times of the year.However, in the Jaguaribe River the occurrence of ovigerous females did not present any relation with the pluviometric configuration, perhaps due to the fact that the State of Ceará is located in a semi-arid region, which probably enabled the species to find a reproductive alternative.
That region of catching is characterized for having only two climatic seasons: the rainy one, whose pluviometric precipitations vary between 400 and 800 mm and dry.Rains that occur during the rainy season are concentrated in a short period that extend from February to May, the so-called "square winterly".During the dry season, that it extends from June to January; the pluviometric precipitations are placed below the average.These months are considered anomalous, therefore as they are situated in the extremities of the dry season, they can also include the rainy season.

Ovarian maturation stages
The four different phases of ovarian development for M. amazonicum females defined in this paper were very similar to the ones described by Porto (1998).
The occurrence of M. amazonicum females with mature ovaries within the group of non-ovigerous during the collection reinforces observations already made by Odinetz Collart (1991a); Bragagnoli and Grotta (1995); Bialetzki et al. (1997) and Porto (1998) that this species is characterized for presenting continuous reproductive activity.
Within the ovigerous group, a higher frequency of females with immature ovaries is expected as they are found in the middle of their gonadal development.However, ovigerous females with mature ovaries were found, although with a low frequency, which indicates that even carrying eggs, the M. amazonicum females immediately after spawning, can initiate a new reproductive cycle, again reinforcing that this species has continuous reproduction.According to Odinetz Collart (1991a), it is possible to find females with their ovaries in different stages of maturation, regardless of the season of the year.

Size of first maturation
In the State of Pará, Silva et al. (2002) found ovigerous M. amazonicum females with total lengths of 3.3 cm, a smaller size than the ovigerous females collected in the Jaguaribe River, Ceará.This fact could be related to the type of trap used, because while in the North region the prawns were captured by means of a "puçá" of 17 cm, in the region studied they were captured by means of a casting net of 20.0 mm.This could possibly explain the difference of the average size of the females of first maturation captured in the Jaguaribe River, approximately 5.0 cm, and the size of the smallest ovigerous females collected in Vigia, Pará by Silva et al. (2002).
Unfortunately, our results cannot be compared to those of Porto (1998), even though he has worked with M. amazonicum females, he used the relative frequency of carapace length to determine the age of the first maturation and not the total length, as suggested by Vazzoler (1982).

Conclusions
The physical-chemical parameters and pluviosity did not affect the population structure and reproduction of M. amazonicum.The occurrence of ovigerous and nonovigerous females with mature ovaries throughout the entire year reinforces the fact that this species possesses a type of continuous reproduction.
who provided helpful comments and suggestions for this manuscript.

Figure 3 .
Figure 3. Monthly relative frequency distribution of maturation ovarian stages for non-ovigerous females of M. amazonicum

Figure 4 .
Figure 4. Monthly relative frequency distribution of maturation ovarian stages for ovigerous females of M. amazonicum.

Figure 5 .
Figure 5. Size of first maturation for M. amazonicum females.