Morphology of the first larval stage of Macrobrachium brasiliense (Heller, 1868) (Caridea: Palaemonidae)

In this paper, we describe and illustrate the morphology of the first larval stage of the prawn Macrobrachium brasiliense. Two ovigerous females were obtained in a stream environment, which belongs to Paraná River Basin, Southeastern of Brazil, and were maintained in laboratory until the time of hatching. The newly-hatched larva bears very advance morphological features, with benthic habits. They had sessile eyes and all appendages, except for the uropods; however, most of the appendages were not fully formed. The description given here is compared with the first larval stage of Macrobrachium species with abbreviated larval development from other localities.

According to García-Dávila and Magalhães (2003), the identification of M. brasiliense is performed based on the examination of adults, preferably the larger males.In this manner, the knowledge about the larval morphology of this species has a fundamental importance for the understanding of their biology, as well as for systematic and taxonomic studies.In this paper, the morphological description and illustrations of the first larval stage of M. brasiliense are provided, and comparisons are made between the first larval stage of this species from another locality (Vega-Pérez, 1984) and between the first larval stage of other species of the genus.

Material and Methods
In December 2009, two ovigerous females were obtained in a stream environment known as "Talhadinho" by locals due to proximity with the city of Talhados, São Paulo State, Brazil (20°47'07"S -49°20'35"W).This stream belongs to the Paraná River Basin, Southeastern of Brazil.The collectors used a sieve (2 mm mesh diameter) near the margins (where there was some aquatic vegetation).The ovigerous females obtained were transported alive to the laboratory (Laboratório de Biologia do Centro Universitário de Rio Preto, UNIRP) in plastic containers with water from the collecting site and a small amount of aquatic plants or litter.
The females were kept isolated in 12 liters aquaria (30x20x20 cm) filled initially with water from the original environment; litter and small pieces of brick were added for shelter.The aquaria were under continuous moderated aeration supplied from an air compressor.One third of the water was replaced twice a week.The ovigerous prawns were fed in excess each afternoon with food for ornamental fish (Tetra Marine Flakes ® and Tetra Color ® ) and bits of shrimp or fish muscle, squid, and bivalves.The leftover food at the following morning was siphoned out.
The newly hatched larvae were immediately fixed in 10% formalin and were transferred to a mixture (1:1) of 70% ethyl alcohol and glycerin.Because of the low number of hatched larvae (female one: 13 larvae and female two: 9 larvae) and the difficulty of collect ovigerous females, only the first larval stage was illustrated and preserved for future studies.Six larvae of each female were dissected for detailed examination and description.
The procedures for dissections and preparation for the illustration was based on Magalhães (1988).The larvae were left in water with 10% alcohol for approximately 20 minutes.Thereafter, it remained transparent for several hours.First stage larvae were dissected under a stereomicroscope and mounted under semi-permanent slides.Morphological characters were examined with a Zeiss Axioscop 2 compound microscope attached to a personal computer using an Axiovision Image Analysis system.A binocular microscope equipped with a "camera lucida" was used for the drawings.Total length (TL) is given as the distance from the tip of the rostrum to the posterior margin of the telson, excluding setae.Carapace length (CL) means the distance from the orbital angle to the posterior margin of the carapace.The entire process of description was standardized according to Pohle and Telford (1981) and Clark et al. (1998) with respect to the quality and terminology.
The two females and the first larval stage obtained of each were deposited in the larvae collection of the Núcleo de Estudos em Biologia, Ecologia e Cultivo de Crustáceos (NEBECC) under the number CAR0001.

Results
The first larval stage of M. brasiliense (Fig. 1) hatch as very advanced larvae, resembling the adults in many aspects.The larvae are benthic as soon as they hatch.In general, they differ from adults by the absence of free uropods, spatuleted telson and unarmed rostrum.In addition, the mouth parts, thoracic and abdominal appendix are not functional.

Discussion
According to Jalihal et al. (1993), the larval development of Macrobrachium brasiliense corresponds to Type II: partially abbreviated development, with all pereiopods uniramous, well developed pleopods, and a fan-shaped telson in first stage.Other characteristics noted by Jalihal et al. (1993) for species with this kind of development (e.g., medium body size, distribution in upper reaches of rivers, and absence of marked sexual dimorphism) fit M. brasiliense well.
The larval characters of M. brasiliense described here suggests a closer relationship with M. nattereri (Heller, 1862) and M. iheringi (Ortman, 1897), both Neotropical species.These species have a morphologically similar first stage with uniramous pereiopods, biramous pleopods, and rounded telson (see Tab. 1).In addition, characteristics mentioned by Bueno and Rodrigues (1995) for M. iheringi from São Paulo State, as an unsegmented antennal scale, the numerous of marginal plumose setae in scaphognathite and six distinct abdominal somites are present in M. brasiliense.Magalhães (1989), comparing the description done by Vega-Pérez (1984), had already mentioned that M. nattereri and M. brasiliense are closely related.They both occur in the Amazon basin and, in many cases even adult specimens are not easily identified.Although their larval development is similar in many respects, some differences can be mentioned in the first larval stage.In M. nattereri, the endopod of antenna, maxillipeds 2 and 3, and the pereiopods are unsegmented, while in M. brasiliense, these structures are clearly segmented.Yet, in the description performed by Vega-Pérez (1984), M. brasiliense has the 6 th abdominal somite clearly separated from the telson, while in M. nattereri this separation is indistinct.Both species have 7 + 7 plumose setae on the posterior margin of telson, but in M. brasiliense this margin is clearly bilobed due to a distinct bulging of the disto-lateral corners (Magalhães, 1989).
However, the first zoea of M. brasiliense studied here, from northwest of São Paulo State, some characteristics resemble those of M. nattereri: the segmentation of the 6 th abdominal somite/telson is indistinct, the posterior margin of telson is not bilobed as was described by Vega-Pérez (1984), and there is a pair of minute naked setae placed in a very faint median notch of telson, which is very similar to M. nattereri.Pileggi and Mantelatto (2010), studying the molecular phylogeny of the genus Macrobrachium based on direct optimization analysis of 16S rDNA, used specimens of M. brasiliense from the São Paulo State (cities of Bauru and Serra-Azul) and M. nattereri from the Amazonas State (Manaus), and observed that the two species belong to sister groups, corroborating the conclusions drawn from larval morphology by Magalhães (1989) and now by the present study.
The wide distribution of M. brasiliense throughout tropical South America suggests that the populations occurring so far apart would exhibit some degree of morphological variation.Since the specimens of M. brasiliense studied by Vega-Pérez (1984) came from Paraná state, and in the present study, from São Paulo State, both Southeastern Brazil (Rio Paraná Basin), it would be interesting to verify how similar the larval morphology of the Central Amazonian population of M. brasiliense is to that one's already studied, as suggested by Magalhães (1989).

Table 1 .
Comparison of the first larval stage for some Neotropical representatives of the genus Macrobrachium with abbreviated larval development already described.