Anais da Academia Brasileira de Ciências ISSN: 0001-3765 [email protected] Academia Brasileira de Ciências Brasil

BOLLA Jr, EDUARDO A.; FRANSOZO, VIVIAN; NEGREIROS-FRANSOZO, MARIA LUCIA Juvenile development of danae Smith, 1869 (Crustacea, , Brachyura, ) under laboratory conditions Anais da Academia Brasileira de Ciências, vol. 86, núm. 1, enero-marzo, 2014, pp. 211-228 Academia Brasileira de Ciências Rio de Janeiro, Brasil

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Anais da Academia Brasileira de Ciências (2014) 86(1): 211-228 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-37652014118912 www.scielo.br/aabc

Juvenile development of Callinectes danae Smith, 1869 (Crustacea, Decapoda, Brachyura, Portunidae) under laboratory conditions

EDUARDO A. BOLLA Jr1, VIVIAN FRANSOZO2 and MARIA LUCIA NEGREIROS-FRANSOZO1

1NEBECC, Departamento de Zoologia, Instituto de Biociências de Botucatu, Universidade Estadual Paulista/ UNESP, Distrito de Rubião Júnior, s/n, Caixa Postal 510, 18618-970 Botucatu, SP, Brasil 2Departamento de Ciências Naturais – Zoologia, Universidade Estadual do Sudoeste da Bahia/ UESB, Estrada do Bem Querer, Km 04, Campus de Vitória da Conquista, Caixa Postal 95, Bairro Universidade, 45031-900 Vitória da Conquista, BA, Brasil

Manuscript received on November 23, 2012; accepted for publication on March 28, 2013

ABSTRACT The juvenile development of Callinectes danae was investigated from megalopae obtained in neuston samples at Ubatuba, São Paulo, Brazil. The individuals were raised in the laboratory under constant temperature (25 ± 1°C), filtered sea water from the collection location (35‰), and natural photoperiod. Newly hatched Artemia sp. nauplii were offered as food on a daily basis and ornamental-fish food was also provided for the juveniles from the 4th stage on. Twelve stages of the juvenile phase were obtained. The main morphological features that allowed recognition of the first juvenile stage were drawn and described. All the subsequent stages obtained were examined and measured, and the main changes in relation to the first stage were recorded. Sexual dimorphism becomes apparent from the fourth juvenile stage onwards. Some appendages and morphological features proved to be of great importance in the identification of species, including the number of segments of the antennal flagellum and the number of setae on the maxilla and on the 1st, 2nd and 3rd maxillipeds. These can probably be used for future comparisons and species identifications. Key words: juvenile stages, morphology, portunid, post-larval, swimming .

INTRODUCTION period (Anger 2001). In the benthic period, early The life cycle of marine brachyurans, in general, has development up to the pubertal molt is characterized two distinct periods, the planktonic and the benthic. as the juvenile phase. This phase comprises a few Each phase has its own particularities, which can be early “undifferentiated” stages (characterized by a reflected in the ’s development, mainly during non-external sexual differentiation of the individual) the change from the larval to the juvenile phase and “immature” stages, during which juveniles can (Kurata 1962). The planktonic period comprises the be sexed by observing the number of pleopods on larval development, which includes a zoea phase with their abdomen. Only in the later immature stages the several zoeal stages, and a final megalopa phase with gonads begin to differentiate, and usually the relative a unique megalopa stage, whose settlement marks growth of certain body parts starts to differ slightly the transition between the planktonic and the benthic between the sexes (Hartnoll 1982). The undifferentiated stages can vary in number, Correspondence to: Eduardo Antonio Bolla Jr E-mail: [email protected] from one in Pachygrapsus transversus (Gibbes, 1850),

An Acad Bras Cienc (2014) 86 (1) 212 EDUARDO A. BOLLA Jr, VIVIAN FRANSOZO and MARIA LUCIA NEGREIROS-FRANSOZO studied by Flores et al. (1998), to eleven stages in In the family Portunidae, juvenile development Sesarma rectum Randall, 1840, described by Fransozo has been described for a relatively small number (1986/87). The undifferentiated stages end with the of species. For the genus Callinectes, only C. pre-pubertal molt followed by the first immature sapidus Rathbun, 1896 and C. ornatus Ordway, stage. The immature juveniles can show a highly 1863 have been studied, both up to the 11th stages variable number of stages and when the individual of the juvenile phase, by Barutot et al. (2001) and reaches sexual maturity at the puberty molt, the adult Bolla Jr et al. (2008), respectively. Ogburn et al. phase begins. As the study of juvenile development (2011) also studied some species of portunids, but in decapods is still incipient, the nomenclature of the only superficial descriptions of the carapace of terms “phase” and/or “stages” is also variable. Some C. sapidus and C. similis were provided. Due to authors use different nomenclatures for the stages in the scarcity of investigations on this aspect, it is juvenile phase in several species. In most cases they difficult to define characteristics that are peculiar do not use the separation between undifferentiated in the juvenile phase for portunids. Only detailed and immature stages. However, we adopted here the studies from laboratory rearing will allow the concept described above. comparison and the establishment of features for Studies of the early life cycle of amplify portunid species. taxonomic knowledge and also provide information This study presents morphological details on their ecology, physiology, phylogeny, and so on and the most significant features that identify (Felder et al. 1985, Anger 2001). A pioneer complete the juvenile phase of C. danae. We also provide study on juvenile development in Brachyura was information to recognize this species in natural accomplished by Shen (1935), who described environments, during its juvenile phase. This Carcinus maenas (Linnaeus, 1758) (Portunidae) information will contribute to species identification up to the ninth stage of the juvenile phase. Studies and culture for economic purposes. in the following decades dealt mostly with the MATERIALS AND METHODS description of the first stage of the juvenile phase. More complete descriptions appeared again during The juvenile development of C. danae was studied the 1980s. The species for which the juvenile using material from the collection of larvae and development has been studied and which occur on juveniles of the “Núcleo de Estudos em Biologia, the Brazilian coast are listed in Table I. Ecologia e Cultivo de Crustáceos (NEBECC)”, The family Portunidae Rafinesque, 1815 is Departamento de Zoologia, Instituto de Biociências, represented on the Brazilian coast by 21 species UNESP, Botucatu, São Paulo, Brazil. This material (Melo 1996, Calado 1996, Carqueija and Gouvêa consisted of juveniles (exuviae and individuals) 1996, Tavares and Mendonça Jr 1996) of which 6 obtained from raising of megalopae captured in belong to the genus Callinectes. The geographical the neuston of Ubatuba Bay (23°26’S; 45°03’W), distribution of Callinectes danae Smith, 1869 Ubatuba/SP. The climate was tropical/subtropical, includes much of the Western Atlantic: Bermuda, with mean temperatures of water surface around Florida, Gulf of Mexico, Antilles, Colombia, 18°C during winter and 29°C during summer Venezuela and Brazil (from the states of Paraíba to (Negreiros-Fransozo and Fransozo 2003). Rio Grande do Sul). This crab occurs from brackish The megalopae were collected using neuston to hypersaline waters, in mangroves and muddy nets (500 µm mesh) in ten minutes of trawling, estuaries, on sandy beaches to the open sea, and at night, from September to February 2005/2006 from the intertidal zone to 75 m depth (Melo 1996). and 2006/2007. After collection and sorting, the

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TABLE I List of species with occurrence in the Brazilian coast, with some stages of the juvenile phase known to date.

Stage of Juvenile the sexual Super Species stages differentiation Authors Families described (based on pleopods) Aethroidea Hepatus pudibundus (Herbst, 1785) 1st to 8th 3rd Hebling and Rieger 2003 Armases rubripes (Rathbun, 1897) 1st to 10th 5th Negreiros-Fransozo et al. 2011 Cyrtograpsus angulatus Dana, 1851 1st to 13th 4th Rieger and Beltrão 2000 Neohelice granulata (Dana, 1851) (described 1st to 8th 3rd Rieger and Nakagawa 1995 Grapsoidea as Chasmagnathus granulata) Pachygrapsus transversus (Gibbes, 1850) 1st to 8th 3rd Flores et al. 1998 Percnon gibbesi (H. Milne-Edwards, 1853) 1st ? Paula and Hartnoll 1989 Sesarma rectum Randall, 1840 1st to 15th 12th Fransozo 1986/87 Majoidea Pyromaia tuberculata (Lockington, 1877) 1st to 9th 2nd Flores et al. 2002 Uca (Leptuca) cumulanta Crane, 1943 1st to 8th 3rd Hirose et al. 2010 Ocypodoidea Uca (Minuca) burgersi Holthuis, 1967 1st to 10th 4th Vieira et al. 2010 Arenaeus cribrarius (Lamarck, 1818) 1st to 3rd ? Stuck and Truesdale 1988 Callinectes ornatus Ordway, 1863 1st to 11th 4th Bolla Jr et al. 2008 Portunoidea Callinectes sapidus Rathbun, 1896 1st to 11th 4th Barutot et al. 2001 Charybdis hellerii (A. Milne-Edwards, 1867) 1st to 3rd ? Dineen et al. 2001 Acantholobulus bermudensis (Stimpson, Martin et al. 1984, M.D. Rodrigues, 1st to 14th 6th 1871) (described as Hexapanopeus heblingi) unpublished data Fransozo and Negreiros-Fransozo Eriphia gonagra (Fabricius, 1871) 1st to 10th 4th 1987 Fransozo and Negreiros-Fransozo Eurypanopeus abbreviatus (Stimpson, 1860) 1st to 7th 4th 1987 Guimarães and Negreiros-Fransozo Eurytium limosum (Say, 1818) 1st to 10th 4th Xanthoidea 2005 Hexapanopeus caribbaeus Stimpson, 1871 1st to 11th 5th R.R.R.Vieira, unpublished data Menippe nodifrons Stimpson, 1859 1st to 8th 4th Fransozo et al. 1988 Panopeus austrobesus Williams, 1983 1st to 15th 4th Hebling et al. 1982 (described as Panopeus herbstii) Panopeus occidentalis Saussure, 1857 1st to 14th 6th M.D. Rodrigues, unpublished data Panopeus rugosus A. Milne-Edwards, 1880 1st to 14th 6th M.D. Rodrigues, unpublished data

? = the authors did not mention or did not get the stage in which the sexual differentiation occurs. megalopae were separated into covered acrylic every second day with filtered and aerated sea containers (30 ml) with filtered sea water (salinity water. After inspection, individuals were fed with 35) from the sampling area, and transported to newly hatched Artemia sp. nauplii ad libitum and, the NEBECC laboratory. The megalopae were for juveniles from 4th stage on, ornamental-fish individually raised at 24 ± 1°C in labeled and food was also provided. Dead individuals and their numbered containers with filtered and aerated respective exuviae were fixed in 80% ethanol and sea water. The containers were inspected daily, glycerin at 2:1, and stage changes and deaths were and debris and exuviae or dead individuals were recorded. These techniques are similar to those removed. The water in the containers was partially used by Guimarães and Negreiros-Fransozo (2005) renewed over one day, and completely replaced and Negreiros-Fransozo et al. (2007).

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All individuals in the different stages were numerous small marginal granules; bisegmented dissected, measured, drawn, and described based peduncle with sparse simple setae; bisegmented on fixed exuviae and dead individuals, using endopod (ventral flagellum) with, respectively, a stereoscopic microscope (Zeiss SV6) or a 3 and 10 simple setae on the proximal and distal compound optical microscope (Zeiss Axioskop segments; exopod (dorsal flagellum) constituted 2, with Nomarski optics), both equipped with a by 7 segments with 0, 14, 10, 8, 5, 0 and 0 long drawing tube, ocular micrometer, and Axiovision aesthetascs from the proximal to the distal segment, image system. respectively. Exopod with 0, 1, 2, 3, 1, 4 and 3 simple At least ten individuals of the first stage of setae from the proximal to the distal segment. the juvenile phase (J1) were dissected, drawn, and Antenna (Fig. 2a) bore a 3-segmented antennal analyzed with regard to their structure and the number peduncle, provided with simple and plumose setae. and types of setae. In addition, at least three specimens Antennal flagellum 10-segmented with 0, 2, 4, 2, 2, of each of the following stages were dissected and 2, 3, 2, 4 and 3 simple setae, all of them terminal. analyzed for evidence of morphological changes Mandible (Fig. 2c) possessed an incised, well over the developmental period. These changes were chitinized blade; palp 3-segmented with 2 serrate recorded in tables, and the most noticeable changes and 14 plumose setae on the distal segment and 1 were illustrated with line drawings. long plumose seta on the middle segment. The terminology adopted for the descriptions Maxillule (Fig. 2e) exhibited a coxal endite with was based on Clark et al. (1998) and Pohle et al. 2 plumose setae, 15 simple setae of several sizes and (1999). Setae types were named according to Pohle 2 serrate setae; basial endite with 4 small simple (1989) and Garm (2004). The setal sequence was setae and 1 long seta on the proximal margin, 6 presented from the proximal to the distal part of the serrate setae, 9 simple, 7 cuspidate and 3 plumose on appendages. Systematic classifications were based distal margin; bisegmented endopod with 3 plumose on Ng et al. (2008). setae and 1 simple seta on proximal segment, and 3 plumose setae and 2 simple setae on the distal RESULTS segment. Two simple setae on protopod margin. During the rearing experiments of C. danae, the Maxilla (Fig. 2d) had a bilobed coxal endite with juvenile development was followed until the 12th 6 plumose setae and 1 simple seta on the proximal stage, when the last specimen died. segment, 2 simple setae and 6 plumose setae on the distal segment; basial endite, bilobed, with 3 plumose MORPHOLOGY OF THE FIRST STAGE OF C. danae setae and 7 simple setae on the proximal segment, The general shape of the first stage (Fig. 1a) was 3 plumose setae and 13 simple setae on the distal similar to that of the adult: carapace flattened segment; endopod with 1 plumose and 4 simple dorso-ventrally, slightly convex, wider than long, marginal setae; exopod (= scaphognathite) with 85 and two small frontal teeth. It also presented: eight plumose marginal setae and 63 simple surface setae. finely serrate lateral spines plus the pair of spines First Maxilliped (Fig. 3a) bore a coxal endite common to portunids; small plumose setae on the with 15 plumose, 10 serrate, 5 simple setae and orbital margin and small granules and sparse simple 3 plumo-denticulate setae; basial endite with 36 setae over the carapace surface, in addition to well- plumose setae, 13 serrate and 9 small simple setae; developed stalked eyes. unsegmented endopod with 8 plumose setae and 4 Antennule (Fig. 2b) had a developed basal simple setae in proximal region, 22 simple marginal segment bearing several plumose and simple setae; setae and 1 sparsely plumose seta on distal region,

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Figure 1 - Callinectes danae. 1st undifferentiated stage: A, dorsal view; B, abdomen (dorsal view); C, sternum (ventral view); pl2-pl5, rudimentary pleopods (from 2nd to 5th abdominal somites). Scale: A, 0.5mm; B, C and pl2-pl5, 0.2mm.

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Figure 2 - Callinectes danae. 1st undifferentiated stage: A, antenna; B, antennules; C, mandible; D, maxilla; E, maxillule. Scale: 0.2mm.

1 small seta on the shaft of this region; bisegmented 16, 4, 11 and 5 simple setae from the proximal exopod with 13 plumose, 2 simple and 5 cuspidate to the distal segment; bisegmented exopod with setae on the proximal segment, 7 plumose and 5 9 plumose setae, 18 simple setae and 8 cuspidate simple setae on the distal segment; epipod well setae on the proximal segment, 3 simple setae and 6 developed, with 58 simple setae. plumose setae on the distal segment; protopod with Second Maxilliped (Fig. 3b) possessed a 3 plumose setae and 6 simple setae; rudimentary 5-segmented endopod with 6, 8, 2, 1 and 0 plumose epipod provided with 3 simple setae. setae from the proximal to the distal segment, 3 Third Maxilliped (Fig. 3c) exhibited a and 4 serrate setae and 0 and 5 cuspidate setae, 5-segmented endopod with several marginal on the 4th and 5th segments, respectively; and 5, spines on the first and second segments, several

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Figure 3 - Callinectes danae. 1st undifferentiated stage: A, 1st maxilliped; B, 2nd maxilliped; C, 3rd maxilliped. Scale: 0.2mm. setae on each segment: 17, 11, 12, 19 and 6 bisegmented exopod with 24 plumose setae and serrate setae, 35, 11, 1, 0, 0 plumose setae, 0, 0, 45 simple setae on the proximal segment and 8 5, 4, 0 plumodenticulate setae and 63, 21, 9, 5, 7 plumose and 2 simple setae on the distal; protopod simple setae from the proximal to distal segment; with 22 plumose setae of several sizes and 44 small

An Acad Bras Cienc (2014) 86 (1) 218 EDUARDO A. BOLLA Jr, VIVIAN FRANSOZO and MARIA LUCIA NEGREIROS-FRANSOZO simple setae; epipod with around 53 small simple Last pair of pereiopods (Fig. 4c) – a spine on the setae on the proximal portion and 15 long simple coxa; propodus and dactyl flattened and paddle- setae on the distal portion. shaped with several marginal plumose setae. Chelipeds (Fig. 4a) were symmetrical, with Thoracic sternites (Fig. 1c) had simple and marginal spines and three strong inner ones on plumose setae sparsely distributed over surface; merus; granules and small spines on external margin concentric and semicircular wrinkle on the 7th of the carpus and propodus; simple and plumose somite (near the coxa). setae sparsely distributed over the appendage Abdomen (Fig. 1b) bore 6 somites, wider than surface, plus long plumose setae on the external long, several sparse simple and plumose setae; the margin of the merus. Second, third (Fig. 4b) and first 5 somites with horizontal ridges; telson smooth fourth pereiopods similar, sparse simple setae and with small simple and plumose setae. Rudimentary several plumose setae present, mainly on the inner pleopods visible ventrally on the 2nd to 5th somites and external margins of the propodus and dactyls. (Fig. 1, PL2–PL5).

Figure 4 - Callinectes danae. 1st undifferentiated stage: A, cheliped; B, 3rd pereopod; C, 5th pereopod. Scale: 0.4mm.

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nd th MORPHOLOGY OF THE 2 TO 12 STAGES OF C. danae The abdomen increased in size during the As the individuals grew in the following stages, growth period, but it changed slightly in shape; the in each successive ecdysis the proportion between sex of individuals could be distinguished only at the carapace width and carapace length varied very 12th stage. As observed in adult swimming crabs, the little. This proportion reached the same as observed male abdomen had an inverted “T” shape and the th in the adult phase, from the 12 stage on (Fig. 5a). female abdomen was semicircular (Fig. 5, J4–J12).

Figure 5 - Callinectes danae. A, carapace development throughout all juvenile stages obtained (numbers represent the stage of the juvenile phase); j4, j8 and j12, development of the abdomen shape of both males and females in 4th, 8th and 12th stages, respectively. Scale: A, 5mm; j4-j12, 2mm.

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th The most remarkable morphological changes, 6 stages (Fig. 6, J5 and J6) no significant changes, nd th th observed as of the 2 stage, were related to the PL1 only increased in size. In the 7 and 8 stages secondary sexual characteristics. The existing (Fig. 6, J7 and J8), PL2 exhibited one reduced rami st rudimentary pleopods in the 1 stage disappeared and PL1 had plumose setae on its base and simple in the 2nd stage. In the 4th stage the pleopods setae along its length. From the 9th stage on (Fig. 7, reappeared, but in different numbers and locations, J9–J12), PL2 was uniramous and both PL1 and PL2 according to each sex, as follows: were gradually larger, with more setae. From the th Male: two pairs of pleopods on the first (PL1) 10 stage on, there was presence of bulges on PL1 th and second (PL2) somite of the abdomen, PL2 distally. Morphology of 12 stage pleopods was the th with one reduced rami (Fig. 6, J4). In the 5 and same as seen in adults.

Figure 6 - Callinectes danae. pl1 and pl2: pleopods of the 1st and 2nd male abdominal somites, respectively, from 4th (j4), 5th (j5), 6th (j6), 7th (j7) and 8th (j8) stages on. Scale: j4, j5 and j6, 0.1mm; j7 and j8, 0.2mm.

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Figure 7 - Callinectes danae. pl1 and pl2: pleopods of the 1st and 2nd male abdominal somites, respectively, from 9th (j9), 10th (j10), 11th (j11) and 12th (j12) stages on. Scale: j9, 0.2mm; j10, j11 and j12, 0.4mm.

Female: four biramous pleopods from the 2nd setae increased, endopod of all pleopods gradually th th to the 5 abdominal somite (PL2, PL3, PL4 and was larger and more segmented. In the 9 stage

PL5); simple setae on endopod of PL2 and PL3 only (Fig. 9, J9), there were simple articulated setae th (Fig. 8, J4). 5 stage pleopods (Fig. 8, J5) were (instead of brush-shaped setae) on the endopod th th larger, with more setae. In the 6 stage (Fig. 8, J6), of all pleopods. From the 10 stage on, pleopods PL2 endopod segmentation begun, a few brush- were very similar to those of adults, with simple th th shaped setae on PL2 and PL3. In the 7 and 8 articulated setae larger and more abundant in later stages (Fig. 8, J7 and J8), number of brush-shaped stages (Fig. 9, J10–J12).

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Figure 8 - Callinectes danae. pl2 to pl5: pleopods from the 2nd to 5th female abdominal somites, respectively, from 4th (j4), 5th (j5), 6th (j6), 7th (j7) and 8th (j8) stages on. Scale: j4, j5 and j6, 0.1mm; j7 and j8, 0.2mm.

In the remaining body structures, some signi­ serrulate harpoon-shaped setae appeared from the 3rd ficant changes occurred in the following: 1) Endopod stage on. Such setae were also found on the epipod of first maxilliped had a hollow in the apical region of the 2nd maxilliped from the 4th stage on, and on the and a foliaceous shape on the internal margin from the epipod of the 1st maxilliped in the 5th stage. 2nd stage on; 2) First segment of the antennal peduncle The main morphological characteristics which had a projection similar to the antennal scale, mainly allow the identification of the first 12 stages of C. from the most advanced stages on. danae are shown in Table II. Other appendages did not show remarkable The vestigial aperture of the female gonopores morphological changes, but the number of setae appeared from the 4th stage on (Fig. 10a, white increased on each segment of the appendages. arrow). In males, it was not possible to verify in On the epipod of the third maxilliped, some denticle- what stage the gonopores appeared.

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Figure 9 - Callinectes danae. pl2 to pl5: pleopods from the 2nd to 5th female abdominal somites, respectively, from 9th (j9), 10th (j10), 11th (j11) and 12th (j12) stages on. Scale: j9, 0.2mm; j10, j11 and j12, 0.4mm.

The abdomen remained sealed throughout the somite) (Fig. 10b, black arrow), which did not stages observed. Detailed inspection revealed two allow the extension of the abdomen. pairs of “closing mechanisms” or “sternal buttons” DISCUSSION on the sternal plates: an anterior one (located on the 5th sternite, at the level of the distal region of the The post-larval development of only a few species 6th abdominal somite) (Fig. 10a, black arrow); and of portunid crabs is known to date. Lebour (1944) another, posterior one (located on the 8th sternite, at presented brief descriptions of the carapace of the the level of the lateral region of the 2nd abdominal first undifferentiated stage of Portunus anceps

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TABLE II Callinectes danae Smith, 1869. The main morphological characteristics for the identification of the first 12 stages of the juvenile phase. The dimensions were taken in millimeters and represent mean values. Numbers in parenthesis mean alternative values with low occurrences.

Characteristics J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 Number of segments 2 2 3 3 3 4 4 4 5 5 5 6 on antennule endopod Number of segments 7 8 9 10 11 12 13 14 15 16 18 (19) 20 on antennule exopod Number of segments 10 10 12 13 18 20 22 24 (23) 29 (30) 32 34 (33) 39 of antennal flagellum Antenna length 1.29 1.42 1.9 2.27 2.96 3.62 4.51 4.98 6.07 6.86 8.4 9.58 Number of setae on the 1 2 2 4 4 4 6 7 (8) 11 (10) 12 12 (13) 13 (14) basis of maxillule protopod Number of marginal setae ±85 ±102 ±148 ±161 ±183 ±229 ±270 ±298 ±325 ±359 ±406 ±425 on maxilla exopod Length of maxilla exopod 0.67 0.77 1.06 1.37 1.59 2.04 2.28 2.64 3.25 3.74 4.39 4.98 Number of apical setae st 11 14 23 ±33 ±38 ±49 ±65 ±73 ±81 ±89 ±101 ±112 on 1 maxilliped Length of basal segment st 0.62 0.74 0.92 1.16 1.36 1.69 1.98 2.18 2.57 2.92 3.55 4.14 of 1 maxilliped exopod Length of basal segment nd 0.64 0.74 0.96 1.23 1.42 1.82 2.12 2.29 2.73 3.2 3.89 4.46 of 2 maxilliped exopod Length of basal segment rd 0.63 0.75 0.92 1.18 1.35 1.71 1.96 2.15 2.62 3.02 3.59 4.11 of 3 maxilliped exopod Length of 2nd pereiopod merus 0.95 1.14 1.64 2.13 2.56 3.37 3.82 4.33 5.38 6.12 7.42 8.91 ± indicates “approximately”.

(Saussure 1858), and found 8 lateral spines in addition to the pair that is common to portunid crabs. Yatsuzuka and Sakai (1980) also described the first undifferentiated stage of P. pelagicus, which showed the same characteristics as P. anceps and also a serrate rostrum. Only C. ornatus (studied by Bolla Jr et al. 2008), C. similis (studied by Ogburn et al. 2011) and C. danae (present study) have been reported bearing a similar serrate rostrum starting from the first undifferentiated stage. In C. sapidus, according to Barutot et al. (2001) and Ogburn et al. (2011), the rostrum is smooth in the first undifferentiated stage, and the spines appear only as of the 6th stage. This feature might be useful as a diagnostic of this species on the Brazilian coast, Figure 10 - Callinectes danae, 4th stage of the juvenile phase: as it is easy to distinguish. Nevertheless, further A, white arrow, the rudimentary female gonopores; black arrow, studies on other species of the genus Callinectes anterior “closing mechanism” position; B, posterior “closing are needed. mechanism” position, pointed by the arrow; C, schematic drawing of the position of the abdomen over the posterior Morphological descriptions of juvenile forms “closing mechanism”. are mainly based on the number of segments and

An Acad Bras Cienc (2014) 86 (1) JUVENILE DEVELOPMENT OF C. danae 225 setae on the body appendages. However, because sapidus, C. ornatus and P. pelagicus, studied up to of the difficulty in discerning characteristics in the 11th, 11th and 3rd juvenile stages, respectively, the morphology of different setae types with such dimorphism is also not observed. This feature only optical microscopy, the study of setae in the probably develops in later immature stages, in juvenile phases has been limited to counting them contrast to representatives of other families, in and noting their location on the animal’s body which the sexual differentiation observed in (Rieger and Beltrão 2000). the abdomen is most pronounced in the early Certain appendages and morphological features immature stages. Examples include Inachus have proven to be highly useful in discriminating dorsettensis (Pennant 1777), a majid studied by species, due to their particularities, which can Ingle (1977); P. transversus, a grapsid studied by be observed with minimal manipulation of the Flores et al. (1998); and C. angulatus, a varunid specimens. These will likely prove to be useful studied by Rieger and Beltrão (2000); in which for future comparisons and species identifications. this differentiation occurs from the 3rd, 6th, and 4th Among these are the following features: number of immature stages on, respectively. segments of the antennal flagellum (i.e., C. sapidus, The presence of the rudimentary pleopods in C. danae and C. ornatus, which have 8, 10 and 10(11) the first undifferentiated stage can also differ among segments, respectively), and the number of setae on species; this feature is observed in C. danae, C. ornatus the maxilla and on the 1st, 2nd and 3rd maxillipeds. and P. pelagicus, but not in C. sapidus. Nevertheless, With regards to the sternal region, the presence except for P. pelagicus which shows rudimentary of wrinkles in concentric semicircles on the 7th pleopods up to the 3rd undifferentiated stage, in all somite of the first undifferentiated stage [present other species studied, these pleopods totally disappear in C. danae and C. ornatus, but not noted by by the 2nd undifferentiated stage. This feature might Yatsuzuka and Sakai (1980) and Barutot et al. be interpreted as a remnant characteristic from the (2001) in P. pelagicus (Linnaeus, 1758) and C. megalopa stage, since they occupy the same location sapidus, respectively], could be interpreted as a on somites of the megalopa pleopods. In the following remnant of the sternal spine that is conspicuous stages, the pleopods appear in accordance with each in the portunid megalopa (Kurata 1975). Such sex (two pairs for males, and four pairs for females), wrinkles are not found in the next stages, reinforcing and the juvenile stage in which such appearance this supposition. Nevertheless, further studies are can be observed varies for each species, as shown needed in order to confirm the origin of this feature. in Table I. In portunids, the pleopods commonly As mentioned by Guinot and Bouchard (1998), appear in the 4th stage of the juvenile phase, as in the anterior abdomen sealing mechanism, located on C. danae, C. ornatus and C. sapidus. the 5th thoracic sternite of C. danae, is in accordance A distinctive and interesting feature with nd with the typical system of sealing found in the regards to the 2 pair of pleopods of males (PL2) is Eubrachyura. However, those authors did not mention the fact that this appendage is biramous until the 8th the closing mechanism (8th sternite). This mechanism stage in C. danae and until the 6th stage in C. ornatus, may be exclusive to portunids, but only through and, at least, until the 7th stage in C. sapidus (as can additional studies of species within and outside the be seen in Barutot et al. 2001). More interesting yet family would this hypothesis be able to be tested. is the fact that only C. sapidus has a biramous first

The sexual dimorphism in the abdominal shape pleopod (PL1) in males. In the remaining portunid of males and females is not evident throughout species studied, the PL1 remains uniramous as of its the juvenile phase observed for C. danae. For C. first appearance in juveniles.

An Acad Bras Cienc (2014) 86 (1) 226 EDUARDO A. BOLLA Jr, VIVIAN FRANSOZO and MARIA LUCIA NEGREIROS-FRANSOZO

Table III presents a comparative analysis, using life cycles, for a better understanding of their life the main morphological characteristics, among the history, or ecological and functional species of the genus Callinectes which occur on issues, since these subjects are interconnected. Such the Brazilian coast and for which their juvenile studies are also important to define characteristics development is known to date. that are peculiar to the juvenile phase, and they could It is of great importance to consider further allow us to identify species for various purposes, studies on the early phases of decapod notably aquaculture and fisheries management.

TABLE III Diagnostic characters which allow the differentiation and identification of the first undifferentiated stage of the known species of the genus Callinectes with regards to juvenile phase.

C. sapidus C. ornatus C. danae JUVENILE I Barutot et al. (2001) Bolla Jr et al. (2008) This study Antennule endopod segments 2 2 2 Antennule exopod segments 7 7 7 Antenna segments 3 + 8 3 + 11 3 + 10 * Mandible palp segments 3 3 3 Maxillule endopod segments 2 2 2 Maxillule endopod (E), basial endite (EB), 4, 2 (E); 23 to 25 (EB); 3, 4 (E); 29 (EB); 4, 5 (E); 30 (EB); coxal endite (EC), protopod (P) setae 15 to 17 (EC); 2 (P) 20 (EC); 3 (P) 19 (EC); 2 (P) Maxilla exopod (Ex), endopod (E), basial 60 to 70 (Ex); 0 (E); 20 to 88 (Ex); 4 (E); 85 (Ex); 5 (E); * endite (EB), coxal endite (EC) marginal setae 23 (EB); 10 to 13 (EC) 22 (EB); 13 (EC) 26 (EB); 15 (EC) 1st maxilliped exopod segments 2 2 2 1st maxilliped endopod segments 1 1 1 1st maxilliped exopod (Ex), endopod (E), 16 to 19 (Ex); 17 (E); 29 (Ex); 35 (E); 32 (Ex); 35 (E); 58 basial endite (EB), coxal endite (EC), 38 to 44 (EB); 17 to 67 (EB); 26 (EC); * (EB); 33 (EC); 58 (Ep) epipod (Ep) setae 21 (EC); 39(Ep) 49(Ep) 2nd maxilliped exopod segments 2 2 2 2nd maxilliped exopod (Ex), endopod (E), 25 to 27 (Ex); 12, 52 (Ex); 27, 8, 14, 44 (Ex); 11, 24, 6, * epipod (Ep) setae 2, 3, 12 to 14 (E) 14 (E); 6 (Ep) 15, 14 (E); 3 (Ep) 3rd maxilliped exopod segments 2 2 2 3rd maxilliped exopod (Ex), protopod (P), 18 to 20 (Ex); 14 to 56 (Ex); 16 (P); 79 (Ex); 66 (P); * epipod (Ep) setae 17 (P); 22 to 24 (Ep) 28 (Ep) 68 (Ep) Number of pleopods 0 4 rudimentary pairs 4 rudimentary pairs *

* = most important characteristics for comparison among species.

ACKNOWLEDGMENTS investigation has been conducted in accordance The authors are grateful to Conselho Nacional with applicable state and federal Brazilian laws. de Desenvolvimento Científico e Tecnológico RESUMO (CNPq) for the first author’s Master of Science scholarship (# 553174/2008-8), to Fundação O desenvolvimento juvenil de Callinectes danae foi de Amparo à Pesquisa do Estado de São Paulo investigado a partir de megalopas obtidas em amostras de (FAPESP) for providing the financial support neuston em Ubatuba, São Paulo, Brasil. Os indivíduos foram during neuston collections (#2004/15194-6), to criados em laboratório sob temperatura constante (25 ± 1°C), all the members of NEBECC for their assistance água do mar filtrada proveniente do local de coleta (35‰) during this study, and to Dr. Janet W. Reid for her e fotoperíodo natural. Nauplii recém-eclodidos de Artemia help with the English review. All sampling in this sp. foram oferecidos diariamente como base de alimento

An Acad Bras Cienc (2014) 86 (1) JUVENILE DEVELOPMENT OF C. danae 227 e também ração de peixes ornamentais foi fornecida a Flores AAV , Marques FPL and Negreiros-Fransozo ML. juvenis a partir do 4º estágio. Foram obtidos 12 estágios 2002. Postlarval stages and growth patterns of the spider crab Pyromaia tuberculata (Brachyura, Majidae) from da fase juvenil. As principais características morfológicas laboratory-reared material. J Crust Biol 22: 314-327. que permitiram o reconhecimento do primeiro estágio Flores AAV , Negreiros-Fransozo ML and Fransozo A. 1998. The megalopa and juvenile development of juvenil foram desenhadas e descritas. Todos os estágios Pachygrapsus transversus (Gibbes, 1850) (Decapoda, subsequentes obtidos foram analisados e medidos, e as Brachyura), compared with other grapsid crabs. principais alterações em relação ao primeiro estágio foram Crustaceana 71: 197-222. Fransozo A. 1986/87. Desenvolvimento dos estágios juvenis registradas. O dimorfismo sexual torna-se evidente a partir de Sesarma (Holometopus) rectum Randall, 1840 do 4º estágio juvenil. Alguns apêndices e características (Decapoda, Grapsidae) obtidos em laboratório. Naturalia morfológicas provaram ser de grande importância para 12: 77-87. Fransozo A and Negreiros-Fransozo ML. 1987. Morfo­ identificação das espécies, incluindo o número de segmentos logia dos primeiros estágios juvenis de Eriphia gonagra do flagelo antenal e o número de cerdas na maxila e no 1º, (Fabricius, 1781) e Eurypanopeus abbreviatus (Stimpson, 1860) (Crustacea, Decapoda, Xanthidae), obtidos em 2º e 3º maxilípedes. Estes, provavelmente, podem ser usados laboratório. Pap Avulsos Zool 36: 257-277. para futuras comparações e identificações de espécies. Fransozo A, Negreiros-Fransozo ML and Hiyodo CM. 1988. 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