Post-larval development and sexual dimorphism of the spider crab Maja brachydactyla (Brachyura: Majidae)

the post-larval development of the majid crab Maja brachydactyla Balss, 1922 was studied using laboratoryreared larvae obtained from adult individuals collected in the ne atlantic. the morphology of the first juvenile stage is described in detail, while the most relevant morphological changes and sexual differentiation are highlighted for subsequent juvenile stages, until juvenile 8. the characteristic carapace spines of the adult phase are present in the first juvenile stage, though with great differences in the degree of development and relative size. the carapace shows a high length/weight ratio, which becomes similar to that of adults at stage 7-8. males and females can be distinguished from juvenile stage 4, based on sexual dimorphism in the pleopods and the presence of gonopores. in addition, the allometric growth of the pleon is sex-dependent from juvenile stage 4, with females showing a positive allometry (b=1.23) and males an isometric allometry (b=1.02).

although the morphological characteristics of early juveniles have not usually been applied in taxonomic studies of crabs, a description of the early instars is necessary to assist in identification of juvenile stages of crabs collected in field studies (ingle and rice, 1984;Guimarães and negreiros-Fransozo, 2005). in addition to elaborating useful identification keys, this information could be important for phylogenetic and phylogeographic studies.
the aim of the present study is to provide a detailed morphological description, including sexual differentiation, of the post-larval development (juvenile stages 1-8) of M. brachydactyla reared in laboratory.

material anD metHoDS
Several adult specimens of M. brachydactyla were collected from the ne atlantic (ría de a Coruña, Galicia, nW Spain; 42º14'n, 8º40'W) and brought to the irta (Sant Carles de la ràpita, tarragona, Spain) in February 2008.once in the laboratory, crabs were kept in 2000 l tanks connected to a recirculation unit at a constant salinity and temperature of 36 and 18±2ºC, respectively, and fed on fresh mussel and frozen crab.
larval rearing (60 larvae l -1 ) was carried out in 35-l, 150-µm mesh-bottomed PVC cylinders (baskets) provided with sufficient aeration and air-lifts to renew the water inside the baskets.the baskets were immersed in 1500-l holding tanks that were kept at a constant salinity and temperature of 36 and 18 ± 2ºC, respectively, under a natural light:dark cycle.larvae were fed Artemia metanauplii (more details in andrés et al., 2007).after 17-20 days of larval culture, 100 juveniles (stage 1) were placed individually in methacrylate compartment boxes (50 x 40 x 45 mm) connected to a recirculation circuit with partial renewal (20%) of the water twice a week.a natural photoperiod (ca.12:12 h light:darkness) was maintained and salinity and temperature were kept at 34 and 20 ± 1°C, respectively.Juveniles were fed a combination of adult Artemia and frozen krill (euphausiacea) once a day. the culture ended when juveniles were sexually differentiated and acquired a morphology similar to adults.the survival until the end of the experiment (juvenile stage 8) was 43%.
exuviae and specimens of each developmental stage were preserved in 70% ethanol or 10% formaldehyde.exuviae were preferred for morphological examinations and measurements; for each stage, 5-20 individuals were used.Dissection and measurements were taken with a nikon SmZ800 stereomicroscope equipped with an image analysing system (analySiS, SiS, münster, Germany).Features of the appendages, after mounting in a polyvinyl medium, were observed under an olympus BH-2 microscope.
Carapace width (CW) was measured as the greatest distance across the carapace measured between the tips of the 2nd or 3rd branchial spines; postorbital spine length (PoSl) was measured as the distance between the tips of the postorbital spines; carapace length (Cl) was measured as the distance between the rostral margin (without rostral spines) and the posterior margin of the carapace (without intestinal spines); and pleon width (PW) was measured as the width of pleonite 6. Crab descriptions follow the basic malacostracan body pattern, starting with the most anterior somite and progressing towards the posterior ones.each appendage is described from the proximal segments towards the distal segment.the long aesthetascs on the antennula and the long plumose setae on the distal exopod segments are drawn truncated.Figures 10 and 11 show only the pleopods of the left side.
the relative growth of body parts was determined using the allometric equation: y = ax b .using the logarithmic equations: log y = log a + b log x, tests for departures from isometry (H o : b = 1) were performed on the slope values obtained using Student's t-test (α = 0.01).reSultS the first juvenile is described in detail and in later juvenile stages only the most relevant ontogenetic changes are described.Balss, 1922 (Figs 1-10)
Pleon (Fig. 9a).Six somites and telson presents, broader than long; dorsoventrally flattened, flexed underneath the thorax and covered in sparsely setae; there is no apparent sex-related dimorphism in the relative dimensions of the juveniles examined; pleopods present in pleonites 2-5, and uropods present in pleonite 6, without setae.

Morphology of juvenile stages 2-8
Figure 2 shows the sequential changes of the carapace shape throughout the stage sequence.Carapace measurements for each stage are given in table 1.Data show that the carapace grows wider at each moult, and the CW/Cl ratio increases in the successive stages, becoming similar to the adult ratio in stage 7-8. the PoSl shows a negative allometric growth (b = 0.817; t = 34.79)while the CW/PoSl  ). the preocular, supraocular, hepatic, and branchial spines increase in size through the stage sequence (Fig. 2).additional spines (secondary spines) appear from stages 7-8, resembling the adult spinulation pattern.the supraorbital spines show lateral spinulation from juvenile stage 2-3.During juvenile ontogeny, the size and setation of the appendages increase.in addition, the number of segments of the antennular endopod and exopod and the antennal flagellum increases (table 1). the distal segment of the exopod of the maxillipeds becomes flagellated from juvenile stage 3, and the number of annulations, which show more differentiation distally, increases progressively with the development of the juveniles (table 1). the merus of the third maxilliped becomes progressively subquadrate and is similar to that of the adult from juvenile stage 5-6 (Fig. 6).
Based on pleopod morphology, sexual differen-  11c).after moulting to juvenile stage 4, females retain the four appendage pairs (located on pleonites 2-5), which become biramous.However, in males the appendage pairs are reduced to two pairs, located on the first and second pleonites (Fig. 9d and 11e). in males, the first pleopod increases in length in successive stages, and a longitudinal groove is present in stage 7 (Fig. 12). the pleopods of females increase in length in successive juvenile stages and show setation from stage 78 (Figs 10 and 12).Gonopores are first visible in juvenile stage 4 in both sexes (Fig. 8). in juvenile males, gonopores closely resemble those of the adult, both in shape and location (coxa of pereiopod 5). in females, gonopores appear as a slight opening in the exuviae, on sternite 6 (Fig. 8  c,d).Sex-related differences can also be detected by examining the PW versus Cl relationship (Fig. DiSCuSSion lebour (1927,1928) briefly described the early juvenile stages of Maja brachydactyla (as Maja squinado), specifically the morphology and size of the carapace in juvenile stages 1-3.However, her description does not correspond to M. brachydactyla. in fact, the carapace shape of the three juveniles that she attributed to Maja does not fit the description of majid species; it is probably the description of a portunid crab.therefore, the present account is the first description of the morphology of early juveniles of the genus Maja, and it cannot be compared with lebour's description.
Several important morphological characteristics used in the identification and classification of adults were not present in the first juvenile stage, but gradually appeared during early juvenile development: mainly the carapace shape and the presence and size of different spines and tubercles on the carapace.However, at the moment, we cannot conclude which juvenile stage may allow us to identify Maja brachydactyla from M. squinado, M. verrucosa and M. goltziana.the morphology of the appendages of the first juvenile stage was similar to that of the megalopal stage (Clark, 1986;Guerao et al., 2008;table 2).although in general the number of setae was higher in the first juvenile stage, some appendages showed a few meristic differences, e.g.antennules, mandibles, maxillules and maxilles.in successive juvenile stages, the number of segments (exopod and endopod of the antennula and antennal flagellum), annulations (flagellated part of the exopod of the maxillipeds), and setation of the appendages increased (table 1), which might be useful for differentiating the juvenile stages.these characteristics are important for the identification of juveniles of other studied brachyuran species (Bolla Jr. et al., 2008).
M. brachydactyla presents separate sexes, and morphological growth differences between sexes have been described in adults and sub-adults (teissier, 1935;González-Gurriarán et al., 1995).Positive allometric growth of the female pleon has been associated with morphological adaptations for reproductive behaviour in adults, and specifically with the capacity for carrying eggs.Dimorphic abdominal growth (Fig. 10) was apparent from the fourth juvenile stage in M. brachydactyla, as was allometry between carapace length and abdominal width (Fig. 13b).Dimorphic abdominal growth has also been reported in juveniles of the other brachyurans species (e.g., negreiros- Fransozo et al., 2003;Guimarães and negreiros-Fransozo, 2005;Vinuesa and Ferrari, 2008).
Sexual differentiation in M. brachydactyla was also determined by the presence and position of the gonopores in juvenile stage 4 (Fig. 8) and the gradual modification of the pleopods in males into gonopods in juvenile stages 4-7 (Figs 11 and 12).Sexual differentiation also appears early in the ontogeny of other species of majoidean crabs (ingle, 1977;Flores et al., 2002). in Pyromaia tuberculata (lockington, 1877) (family inachoididae) the development with undifferentiated sexes is much abbreviated: pleopod differentiation was found in juvenile stage 2 (Flores 1 and 2, and on segment 3, but here they were very minute and vestigial.in I. dorsettensis, the pleopods on segments 2-5 were reduced to minute buds in the second juvenile stage, which is also one juvenile stage before sexual differentiation (one juvenile stage earlier than in M. brachydactyla).

taBle 1 .
-morphometrical and morphological characteristcs of early juvenile stages of Maja brachydactyla Balss, 1922 useful for their identification.