The complete larval development of the crab Pilumnus spinifer (Brachyura: Xanthoidea: Pilumnidae) reared in the laboratory

The complete larval development of the crab Pilumnus spinifer from the western Mediterranean was obtained in the laboratory. All four zoeal stages and the megalopa are described and illustrated. The morphological characters of the larvae of Pilumnus spinifer are compared with those of other known larvae of the genus. The zoeae of P. spinifer show the rostral spine longer than the antennule (excluding aesthetascs); short lateral spines present on the carapace, and the mediolateral processes are present only in abdominal somites 2 and 3. The megalopa is similar to that of other Pilumnidae species. The morphology of the larval stages shows very similar characteristics to that of those of P. hirtellus and P. dasypodus, among the described stages of the family.

The taxonomy and systematics of the species of the genus Pilumnus are still problematic (d'Udekem d'Acoz 1999;Clark and Ng 2004a). Five species are usually recognized in north-eastern Atlantic and Mediterranean waters (Zariquiey-Á lvarez 1968), with one or two Indo-Pacific lessepsian immigrants into the Mediterranean (d'Udekem d'Acoz 1999; Galil et al. 2002), one Indo-Pacific isolated record in the north-eastern Atlantic and one yet undescribed new species (d'Udekem d'Acoz 1999).
The present study aims to describe the morphology of the complete larval development of Pilumnus spinifer, in larvae hatched from an ovigerous female captured in the western Mediterranean, and to compare its larval features with those known for other species of Pilumnus. This constitutes the second larval development description, after that of P. hirtellus (Lebour 1928;Salman 1982;Ingle 1983) for a species of Pilumnus in northeastern Atlantic and Mediterranean waters.

Materials and methods
A single Pilumnus spinifer ovigerous female was obtained, associated with a large soft-bodied sponge, by bottom trawling on muddy bottoms of the continental shelf off Punta Entinas (Almería) in the western Mediterranean (36u38.19N, 2u46.99W), from a depth of 53-58 m on 10 May 2004. Sampling was performed within the frame of the EU demersal fisheries research programme ''MEDITS'' on board B/O Cornide de Saavedra.
The crab was placed in a thermally insulated plastic container (60635630 cm) on board the ship, containing well-aerated filtered sea water, changed daily. The ovigerous crab was transferred to the laboratory on land on 24 May 2004. The incubation and larval rearing was carried out in the laboratory at a salinity of approximately 37.5 and kept at 25¡1uC and an artificial 12 h light: 12 h dark cycle. The larvae were fed freshly hatched nauplii of Artemia sp. Water and food were changed daily. Exuviae and specimens of each developmental stage were preserved in 70% alcohol.
A binocular microscope equipped with an ocular micrometer was used for the dissection and measurements of individuals (5-10 individuals of each larval stage were measured). A microscope was used for the determination of the setal formula and measurements of the appendages. The following measurements were taken: rostro-dorsal length (RDL) as the distance between the tips of the dorsal and rostral spines; carapace length (CL) from the base of the rostral spine to the posterolateral carapace margin; distance between the tips of the lateral spines (CW); furcal length (FL) from an imaginary line across the base of the outer lateral spine of the telson to the furcal tip; length of the outer lateral spine of the telson (OLS); length of the outer minute lateral spine of the telson (OLM); length of the telson dorsal spine (DS). For the megalopa, carapace length (CL) was measured as the distance from the frontal margin to the posterior margin of the carapace; carapace width (CW) as the greatest distance across the carapace.
All drawings were made with the aid of a camera lucida and microscope photography. The long aesthetascs of the antennules and the long plumose setae on the distal exopod of the maxillipeds and pleopods are not fully illustrated and are drawn truncated instead. Larval descriptions followed the basic malacostracan body pattern, and setal armature on appendages is described from proximal to distal segments and from endopod to exopod according to Clark et al. (1998). When the type of seta is stated, terminology has followed Ingle (1992) and Garm (2004).
The female from which these larvae hatched plus samples of all larval stages have been deposited in the Biological Collections of Reference of the Institut de Ciències del Mar (CSIC) in Barcelona (codes: ICMD 2/2005 for the female; ICMD 3-6/2005 for the zoeal stages; ICMD 7/2005 for the megalopae).

Results
The larval development of Pilumnus spinifer consisted of four zoeal stages and one megalopa. The development through the zoea I stage lasted (mean¡SD; initial n5100) 4¡0.8 days, the zoea II stage took 3.9¡1.1 days, the zoea III 4.1¡0.9 days, and the zoea IV 4.2¡0.8 days. The first zoeal stage is described in detail, while in subsequent stages only differences and changes are described.  Carapace ( Figure 1A, B). Globose, smooth, without tubercles. Dorsal spine present, welldeveloped, markedly curved posteriorly, with sparsely minute protuberances ( Figure 1A 1 ). Rostral spine smooth, thin and shorter than dorsal spine, longer than antennule (excluded aesthetascs) and shorter than antenna ( Figure 1B). A single thin and short lateral spine. A pair of simple setae on posterodorsal region. Posterior and ventral margin without setae. Posterolateral margin of carapace with numerous acute small spines ( Figure 1A 2 ). Eyes sessile.
Antenna ( Figure 4A). Longer than rostral spine. Protopodal process and exopod approximately equal in length. Protopodal process with two rows of spines of different size along distal half. Exopod unsegmented with one long and one smaller seta arising near mid-length and two rows of spines of different size along distal half. Endopod absent.
First pereiopod. Present as small bud.
Sternum ( Figure 13I). Maxillipeds and cheliped sternites fused with six setae plus one pair of small processes. All sternal sutures are interrupted medially.

Discussion
The zoeal morphology of the species of the family Pilumnidae is more variable than in other Xanthoidea families (Rice 1980;Ko 1994aKo , 1994bKo , 1995Clark and Ng 2004a). Characters that are usually not very variable within a family show in the Pilumnidae a great variability between genera and even within species of the same genus, such as number of zoeal stages (zero to four), presence or absence of the carapace lateral spines, presence or absence of the dorsolateral abdominal processes on somites 4-5, and length of the carapace rostral spine (long, short, or vesigial) (Lim and Tam 1981;Martin 1984;Ko 1994aKo , 1994bKo , 1995Spivak and Rodríguez 2002;Ko and Yang 2003). This has given rise to the consideration that a revision of the genus Pilumnus is necessary, since based on larval characteristics, such as number of zoeal stages, among others, there appear to be reasons to reassign species to different, new genera (Clark and Ng 2004a).
The main characteristics present in all the described zoeae of the family Pilumnidae are: (1) the exopod of the antenna is more or less equal in length to the spinous process of the protopod; it is distally bilaterally spinulated with one long and one smaller seta arising near mid-length; (2) the endopod of the maxillule always bears 1, 6 setae; and (3) telson with three pairs of spines. Additionally, some rather constant characteristics are: (4) the endopod of the maxilla with eight setae (seven in Pilumnopeus makiana); (5) setation of endopod of first maxilliped is 3, 2, 1, 2, 5 in zoea 1 (3, 2, 1, 2, 6 in Benthopanope eucratoides (Stimpson, 1858) (Rice 1980;Martin 1984;Ko 1994aKo , 1994bKo , 1997Ko and Yang 2003;Clark and Paula 2003). Ko (1994bKo ( , 1997 assigned the Pilumnidae zoeae to five groups based on the characteristics of the carapace spines and abdominal dorsolateral processes: (1) the genus Heteropanope, (2) Heteropilumnus and Pilumnopeus, (3) Actumnus and Pilumnus, (4) Parapilumnus, and (5) Benthopanope (see Ko 1994b;Ko and Yang 2003). The group 3 species (Pilumnus and Actumnus) share the following characteristics: (1) carapace with lateral spines; (2) carapace with curved (hook-like or slightly) dorsal spine; (3) a 0, 1, 6 setation of the second maxilliped endopod, and (4) rostral spine between 0.5 and 2.0 times the length of antennula, excluding aesthetascs (Sandifer 1974;Ko 1994bKo , 1997Spivak and Rodríguez 2002). The zoeal morphology of Pilumnus spinifer herein described completely fits the characteristics of Ko's group 3. Table I compares the morphological and meristic characters that may differ between the different larval stages of the known larval stages described for the species of the genus Pilumnus. Unfortunately, some of the descriptions do not show the sufficient level of detail or accuracy to compare with total reliability all the necessary characters, and therefore some of these characters, such as the setation of the zoeal carapace which sometimes is present in the figures but not in the descriptive text, are not shown in Table I. The zoeal morphology of P. sayi is similar to that of P. dasypodus, but is somewhat confused and therefore the characters of this species are not shown in the table; however an important difference has been described for the zoeae of these species: a pair of dorsolateral spines is present on abdominal segments 4 and 5 in the zoeal stages of P. sayi, which are absent in the zoeal stages of P. dasypodus (Bookhout and Costlow 1979).
The zoeae of Pilumnus spinifer resemble those of P. hirtellus, P. dasypodus, P. scabriusculus, and P. limosus by having four zoeal stages and by showing mediolateral processes only on abdominal somites 2-3 ( Table I). The zoeae of P. reticulatus, P. minutus, and P. sayi can be clearly differentiated from those of P. spinifer by presenting mediolateral processes on abdominal somites 2-5 (Table I). Pilumnus minutus and P. vespertilio can be well differentiated from P. spinifer by the reduced length of the rostral spine, which is shorter than the antennule (Lim and Tan 1981;Ko 1994aKo , 1994bKo , 1997Spivak and Rodríguez 2002).
Some of the variability reported in some zoeal characteristics may not be so since recent descriptions have shown that some characters may have been overlooked due to observation difficulties. Thus, Lim and Tan (1981) did not observe the occurrence of the minute lateral spines on the base of the telson of P. vespertilio, which were however observed by both Terada (1990) and Clark and Paula (2003), and showed the occurrence of nine setae on the basis and of two setae on the first endopod segment of the first maxilliped. The works of Terada (1990) and Clark and Paula (2003) showed in this species  Sandifer 1974, Bookhout andCostlow 1979 c ;P. scabriusculus, Terada 1990;P. limosus, García-Guerrero et al. 2005;P. verpertilio, Lim and Tam 1981, Terada 1990d , Clark and Paula 2003P. sluiteri, Clark and Ng 2004af , P. kempi, Siddiqui and Tirmizi 1992, Clark and Ng 2004aP. Iongicornis, Clark and Paula 2003. g In all zoeal stages.
the characteristic setation of the genus (10 and three setae, respectively). Additionally, in the original description of P. kempi, a species with abbreviated development, the zoea 1 shows 11 setae on the basis of the first maxilliped; however, the figure corresponding to zoea 2 showed 10 setae. The recent redescription of P. kempi by Clark and Ng (2004a) shows 10 setae in the two zoeal stages. Spivak and Rodríguez (2002) mention that the presence of the posteromarginal minute spines on abdominal somites 2 to 5-6 may be important in the identification of Pilumnus zoeae (according to these authors, present only in P. reticulatus, P. kempi, and P. hirtellus). However, it is probable that this character may be present in all the species of the genus. This character has been described in P. hirtellus, P. reticulatus, P. kempi, P. dasypodus, P. spinifer, P. longicornis, P. sluiteri, and P. limosus (Sandifer 1974;Salman 1982;Siddiqui and Tirmizi 1992;Spivak and Rodríguez 2002;Clark and Paula 2003;Clark and Ng 2004a;García-Guerrero et al. 2005). In P. minutus, it is not reported in the text, but it is clearly present in the figures (see Ko 1994a, Figures 1I, 2F, 3G). In P. vespertilio and P. sayi it is not described by Lim and Tan (1981) but Clark and Paula (2003) reported the occurrence of this character in the zoea 1 of P. vespertilio. Additionally, this character also occurs in other genera of the family Pilumnidae, such as in Parapilumnus trispinosus Sakai, 1965, Benthopanope indica (De Man, 1887), Pilumnopeus granulatus Balss, 1933, Eurycarcinus natalensis (Krauss, 1843), and Actumnus setifer (De Haan, 1835) (Ko 1994b(Ko , 1995(Ko , 1997Clark and Paula 2003;Clark and Ng 2004b).
Several other differences among Pilumnus zoeae were observed regarding the setation formulae (Table I). In general, differences in setation are small, except for P. vespertilio, P. kempi, and P. sluiteri which present an abbreviated larval development (three, two and two zoeal stages, respectively; see discussion by Clark and Ng 2004a). The heterochronic processes linked to the reduction of stages need to be taken into account when comparing the morphology of stages among species with different numbers of larval stages.
Concerning the megalopae, there is no single character or group of characters that can be used to clearly differentiate Pilumnus megalopae from those of other Pilumnidae genera. The megalopal morphology of the known species of the genus Pilumnus is similar (see Table II), except for P. novaezealandiae and P. lumpinus, which have not been included in Table II since most of the setation reported in these species is figured but not specifically described in the text. However, the larval development of these species is abbreviated: the megalopa hatches directly from the egg and the carapace and appendices show important differences from the rest of species shown in Table II (Wear 1967;Martin 1988).
In the north-eastern Atlantic and Mediterranean waters, the zoeal morphology of species of the genus Pilumnus is only known for P. hirtellus (Salman 1982;Ingle 1983Ingle , 1992 and P. spinifer, but at least five species are recognized to occur in this biogeographical area (Zariquiey-Á lvarez 1968;d'Udekem d'Acoz 1999). The only useful characteristics to distinguish the zoeae of P. spinifer from those of P. hirtellus is the length of the rostral spine. The rostral spine of P. spinifer does not clearly reach the aesthetascs and the ratio length of rostral spine/length of antennule (excluding aesthetascs) is approximately 1.6-1.7. The rostral spine of P. hirtellus clearly surpasses the aesthetascs and the ratio reaches values of around 2.0. Also, rostro-dorsal length (RDL) is greater in P. hirtellus than in P. spinifer (see Table I).
on the ischium of the first pereiopod (see figure 11D). However, in most of the published descriptions, this information is not available or is incomplete (see Table II). The megalopa of P. spinifer can be differentiated from that of P. hirtellus in the morphology of the posterolateral processes of the abdomen of somite 5, which overlaps somite 6 in P. spinifer, whereas in P. hirtellus the margin is rounded and does not overlap with abdominal somite 6 (Ingle 1983(Ingle , 1992.