Abbreviated larval development of Tunicotheres moseri ( Rathbun , 1918 ) ( Decapoda : Pinnotheridae ) , a rare case of parental care among brachyuran crabs *

1 Laboratory of Carcinology, Universidad de Oriente, Apartado 147, Porlamar, Isla de Margarita, Venezuela. 2 Instituto de Ciencias Marinas de Andalucía (CSIC), Polígono Río San Pedro, s/n. Apdo. Oficial, 11510 Puerto Real, Cádiz, Spain. E-mail: jose.cuesta@icman.csic.es 3 Department of Biology, Laboratory for Crustacean Research, University of Louisiana at Lafayette, P.O. Box 42451, Lafayette, Louisiana 70504-2451, USA.


INTRODUCTION
The family Pinnotheridae de Haan, 1833 is a somewhat heterogeneous group of small crabs living as symbionts with other invertebrates.In the last ten years this family has been the object of several studies based upon larval and adult morphology (Marques and Pohle, 1995;Campos, 1996Campos, , 1999;;S ˇtevc ˇic ´, 1996;Pohle andMarques, 1998, Campos andManning, 2000).More recently, two of the present authors have focused efforts on combining molecular genetic data with findings from comparisons of larval and adult morphology in the course of a broad-based study of systematics and phylogeny within the Pinnotheridae (Cuesta et al., 2001, 2002, Cuesta andFelder, in prep.).The preliminary results of these projects underscore the polyphyletic nature of the family Pinnotheridae as presently composed and make clear the need for a revised classification based on both molecular analysis and re-evaluations of larval and adult morphology.
The genus Tunicotheres was erected by Campos (1996) for a single species, Pinnotheres moseri Rathbun, 1918, originally described on the basis of female specimens from Jamaica (type locality) and Florida.The apparent male of this species was found recently in Laguna La Restinga (Isla de Margarita, Venezuela) inside the black tunicate, Phallusia nigra, as reported by Hernández and Bolaños (1995).As is now known, the distribution of this species is believed to be restricted to Jamaica, the west coast of Florida and Isla de Margarita (Venezuela).Specimens have been found living as commensals within the ascidians Phallusia nigra Savigny, Molgula occidentalis Traustedt, 1882, Polycarpa spongiabilis Traustedt, 1883, and Styela plicata (Lesueur, 1823) (Goodbody, 1960;Roberts, 1975;K. Strasser, University of Tampa, pers. comm.).
This species presents a rare case of post-hatching parental care in a marine decapod crustacean.All larval stages are contained within the space delimited by the flexed abdomen and sternum of the female, hereafter termed the abdominal enclosure.The megalopa moults to the first crab stage while still contained within the abdominal enclosure, and the first crab is the first instar to leave the abdomen of the female and exit the atrial cavity of the host, thereafter potentially dispersing to a new host.
In the present study, the complete larval development of T. moseri from Isla de Margarita (Venezuela) is described and fully illustrated.Where possible, comparisons are made with developmental stages described by Goodbody (1960) for a Jamaican population of the species.Interspecific comparisons are made with known larvae of other Pinnotherinae.

MATERIAL AND METHODS
Thirty-two ovigerous females were collected between 1995 and 2001, all from the atrial cavity of the host tunicate, Phallusia nigra, in Laguna La Restinga, Isla de Margarita, Venezuela.The females were transferred live to the Laboratory of Carcinology, Universidad de Oriente, Boca del Río (Isla Margarita, Venezuela).The egg masses were removed from nine ovigerous females and cultured in vitro.The remaining ovigerous females were maintained in aerated aquaria at 23-27°C and 36-38 psu salinity, so development of the eggs could proceed while egg masses remained intact in the abdominal enclosures of the females.Larvae hatched from in vitro egg cultures were reared at 23-27°C and 36-38 psu salinity in gently aerated beakers (120 ml, ca. 10 larvae per beaker).Water was changed and freshly hatched nauplii of Artemia were added (ad libitum) after each daily examination of cultures for mortalities and evidence of moulting.Control groups of larvae were reared under exactly the same conditions, but without feeding, to ascertain any effects of starvation.Samples of larvae and exuviae were fixed in a 4% neutralised solution of formaldehyde in seawater.
The two zoeal stages, the megalopa and first crab were also harvested from the abdominal enclosures of the aquarium-held females for comparison with the stages reared from in vitro egg cultures.Different larval stages, along with the first crab stage, were sometimes taken simultaneously from the same female.
Dissections were made under a Leika MZ8 binocular dissecting microscope.Drawings and measurements were made using an Olympus BH-2 compound microscope equipped with a camera lucida.Semi-permanent and permanent slide mounts in polyvinyl lactophenol were made of whole larvae and dissected appendages.All measurements were made with an ocular micrometer.Drawings were based on examinations of no less than 5 larvae for each feature, but more than 20 larvae of each stage were dissected and examined to determine setal variability.Size measurements were based on 10 larvae per stage from 3 different hatches.For zoeal stages, rostro-dorsal length (rdl) was measured from the tip of the rostral spine to the highest point on the carapace; carapace length (cl) was measured from the base of the rostrum to the posterior margin of the carapace; and carapace width (cw) was measured as the greatest distance across the carapace.In the megalopa stage, carapace length (cl) was measured from the base of the rostrum to the posterior margin of the carapace, and carapace width (cw) was measured as the maximum width of the carapace.
The long setae on the distal exopod segments of the first and second maxillipeds were drawn truncated.Descriptions and figures were arranged according to the standards proposed by Clark et al. (1998).Samples of all larval stages and one parental female of Tunicotheres moseri from the Venezuelan population were deposited at the Smithsonian Institution National Museum of Natural History, Washington, D.C. (USNM 1020583) and at the University of Louisiana-Lafayette Zoological Collection (ULLZ 5578).

RESULTS
Hatching of the eggs cultured in vitro was followed by two morphologically distinct zoeal stages and a megalopa (Table 1).Development to the megalopa was completed in a minimum of 3 days from hatching.All zoea I stages appeared to moult to zoea II, with no evidence of direct moulting from zoea I to the megalopa.opmental stages reared within the abdominal enclosure of females.There were no differences in survival or stage durations between larvae cultured under starvation and those fed nauplii of Artemia.Larvae appeared to carry large masses of yolk in all stages.There was no evidence of a source of food for larvae developing in the abdominal enclosure of females.It thus appeared that the larval stages were either non-feeding or at least facultatively lecithotrophic.
Ovigerous females maintained in aquaria never released larvae into the water column, as none of these stages were ever observed within the tanks.The first crab instar was the only stage observed in the aquaria.
The first zoeal stage of Tunicotheres moseri was described completely, whereas only differences were described in detail for subsequent stages.Mor-phological differences were not found between larvae cultured in vitro and those obtained from female abdominal enclosures.For some larval appendages the number of setae varied.In these cases, the typical number was illustrated, but ranges were indicated in the text.
Telson (Fig. 7A,B): Telson bifurcated with 3 pairs of short serrulate setae on posterior margin.Telson base, proximal to bifurcation, 2.5 times longer than furcal branches.Each furcal branch with small dorsal spine proximally and covered with spinules distally.

DISCUSSION
Tunicotheres moseri exhibits highly abbreviated larval development in terms of both the number of zoeal stages and the duration of development.The number of zoeal stages for pinnotherid species described to date ranges from 3-5, with 3-4 zoeae being typical of species in the subfamily Pinnotherinae.The shortened larval history, together with the peculiar retention of all larval stages by the female and the presence of a large yolk mass in all larval stages, strongly suggest that these larvae are lecithotrophic.As previously noted by Goodbody (1960), this type of abbreviated development and parental care in an obligate commensal of ascidians probably represents an advantageous strategy to avoid dispersing too far from host populations.
The mouthpart appendages of the larvae are not well-developed, much as in the case of other lecithotrophic species of brachyurans (Hartnoll, 1964;Soh, 1969;Rabalais and Cameron, 1983;Taishaku and Konishi, 2001), and this may also explain the unusually great variability in setal formulae for these appendages.Therefore, it is difficult to compare the setal patterns of anterior appendages of T. moseri with those in larvae of other Pinnotheridae.However, characters of the antenna and telson suggest that T. moseri does not belong within the Pinnotherinae.In the zoeal stages of Pinnotherinae s.s.(sensu stricto, as per Cuesta et al., 2001), the antenna is absent or reduced to small setae, whereas in T. moseri it is present as a small but well-developed protopod with two rows of spinules.The zoeal telson in T. moseri differs very strikingly from Pinnotherinae s.s. in that it is furcate and bears two small dorsal spines instead of the peculiar trilobate trapezoidal shape that typifies zoeae of the Pinnotherinae.These two zoeal characters place T. moseri close to the genera Calyptraeotheres, Clypeasterophilus, Dissodactylus, Pinnaxodes, and Tumidotheres, composing an intermediate group between Pinnotherinae s.s. and Pinnothereliinae.Campos (2000) has, on the basis of adult morphology, also rediagnosed the subfamily Pinnotherinae and concluded that the genera Calyptraeotheres, Clypeasterophilus, Dissodactylus, Fabia, Holothuriophilus, Juxtafabia, Ophistopus, Parapinnixa, Pinnaxodes and Tumidotheres do not belong there.We find that Tunicotheres moseri likewise does not exhibit the complete set of adult characters proposed by Campos (2000) for defining the true Pinnotherinae, viz: soft carapace, slender and atrophied walking legs, the ischium and merus of third maxilliped indistinguishably fused and the presence of a tubercle on the basal antennal segment.Finally, preliminary comparisons of mitochondrial DNA sequences support removal of T. moseri from the Pinnotherinae s.s. and clarify its relationship with some other Pinnotherinae genera, as will be addressed in a forthcoming paper (Cuesta and Felder, in prep.).
A comparison of the present larval description of T. moseri with the first brief larval description of this species by Goodbody (1960) shows two distinct dif-ferences.Goodbody described only a single zoeal stage, while we found two zoeal stages, both in larvae we cultured in vitro and among specimens removed directly from the abdominal enclosures of the females.Goodbody (1960) also described hatching processes and natatory behaviours in the first zoea that were never seen in the present study.We never observed hatching, and facultative swimming was observed only in those larvae hatched from eggs cultured in vitro.Despite the apparent adaptation of early development to the confines of the parental female's abdominal enclosure and thus limited area for locomotion, the larvae did not lose natatory capacity.How this swimming behaviour might be inhibited or initiated in larvae that hatch and develop inside the abdominal enclosure is unknown at present.
The duration of the zoeal stage described by Goodbody (1960) falls into the range recorded for the first zoeal stage in the present study, between 24 and 36 h.The zoea I illustrated by Goodbody (1960: 471, Fig. 1) exhibits characters typical of a last zoeal stage, similar to those of the zoea II described in the present paper, but appears to have only four natatory setae characteristic of a zoea I.The latter pattern at least suggests that no preceding stage was overlooked by Goodbody (1960), but could also indicate that the previously described larvae exhibit a different pattern in natatory setae during zoeal development.Unfortunately, these earlier larval descriptions are not detailed for any stage, and no further comparisons can be made.We cannot exclude the possibility that the specimens described by Goodbody (1960) from Jamaica could in fact represent a different species from populations reported in the present paper, especially given the limited potential for larval dispersal and thus apparent high potential for endemism.
Preliminary comparisons of mtDNA sequences for specimens of putative T. moseri taken in Venezuela and Florida suggest that at least these two are in fact different species (Cuesta and Felder, in prep.), but it remains to be determined whether either of these two populations is conspecific with topotypes from Jamaica.Sequencing of mtDNA from Jamaican specimens of T. moseri, along with further studies of larval morphology for both the Florida and Jamaican populations, should do much to clarify the relationships of these three possibly isolated populations.
The possibility of yet another unrecorded population of T. moseri could be inferred from the direct development described for "Pinnotheres sp." by Kurata (1970: 229).According to Kurata (1970) a first young stage "emerged from the brood pouch of the female", and the brief description of this stage and the illustration clearly match the first crab of T. moseri.The ovigerous female was collected at Sponge Reef off Sapelo Island (Georgia, US), but no data are provided regarding the host.Unfortunately the material is no longer extant (R. Heard, pers.comm.) and we therefore cannot identify the parental female.We conclude that this record must represent either another population of T. moseri or a population of another closely related species with a similar larval history.

TABLE 1 .
-Dimensions (rostro-dorsal length, rdl, carapace width, cw, and carapace length, cl; mean ± SD in mm) for zoeae and megalopa of Tumidotheres moseri, along with day of first appearance(F)and duration of each stage (D) in days.