Chaetotaxy and setal diversity of grooming legs in species of porcelain crabs (Crustacea: Anomura: Porcellanidae)

The morphology of the fifth pereiopods was studied under scanning electron microscopy in ten species of porcelain crabs for chaetotaxy and setal diversity, namelly Megalobrachium pacificum, Megalobrachium roseum, Pachycheles grossimanus, Petrolisthes armatus, Petrolisthes tuberculatus, Pisidia brasiliensis, Pisidia longicornis, Polyonyx gibbesi, Porcellana platycheles and Porcellana sayana. Six setal types were identified: simple, pappose, sickle-shaped serrate, straight serrate, club-shaped and tooth-like cuspidate. Porcelain crabs can differ in the fifth pereiopod setal morphology, chaetotaxy and setal density, even among species within the same genus. The absence of sexual dimorphism of the grooming legs in porcelain crabs suggests that grooming eggs requires no particular grooming apparatus in females and that male and female are equal in grooming efficiency. Key-Words. Fifth pereiopod; Porcellanid crabs; Seta; Setal types.


INTRODUCTION
Many decapod crustaceans use at least one pair of thoracopods for grooming the body. In anomuran crustaceans, the fifth pereiopod is chelate in males and females and differ significantly from other pereiopods by being distinctly morphologically reduced, markedly flexible, and often carried underneath the carapace. The fifth pereiopod is a cleaning appendage with cuticular outgrowths used to brush off fouling organisms and residues from gills, eggs and the general body surface (Bauer, 1977(Bauer, , 1981(Bauer, , 1989Martin & Abele, 1988;Forster & Baeza, 2001). Grooming may also be effective in the removal of larvae and infective stages of rhizocephalan parasites of the branchial chamber in porcelain crabs (Ritchie & Høeg, 1981).
It has been abundantly shown by others that sensory and respiratory structures as well as developing eggs may suffer severe fouling and agglomeration of residues from water when prevented from grooming (Bauer, 1977(Bauer, , 1981(Bauer, , 1989Holmquist, 1989;Pohle, 1989;Keiler & Richter, 2011). Different setal types are used for different mechanical functions (e.g., Schembri, 1982;Fleischer et al., 1992;Garm, 2004a, b), such as brushing, combing, and scraping. Thus, setal diversity enhances grooming and cleaning efficiency of the grooming structures.
The morphological diversity of setae and chaetotaxy (setal arrangement) on the groom-ing apparatus of the distal segments of the fifth pereiopod in the Anomura have been the focus of several studies, but less commonly so in the porcellanid crabs with ten species in five genera to date (Table 1). In this paper, we examine the chaetotaxy and setal diversity on the grooming apparatus of the fifth pereiopod in ten porcelain crabs species related to six genera. Table 1. Porcelain crab species studied to date for grooming leg setation.

MATERIAL AND METHODS
Several adult males and females from ten species of porcellanid crabs, in six genera, were obtained from the collections of the Museu de Zoologia, Universidade de São Paulo (MZUSP) and their fifth pereiopods (P5) were dissected using a stereomicroscope (Appendix 1). The specimens had been previously stored in 70% ethanol. The P5 were rinsed in a weak solution of dishwater detergent in distilled water prior to being cleaned mechanically by ultrasound (~5 minutes) and then by soft brushing while immersed in distilled water to remove excess fixative.

Comparative gross morphology of P5
In porcelain crabs the 7-segmented P5 is much smaller and remarkably more slender and flexible than P2-P4 and truly chelate in males and females. There is no sexual dimorphism of the chelae. The coxa and basis is followed by an endopodite with five distinct segments: ischium, merus, carpus, propodus and dactylus (Figs. 1A; 2A; 3A; 4C; 5A, G; 6A, E; 7A, D).
The merus of the P5 is by far the longest segment in the leg. The carpus can be slightly shorter than merus, as in Pachycheles grossimanus, Petrolisthes armatus, P. tuberculatus, Pisidia longicornis and Porcellana platycheles (e.g., Figs. 4C, 5G), or distinctly shorter than merus in Megalobrachium pacificum, M. roseum, Pisidia brasiliensis, Polyonyx gibbesi and Porcellana sayana (e.g., Fig. 6A, E). However, while in Megalobrachium pacificum, M. roseum and Porcellana sayana, the fixed and the movable fingers leave a very distinct gap when the chela is closed (e.g., Figs. 2F; 7E), no such is the case in Pachycheles grossimanus, Petrolisthes armatus, Petrolisthes tuberculatus, Pisidia brasiliensis, P. longicornis, Polyonyx gibbesi and Porcellana platycheles, in which the fingers close completely without an apparent gap (e.g., Figs. 4F; 6B). Expectedly, true chelae pick and remove fouling and debris more easily than subchelae, and gaping chelae can fit closely and hold more firmly larger fouling objects and residues than non-gaping chelae. Bauer (1989) reported cheliped picking at the gills in Petrolisthes galathinus, and Fleischer et al. (1992) commented on the role played by chela in dealing with objects not removed by the grooming setae. Softness and thickness of the gill filaments in Petrolisthes spp. play a role in allowing insertion of the chela in between the narrowly spaced gill filaments (Høeg, 1985;Fleischer et al., 1992).

Morphological adaptations of P5 to grooming
The P5 coxa and basis are united by a very large arthrodial membrane (Figs. 1A; 4C; 5A, C), which allows for a tight fold of the leg against the coxa, lending great mobility to the grooming leg. The male gonopore (Fig. 6A) located on the coxa (see also Guinot et al., 2013: 14) is large, operculated, and without setae. The basis is very short and fused to ischium (Figs. 1A; 2A; 4C; 5A, G; 6A, E; 7A, D), with suture line visible (Fig. 3E), so that the basis-ischium form a functional unit lending extra strength to the leg movement. The ischium and merus are not fused together (Figs. 1A; 4C; 5G; 6E; 7A, D). The merus and carpus are disproportionately long, an adaptation to reach more distant parts of the body. The merus is excavated on its ventral surface so to allow for a tight fold of carpus ( Fig. 6A, E). The merus and carpus are distinctly slender, dorsoventrally flattened and curved ( Fig. 4C) so to fit the space between the carapace and the gills and match closely the surface curvature of the body underneath the carapace. The hinged articulation and the extensive area of arthrodial membrane between merus and carpus lend extra mobility to the grooming leg (Fig. 5G).
The flexibility of the pereiopods is enhanced by the fact that the movement of any two given contiguous segments is alternate and opposite to each other, so that if one given segment moves horizontally the next contiguous segment will move vertically so as to widen the moving range. The direction of the movement of a particular leg segment (whether horizontal or vertical) is given by the position of its particular condyles. Lateral to lateral articular condyles only permit dorsoventral movements, whereas dorsoventral articular condyles impose lateral to lateral movements. The pereiopod dactylus, for instance, articulates with the propodus through a pair of lateral to lateral articular condyles and, therefore, the dactylus can only execute vertical movements. The propodus articulates to carpus through a pair of dorsoventral articular condyles, which in turn articulates to merus through a pair of lateral to lateral articular condyles, and so forth. The articular condyles thus alternate from lateral to lateral to dorsoventral from dactylus to coxa, so that the leg segments move in respect to each other in an alternate fashion -vertical, horizontal, vertical, and so on. In P5, however, the articulation dactylus-propodus is rotated inward so that the articular condyles assume an almost dorsoventral position (instead of lateral to lateral as seen in P2 to P4; see Table 2) and, therefore, the chela is flipped onto its side, an adaptation for easy sliding into the constricted space between the carapace and the gills. Furthermore, whereas the thoracic somites I-VII are fused together and are immovable in relation to one another, in anomurans the last thoracic somite (VIII), which carries the P5, is not fused to the precedent somite (VII). The somite VIII is connected to somite VII through a large membranous area thus being movable and lending additional mobility to the grooming leg (Fig. 8A).
The grooming leg is furnished with a terminal, weak chela in which the movable dactylus is about 1/3 to 1/4 length of the propodus. The chela is very small being only 1/5 to 1/7 of the maximal length of the carpus; the fingers have a duckbill shape distally (Figs. 2E; 3F; 4F; 5B, E; 6B, F-G; 7F). In resting position the grooming leg is folded in Z-form outside of the branchial chamber ( Fig. 8B), but when extended inside the branchial chamber the terminal chela can reach far forward to the entire branchial region (Fig. 8C).

Chaetotaxy and setal diversity on the grooming legs
Generally, the dorsal surface of the P5 coxa and the subdistal end of basis-ischium have one small tuft of pappose setae each (Figs. 1A, C; 2B; 3D). The P5 merus has very scattered, minute pappose setae (Fig. 1D), while the distal parts of carpus, propodus and the entire dactylus are densely setose (Fig. 1A). Not surprisingly, the distal parts of carpus, propodus and dactylus also concentrate most of the setal diversity (Figs. 1A; 2E-F; 3F; 4F; 5C; 6B, F-G; 7E-F), since these are the parts of the grooming leg that most easily reach almost everywhere in the gill chamber and various areas of the body in general. Six distinct setal types were found in the P5 of the studied species: straight serrate setae, sickle-shaped serrate setae, pappose setae, simple setae, club-shaped setae, and tooth-like cuspidate setae. Setal distribution among species is summarized in Table 3 (Fleischer et al., 1992;Forster & Baeza, 2001). Multiple rows of these setae were found along the inner and outer margins of the chelae on both dactylus and propodus. The club-shaped setae were referred to as curved setae and small simple setae in Petrolisthes violaceus and Porcellana platycheles by Forster & Baeza (2001) and Keiler & Richter (2011), respectively. Shaft usually long, slender, without terminal pore. Setules long, randomly arranged along the entire length of shaft lending feather-like appearance to setae, as also seen in plumose setae (Fleischer et al., 1992;Forster & Baeza, 2001;Garm, 2004b). In all the species studied the pappose setae were scattered arranged on the coxa and formed a dense tuft on the ventrodistal surface of ischium near the joint to the merus. These setae were also present as a fringe along the lateral margin of the merus. Fleischer et al. (1992) and Forster & Baeza (2001: 608, fig. 1) recorded the presence of plumose setae in the ischium near the joint to the merus in both Petrolisthes cabrilloi and Petrolisthes violaceus, respectively. However, in P. cabrilloi and in P. violaceus the setules on the shaft are located randomly throughout the shaft, instead of arranged into two rows on the opposite sides of the shaft. For this reason, we interpret these setae to be pappose instead of plumose.
Sickle-shaped serrate setae (S1), Figs. 1A, G; 2A, E; 3A, F, G; 4C, F; 5A, C, D; 6A, B, F; 7F: Shaft very long, curved, with double row of spaced denticles; distal edge (tip of seta) slender, twisted, devoid of denticles (Fleischer et al., 1992;Forster & Baeza, 2001;Keiler & Richter, 2011). This setal type was found on the ventral surface of propodus, with a larger portion present on the proximal margin, and a smaller portion on the distal margin. The sickle-shaped serrate setae are always in contact with the gills as a result to their position on the propodus (e.g., Fig. 4F). Fleischer et al. (1992) commented that sickle-shaped serrate setae can be used for scraping and their curved, serrated part can reach difficult-of-access body areas (e.g., between gill filaments) thus enhancing grooming efficiency.  (Fleischer et al., 1992;Forster & Baeza, 2001;Keiler & Richter, 2011). Straight serrate setae were present in large numbers on the ventral and dorsal surfaces of propodus and dactylus. The carpus bore a tuft of straight serrate setae ventrodistally. The strong denticulation of the straight serrate setae (Fig. 4E) are used for combing and grooming (Schembri 1982;Watling, 1989). This setal type was named as straight long setae in Petrolisthes violaceus and composite serrate . Each seta is divided into an inner and an outer part with a terminal pore ( Fig. 1H; 6D) (see also Fleischer et al., 1992). In the literature this seta has also been called "teeth setae" in Petrolisthes cabrilloi and "tooth-shaped projections" in P. violaceus by Fleischer et al. (1992: 428, fig . 3F) and Forster & Baeza (2001: 608), respectively.

Comparative chaetotaxy
Porcelain crabs can differ in P5 setal morphology, chaetotaxy and setal density, even among species within the same genus. The tooth-like cuspidate setae around the chelae varies in morphology according to species. In Megalobrachium pacificum, Pachycheles grossimanus, Petrolisthes armatus, Pisidia brasiliensis, P. longicornis and Porcellana platycheles, the tooth-like cuspidate setae were straight truncate and provided with no distinct terminal projections (Figs. 1H; 3H; 4A, B, G, H; 5H; 6C, D; 7C), whereas in Petrolisthes tuberculatus the distal margin of the toothlike cuspidate setae was also truncate, but furnished with a strong, broadly curved, sharp, single mesial spine (Fig. 5F). Contrary, in Megalobrachium roseum, Polyonyx gibbesi and Porcellana sayana the distal margin of the tooth-like cuspidate setae was rounded, provided with minute, unevenly sized, slightly acuminate teeth, but never formed a strong spine as in Petrolisthes tuberculatus (Figs. 2H; 6H; 7H). Fleischer et al. (1992) found almost no interspecific differences in the morphology of P5 in Petrolisthes spp. and only little differences between Petrolisthes spp. and Pachycheles spp. However, Petrolisthes cinctipes and P. galathinus (from Fleischer et al., 1992: 4B-C, respectively) differ from the single-spined P. tuberculatus (Fig. 5F) in having a pair of terminal spines in the tooth-like cuspidate setae, whereas Petrolisthes cabrilloi has no terminal spines (Fleischer et al., 1992: 4D). Mediterranean Sea specimens of Porcellana platycheles were reported to have a pair of terminal spines in the tooth-like cuspidate setae (Keiler & Richter, 2011: fig. 6F), while the southwestern Atlantic specimens studied herein are devoid of any terminal spines (Fig. 7C). Keiler & Richter (2011) reported artificial abrasions in Porcellanidae spp. In the southwestern Atlantic specimens of P. platycheles tooth-like cuspidate setae are very similar in size and shape to one another, suggesting that their feature is not the result of abrasion. A moderate dense fringe of pappose setae were present on the lateral margin of merus in Megalobrachium pacificum, Pachycheles grossimanus, Petrolisthes armatus, Pisidia brasiliensis, Polyonyx gibbesi, Porcellana platycheles and P. sayana (Figs. 1A, D; 4C; 7A), whereas pappose setae are scarce and minute on the lateral margin of merus in Megalobrachium roseum, Petrolisthes tuberculatus and Pisidia longicornis (Fig. 5A, G).
In the Petrolisthes spp. and Pachycheles spp. studied by Fleischer et al. (1992), simple setae (termed smooth setae by Fleischer et al., 1992) were restricted to the chelae margins, whereas in Petrolisthes armatus and P. tuberculatus and Pachycheles grossimanus, but also in Megalobrachium roseum, M. pacificum, Pisidia brasiliensis, P. longicornis, Polyonyx gibbesi, Porcellana platycheles and P. sayana (present study), simple setae were found additionally in tufts on the P5 coxa (e.g., Fig. 1A, B) and scattered over the leg. Fleischer et al. (1992) suggested that the porcellanid P5 ground pattern is fitted to deal with a variety of situations and removal of fouling and foreign objects and even the removal of parasites requires no particular adaptations. In addition, we submit that the absence of sexual dimorphism of the grooming legs in porcelain crabs suggests that grooming eggs requires no particular grooming apparatus in females and that male and female are equal in grooming efficiency.