First record of the hyperparasite Liriopsis pygmaea (Cryptoniscidae, Isopoda) from a rhizocephalan parasite of the false king crab Paralomis granulosa from the Beagle Channel (Argentina), with a redescription

The Cryptoniscidae are epicaridean isopod parasites or hyperparasites of other crustaceans. Liriopsis Schultze in Müller, 1859, one of the genera included in this family, now contains two nominal species: L. pygmaea (Rathke, 1843) and L. monophthalma (Fraisse, 1878). Both of these species infest rhizocephalan cirripeds, which are in turn parasites of hermit crabs. Among the false king crabs, Paralomis granulosa (Jacquinot, 1847), captured commercially in the Beagle Channel in 1996–1998, we found 31 specimens of the rhizocephalan Briarosaccus callosus Boschma, 1930 infested by one or more specimens of L. pygmaea. Neither L. pygmaea nor L. monophthalma has been reported previously for the southern seas. Although unidentified isopod hyperparasites have been found on B. callosus infesting other lithodids from Crozet Islands, South Georgia Island and Canadian Atlantic waters, this is the first time that one of these hyperparasites has been identified as a member of the genus Liriopsis. Since the differences between L. pygmaea and L. monophthalma remain obscure, the epicaridium and cryptoniscus larvae and three females stages of L. pygmaea are herein described from the material collected in the Beagle Channel.


Introduction
Cryptoniscid isopods belong to the suborder Epicaridea and are parasites or hyperparasites of a great variety of other crustaceans (Grygier and Bowman 1990). They are protandrous hermaphrodites, functional males when young and later changing into females. Eggbearing females lose their isopod appearance and take a sac-like shape (Caullery 1908). Grygier (1993) reviewed the nomeclature of the family Cryptoniscidae. The genus Liriopsis Schultze in Mü ller, 1859 includes two nominal species, L. pygmaea (Rathke, 1843) and L. monophthalma (Fraisse, 1878); however, the differences between these two species remain obscure.
A total of 29 570 specimens of Paralomis granulosa were collected in the Beagle Channel (about 54u549S, 67u129W), Tierra del Fuego, Argentina, at 10-50 m depth, from 1996 to 1998 (for further sampling details, see Roccatagliata and Lovrich 1999).
Only 78 specimens of P. granulosa were infested by B. callosus, with one (occasionally two) rhizocephalan externae under their abdomens. The externae were removed from the crab, fixed in 5% formalin and dissected under a stereomicroscope for stages of isopod hyperparasites. In 31 of the 85 externae dissected, one or more stages of a cryptoniscid isopod, Liriopsis pygmaea, were recovered. All the dissected externae and the recovered isopods were preserved in 70% ethanol.
Almost all of the cryptoniscus larvae and all of the early subadult females were found inside the mantle cavity of the externae. On the other hand, a few cryptonisci, all the advanced subadult females (excepting one aberrant female that was found within the mantle), and all of the adult females were attached to the outer wall of the externae. A few epicaridium larvae taken from the brood pouch of a female, collected on 23 March 1997, were used for the description of this larval stage.
Cryptoniscus larvae and functional males are not distinguishable by their external features, both retain the typical isopod morphology. The term 'cryptoniscus larva' is used in this survey to refer to both stages.
Appendages were dissected and stained with a saturated solution in 70% ethanol of Chlorazole Black E. Drawings were made with the help of a camera lucida. Material for SEM was cleaned with a diluted solution of TritonH X-100 detergent and exposed to ultrasonic treatment for a few seconds.
The total length of the epicaridium and cryptoniscus larvae was measured along the midline from the anterior edge of the cephalon to the tip of the pleotelson.
Most of the material studied has been deposited in the Museo Argentino de Ciencias Naturales 'Bernardino Rivadavia' .
The abbreviation 'Cr' refers to the cryptoniscus larva, 'SAR 1 ' to the early subadult female, 'SAR 2 ' to the advanced subadult female and 'AR' to the adult female.
First antenna. Peduncle with two articles, basal article with three plumose setae. Rami with two and three distal setae, respectively (articulation with peduncle observed for one ramus only), two aesthetascs arising between rami.
Second antenna (Figure 2a). Peduncle with three articles; distal article longest, with three plumose and one small simple setae, distally. Flagellum with three articles gradually decreasing in length distally, third article with three long lamellae (only one drawn) and five setae (at least some of them plumose), largest seta as long as antenna.
Uropod (Figures 1b, 2d). Uropod widely covered dorsally by pleotelson lamella. Peduncle with one long plumose seta on outer distal angle. Exopod placed dorsally and anteriorly to endopod; distal end with two plumose setae. Endopod slightly shorter than exopod, with two large setae curved downwards and one small straight seta.
Body (Figures 3a, b, 4a). Body flattened dorsoventrally, gradually narrowing posteriorly, dorsal surface with faint (visible only under high magnification) transverse striations (not drawn). Cephalon slightly longer than half its width, frontal edge almost semicircular. Eyes very distinct, consisting of a single lens encircled by reddish pigment. All pereonites with a couple of minute setae dorsally; third and fourth pereonites slightly shorter than remaining ones. Pleon (including pleotelson) somewhat longer than the first five pereonites together; first to fourth pleonites with mid-ventral lobe between pleopods, becoming gradually less developed from first ( Figure 3e) to fourth and disappearing on fifth. Pleotelson with posterior margin rounded and entire, slightly exceeding peduncles of uropods.
First antenna (Figure 4b). Peduncle with three articles. First article posteriorly expanded; caudal end rounded, with three setae (one plumose, two simple); anterior end projected medially into large spur-like tooth (either exposed, Figure 3d, or partially covered by median plate, Figure 3c), and laterally with three setae (one plumose, two simple). Second peduncular article, ornamented with delicate ridges bounding polygonal areas ( Figure 3d); small tooth halfway along its anterior margin ( Figure 3d); distal end with row of four setae (two plumose, two simple) anteriorly and two setae (one plumose, one simple) posteriorly. Third peduncular article partly hidden by second one; anterior margin with long simple seta; distal margin with numerous aesthetascs (only most ventral ones drawn). Ventral flagellum with three long distal setae, dorsal flagellum with four long distal setae and two basal aesthetascs.
Second antenna (Figure 4c). Second antenna scarcely extending beyond fourth pereonite (including distal setae). Peduncle with four articles, first two articles with fine striae. First article largest and unarmed, remaining ones gradually increasing in length and with setae, namely second article with one simple, third article with two plumose and one simple, and fourth article with four plumose (only ventral ones shown) and one simple setae. Flagellum with five articles, approximately 0.70 times as long as peduncle, with 2-1-2-1 short simple setae on first four articles (only ventral ones shown); fifth article with four unequal distal setae.
Pereopods. First pereopod (Figure 4d): basis with strong distal condyle flanked by two transparent lamellae. Ischium 0.65-0.73 times as long as basis. Merus with seta-like projection anteriorly and two feeble simple setae posteriorly. Carpus, armed on posterior margin with distal tiny spine between two unequal setae, proximal one hardly noticeable. Propodus slightly shorter than basis, broadly articulated with carpus, postero-distal angle with a depressed area limited proximally by a blunt process bearing two unequal spines (see detail). Dactylus fitting into depressed area of propodus, anterior margin slightly serrated, with one stout spine and one delicate seta distally. Second pereopod similar in shape to first, but slightly larger. Third (Figure 5a) to fifth pereopods alike, but slightly increasing in length posteriorly. Basis length roughly equal to that of propodus. Ischium 0.76-0.81 times as long as propodus, biceps-like, distally with two transparent lamellae flanking merus. Merus with one thick and one feeble setae, distally. Carpus with strong spine on distal-posterior angle. Propodus with three spines in distal depressed area, posterior margin with fringe of minute setae. Dactylus apparently fused with distal spine (no articulation discernible at high magnification), both together approximately half as long as propodus, posterior margin with fringe of minute setae, distal end bifid.
Sixth pereopod (Figures 3e, 5b): ischium approximately half as long as basis. Merus with one thick and one feeble setae, distally. Carpus approximately as long as merus. Propodus almost globular, approximately 0.75 times as long as ischium. Dactylus (probably the dactylus itself plus a completely fused distal spine) extremely large and styliform, almost twice as long as basis. Seventh pereopod (Figures 3e, 5c) as sixth except for: shorter thick seta on merus (only the thicker basal part of this seta remains).
Pleopods. All five pleopods alike (Figure 5d). Basal segment with two coupling setae on inner margin, these slightly longer than endopod. In first four pleopods both rami with five plumose setae distally, outermost setae of exopods clearly shorter than remaining ones. Fifth pleopod as previous ones, except endopod with only three distal setae.
Uropod (Figure 5e). Peduncle and endopod combined approximately as long as pleotelson. Peduncle widely covered by pleotelson, outer margin with two unequal simple setae distally, longer one reaching end of exopod. Exopod approximately 0.80 times as long as endopod. Endopod with six delicate plumose setae proximally on dorsal surface, inner margin with fringe of minute setae, distal end with five plumose and three simple setae, two of latter longer than others, 1.5 and twice as long as article, respectively. Exopod, with five simple setae at distal end, longest seta approximately three times as long as article.
Whole isopod inside mantle cavity of rhizocephalan externa. Body (Figure 6a, b) orangereddish (same colour as rhizocephalan ovary), consisting of large sac with small flat disc tilted over it. Appendages absent. Sac with five or six lateral lobes and pyriform vesicle (heart?) visible through cuticle. Disc with irregular concentric folds, detached from sac on one side but continuous with it on the other. Apparently, sac develops as internal anchor and disc becomes external brood sac in latter stages.
Anterior part of isopod protruding from mantle cavity of rhizocephalan externa. Body (Figure 6c) divided by constriction into two parts: rhomboidal caudal anchor, immersed in mantle cavity, and a freely exposed anterior subspherical sac. In some specimens developing adult visible beneath subadult cuticle. These developing adults with incipient brood pouch on ventral surface, visible as elongated area bounded by two folds and with rudiments of anterior finger-like processes and caudal digitations at its extremities.
Anchor with definitive form and sac exhibiting long ventral slit, through which the epicaridium larvae are expelled. Body (Figure 6d, e) divided by narrow neck into anchor deeply embedded in host's body and subspherical part (brood pouch) protruding from host's mantle wall. Anchor part: dorsal surface convex, with three parallel hollow bars; ventral surface concave, with tiny central papilla (called 'vésicule rectale' by Caullery, 1908, in his description of Liriopsis monophthalma). Heart visible through translucent cuticle as rounded sac below this papilla. Brood pouch: dorsal surface circumvallated by four lines, with five bunches of longitudinal muscles between them (muscles only evident in spent females). Ventral surface with longitudinal mid-line groove in ovigerous females (visible as slit in spent females). Mid-line furrow (slit): anterior end with finger-shaped process fitting between thick margins ( Figure 6f); posterior end with six internal digitations, only visible by stretching pouch and peering through slit (Figure 6g).

Discussion
The genus Liriopsis contains two nominal species: L. pygmaea and L. monophthalma (see Grygier 1993). Rathke (1843) described Liriope pygmaea, the type species by monotypy of the genus, based on a few cryptoniscus larvae. In his description, Rathke mentioned that the last (seventh) pair of pereopods of the larva was shorter and thinner than the previous ones and ended in a stylet-like article (see also his Figure 11 in pl. I). Sars (1899) presented a more detailed description with illustrations of the cryptoniscus larva; he noticed that the last two pairs of pereopods were very small, with the propodus almost globular and the dactylus straight and styliform. More recently, Nielsen and Strömberg (1973) published SEM photographs of the two pereopods (see their Figures 36, 37) confirming Sars' description.
Our cryptoniscus larva differs from previous descriptions of L. pygmaea in the following details: (1) Sars (1899) stated that the large, linguiform expansion of the first antennular article had no traces of setae or spines; however, these setae are present both in an SEM photographs presented by Nielsen and Strö mberg (1973: Figure 6) and in our specimens (see Figure 4b).
(2) Sars (1899) mentioned and illustrated (see his figure '1C.' in pl. 100) two teeth on the frontal edge of the second article of the antennule, and Nielsen and Strö mberg (1973: Figures 6, 7b) provided SEM photographs of these two teeth. In contrast, our larvae have only one tooth there (see Figures 3d, 4b).
(3) Sars (1899) stated that the exopod of the uropod scarcely attains half the length of the endopod (see his '1Urs.' in pl. 100), but in our specimens the exopod is approximately 80% as long as the endopod (see Figure 5e). Fraisse (1878) described and illustrated the cryptoniscus larva of Cryptoniscus monophthalmus. Later, Bonnier (1900) transferred this species to the genus Liriopsis, where it has since remained. According to Fraisse (1878), this larva has only the last (seventh) pereopod different from the others, with a fusiform propodus, of about the same length as the dactylus (see his Figures 45 and 54a in pl. XV). On the other hand, Caullery (1908) noticed that the last two pairs of pereopods (sixth and seventh) of L. monophthalma were quite similar to those of L. pygmaea, i.e. both have a subglobular propodus and a sword-like dactylus (see his figures B10-11). Since these two available descriptions are inadequate and also contradictory, we cannot establish with certainty the differences between the cryptoniscus larvae of L. monophthalma and L. pygmaea. Lilljeborg (1859) described the epicaridium of L. pygmaea. Our specimens mainly differ from that description in having a biramous antennule and an anal tube distally. For L. monophthalma, Fraisse (1878) reported a bifid antennule but he did not mention an anal tube. Caullery (1908) revealed that L. monophthalma had an anal tube but unfortunately he did not illustrate it. Epicaridium larvae are very small and some anatomical aspects are hardly visible even at high magnification, therefore, some structures could have been erroneously reported or omitted in earlier descriptions. Caullery (1908) described several juvenile females of L. monophthalma, all of them free in the mantle cavity of their host. The earliest stage is similar to the male except for having only two pairs of appendages, the antennules and the second(?) pereopods; the following stages become gradually broader. Despite the fact that we have recovered hundreds of tiny cryptoniscus larvae from our dissected rhizocephalan externae, no juvenile females have been found.
The stage herein referred to as SAR 1 resembles Caullery's description of L. monophthalma (1908: figure G3). However, the anterior end of the early subadult female of L. monophthalma is more or less conical, whereas that of our SAR 1 carries a flat apical disc. This disc, by increasing many-fold in size, is likely to become the large brood pouch of the AR. The developing AR is sometimes visible through the cuticle of the SAR 2 , confirming that the latter effectively precedes the former.
The AR of L. pygmaea was satisfactorily described by Lilljeborg (1859). In addition, Fraisse (1878) briefly described the AR of L. monophthalma, but did not mention any feature distinguishing it from the female of L. pygmaea. Lilljeborg (1859) had the opportunity to observe living specimens of L. pygmaea showing pulsating movements of the brood sac. As was suggested by Caullery (1908), these contractions seem to be generated by the dorsal bundles of muscles, which have been represented in Figure 6d of the present paper.
The differences between L. pygmaea and L. monophthalma are obscure. The latter species is apparently restricted to the Mediterranean Sea, whereas L. pygmaea appears to have a broader geographic distribution. Because of the lack of detailed descriptions of these two species, and in order to avoid future nomenclatural problems, we provisionally identify our specimens as L. pygmaea, the type species of the genus. It is hoped that the present descriptions, together with the study of other relevant material, will allow a thorough revision of this genus in the future.