H. Glenner á J. T. Hùeg á J. J. O'Brien á T. D. Sherman

Invasive vermigon stage in the parasitic barnaclesLoxothylacus texanus and L. panopaei (Sacculinidae): closingof the rhizocephalan life-cycle

Received: 12 August 1999 /Accepted: 3 November 1999

Abstract The parasitic barnacles, Rhizocephala, areunique in Crustacea by having an entirely endo-para-sitic phase inserted into their lifecycle. A cypris larva,remarkably similar to the cypris of conventional acornand goose barnacles (Thoracica), settles on the crusta-cean host and develops an infective stage, the ken-trogon, underneath the exuviae of the cypris. Thekentrogon penetrates the integument of the host by ahollow cuticle structure, the stylet, and injects theparasitic material into the hemocoelic ¯uid of the host.Although advanced stages of the internal developmenthave been found and described several times, the na-ture of the originally injected parasitic material hasremained obscure for decades. Recently, however, itwas shown that the parasitic material was injected bythe kentrogon in the form of a motile, multi-cellularand vermiform body. The present study demonstratesthat the vermiform stage is an instar which forms theonly and direct link between the kentrogon and thematuring internal parasite. The vermiform instar, orvermigon, is at all times clothed in a cuticle, containsseveral types of cells, including epidermis and the an-lage of the later ovary, and stays intact while growinginto the internal parasites with rootlets.

Introduction

The Rhizocephala are barnacles that parasitize othercrustaceans and exercise a profound control over theirhosts by a�ecting them at morphological, physiologi-cal, and behavioural levels (O'Brien and van Wyk 1985;O'Brien and Skinner 1990; Hùeg 1995; Hùeg andLuÈ tzen 1995). Paramount among the numerous chan-ges induced in the hosts is parasitic castration. Gonadsof parasitized crabs do not mature and in consequenceparasitized hosts do not reproduce (Reinhard 1956;Kuris 1974). The end e�ect could be described eithercolloquially as transforming the host into a robot thatonly serves the needs of the parasite or, more appro-priately, as creating an organism possessing the phe-notype of the host and the genotype of the parasite(O'Brien 1999).

Studies of rhizocephalan life-cycles have taken on anew signi®cance in terms of their potential for resourcemanagement. Sacculinid rhizocephalans, which infestbrachyuran decapods, have been suggested as biologicalcontrol agents against introduced species of crabs, suchas the green crab, Carcinus maenas, which are perturbingmarine ecosystems throughout the world (La�erty andKuris 1996; Kuris 1997). These parasitic castrators areattractive as biological control agents because there isevidence to suggest that they may be speci®c in theirchoice of hosts; consequently, they might not present athreat to native crab species if introduced to control thepest crab (papers in Thresher 1997).

The life-cycle of sacculinids is direct; there is only onehost. Nauplii, released regularly from the adult para-sites, develop into typical cypris larvae. Sexes are sepa-rate in the Rhizocephala. Female cyprids settle on avulnerable host and metamorphose into a kentrogon,which develops a hollow cuticular stylet with which itpenetrates into the host and injects parasitic material.However, the nature of the injected material and theearliest internal development has until very recently re-mained obscure.

Marine Biology (2000) 136: 249±257 Ó Springer-Verlag 2000

Communicated by L. Hagerman, Helsingùr

H. Glenner (&)Department of Evolutionary Biology, Zoological Institute,University of Copenhagen, 15 Universitetsparken,DK-2100, Copenhagen, Denmark

J. T. HùegDepartment of Zoomorphology, Zoological Institute,University of Copenhagen, 15 Universitetsparken,DK-2100, Copenhagen, Denmark

J. J. O'Brien á T. D. ShermanDepartment of Biological Sciences, LSCB Rm. 124,University of South Alabama,Mobile, Alabama 36688, USA

The kentrogon stage was ®rst described by Delage(1884), but its anatomy remained entirely unknown untilHùeg (1985) gave a complete TEM-based description ofthe kentrogon of Lernaeodiscus porcellanae, and itsformation from the female cypris. While Hùeg (1985)did not actually observe the injection of a parasite intothe host, this crucial part of the life-cycle was ®nallydocumented by Glenner and Hùeg (1995). These authorsstudied the sacculinid Loxothylacus panopaei parasiti-zing the mud crab Rhithropanopeus harrisii and observedthe infection of the host in live material using video re-cordings followed by a TEM analysis (Fig. 1). Femalecyprids of L. panopaei settle in the gill chamber just asis the case in Lernaeodiscus porcellanae. Followingpenetration of the stylet, the Loxothylacus panopaeikentrogon expels a large, vermiform body into the host,which after an initial quiet period passes through a

phase of undulatory body motions. Glenner and Hùegreported that between 8 and 10 h following injection, thevermiform bodies begin to disintegrate and liberate nu-merous large cells exhibiting amoeboid motions. Theytook this as evidence that the ``amoeboid'' cells corre-sponded to the single invasion cell, which Hùeg (1985)suspected to be the only element passed into the hostfrom the Lernaeodiscus porcellanae kentrogon. Thedi�culty with this explanation revolves around howsuch a stage with naked, undi�erentiated cells couldhave become intercalated into the life-cycle of an oth-erwise highly evolved crustacean. In addition, Glennerand Hùeg (1995) did not observe the injected parasitesin situ in the host, but instead in preparations culturedin arti®cial sea water, and thus could not observe theparasite for any extended period of time.

The purpose of the work presented here was to ex-amine the vermiform stage in its natural environmentwithin the tissues of the crab. We achieved this by in-festing megalopa larvae and subjecting them to light andelectron microscopy and by observing recently injectedparasites in vivo within the crab. This approach dem-onstrates that the vermiform body never breaks up butinstead develops directly into a single internal parasitewhile always being clothed in a cuticle.

Materials and methods

Rearing and infection experiments

Mud crabs, Rhithropanopeus harrisii, parasitized with the sacculinidrhizocephalan Loxothylacus panopaei were sampled near the DukeUniversity Marine Laboratory, Beaufort, NC, USA. Larvae re-leased from the parasite were cultured to the cypris stage andbroods of female cyprids were used for infection experiments(Glenner and Brodin 1997). Larvae of R. harrisii were reared to themegalopa stage as in Forward and Wellins (1989) and then exposedto female L. panopaei cyprids. Larvae of Loxothylacus texanus werereared in a comparable fashion and female larvae used to infestblue crabs, Callinectes sapidus. In this parasite species the cypridssettle on the external body surface, where they attach at the base ofsetae, especially on the appendages and on intersegmental mem-branes. Numerous cyprids attached to the last, ®fth, pair ofthoracopods (the paddle).

Video recordings

Using light microscopy on live Callinectes sapidus immobilized toglass microscope slides by rubber bands, we observed the devel-opment of parasite larvae attached to the paddle and made videorecordings of the injected vermiform parasites in situ within thetissues of the limb.

Immunostaining

A few limbs of Callinectes sapidus containing vermiform parasitebodies were removed, and stained for actin using TRITC-phalloi-din (®nal concentration of approximately 20 lM) or for nuclei withthe addition of Hoechst 33258 (®nal concentration of approxi-mately 1 lg ml)1) in the solution containing crab tissue. Stainingwas performed after having opened the cuticle to allow observationand provide access for the stain. The stained preparations werethen observed with a Leitz epi¯uorescence microscope.

Fig. 1 The life-cycle of Loxothylacus panopaei parasitizing the mudcrab Rhithropanopeus harrisii. Nauplii, released by the externalreproductive body, develop into cyprids. The female cyprid settles inthe gill chamber of the crab and metamorphoses (moult) into akentrogon, which penetrates the integument and injects a vermigoninto the blood spaces. The vermigon migrates through the crab andgrows into an internal parasite, which eventually erupts with anexternal reproductive body. The cells of the primordial ovary (arrows)can be traced as a globular body of cells already from the kentrogon.All stages in the parasite life-cycle, including the early vermigon, areenveloped by epidermis and cuticle. Male parts of life-cycle excluded.Modi®ed from Glenner and Hùeg (1995); drawn by Beth Beyerholm(ce cement; st stylet; gl gill)

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Light and electron microscopy

Megalopa larvae were ®xed for TEM as in McDowell and Trump(1976) at various time intervals following exposure to the parasitelarvae. Fixed megalopae were rinsed in bu�er, dehydrated withdimethoxypropane and embedded in TAAB 812. Serial sectionswere ®rst cut at 2 lm, stained with toluidine blue and mounted onmicroscope slides. All sections of a series were observed through aLeica DM RXA, and photographs of sections stored digitally inLIDA (Leica Image Database) on a PC system. We also photo-graphed selected sections on 35 mm negative ®lm. This allowed usto detect, count and follow vermigons in a fast and easy manner.Exceptionally informative sections were extracted from the slidesand remounted on an TAAB 812 block for TEM analysis as fol-lows: we carefully removed the coverslip and then used fresh, un-polymerized TAAB 812 to glue an elongate piece of resin onto thesection in question. Subsequently we immersed the slide in liquidnitrogen enabling removal of the slide from the resin block byshaking. The section then adhered to the top of the resin block andcould be remounted for ultrathin sectioning for TEM. This tech-nique enabled us to scan through very long series of 2-lm-thicksections in a fast and easy manner using light microscopy and tostudy selected sections with internal stages using TEM.

Results

Observations on Loxothylacus panopaei

We observed numerous parasite larvae settled on thegills of Rhithropanopeus harrisii on sections of meg-alopae ®xed 2 to 4 d after exposure to parasite cyprids.Sometimes the parasite larvae occurred in such numbersthat they almost occluded the chamber. Numerousvermiform bodies, henceforward called vermigons,occurred within the blood spaces of the gills having beeninjected by the attached kentrogons (Fig. 2A, B).Occasionally the parasite larvae could actually cause thecarapace covering the branchial chamber to bulge out asis sometimes seen in decapods infested with isopod gillparasites.

From the positions of the vermigons in megalopae®xed at increasing time intervals after infection it followsthat they are initially located in branchial sinuses,whence they migrate through the heart and thence intoother body parts. Fig. 2D shows two vermigons in theprocess of migrating from the pericardium and into theheart through ostia. The vermigons were always lyingfree in the branchial blood sinuses without any signs ofhaving attached to the wall. Eventually the vermigonsmigrate to many parts of the body and we even detectedone lodged in the blood spaces of an eyestalk. Duringthis migratory phase the distinct spiralling body de-scribed by Glenner and Hùeg (1995) remains clearlyvisible extending the entire length of the vermigon(Fig. 2C, arrows).

The vermigons located in the blood spaces of the gillsand the heart had a size and morphology comparable tothat of recently injected ones (Glenner and Hùeg 1995).Those present elsewhere in the body were mostly larger,sometimes considerably so, and showed clear signs ofgrowth and maturation. The exceedingly thin ``sheath''surrounding the very recently injected vermigon

(Glenner and Hùeg 1995) remains intact at all times, butit increases in thickness and reveals itself to be a truecuticle, which was seen with LM on 2-lm sections(Fig. 2C, E).

At all stages the vermigon consists of at least threecellular components: an outer epidermis, a subjacent layerof large cells (the ``amoeboid cells'' of Glenner and Hùeg1995), and a very distinct globular body of small cells(Fig. 2C). The globular body has a diameter of half thewidth of the vermigon and is situated midway along thelength of the vermigon. With size and age these threecellular components become increasingly distinct fromeach other; the ultrastructural details of this growth pro-cess will be published elsewhere (Glenner in preparation).

The oldest vermigon bodies developed projectionsthat represent the earliest part of the rootlet system bywhich rhizocephalans absorb nutrients from their hosts.The cuticle surrounding these stages has increased inthickness and can easily be seen in the light microscope,although it still remains a delicate epicuticle (Fig. 2F).The thickness of this cuticle is only slightly thinner thanthe so-called homogenous layer of the cuticle coveringthe fully developed rootlet system of later stages (Hùeg1992). The above-mentioned globular body has devel-oped into the primordium of the external reproductivesac (externa). This primordium, very confusingly called``nucleus'' in much older literature, consists of a centralanlage to the ovary surrounded by an incipient but quitedistinct mantle cavity and its accompanying epithelia. Itis this primordium that will eventually emerge as theexterna beneath the abdomen of the host, while therootlet system remains within the crab. In the entirevermigon body, the tissue beneath the epidermis hasloosened up leaving large, sinusoidal spaces (Fig. 2E).At this stage, our internal Loxothylacus panopaei speci-mens are comparable in degree of development to thoseRubiliani et al. (1982) observed with light microscopy inthe sacculinid Sacculina carcini. The succeeding internaldevelopment is quite well documented in several species(Hùeg and LuÈ tzen 1995).

Observations on Loxothylacus texanus

Despite a supposed close relationship between L. pan-opaei and L. texanus, the latter has not been observed toattach in the branchial chamber, but instead upon theappendages just as in the ``classical'' rhizocephalanSacculina carcini. After exposure of Callinectes sapidusto female cyprids, we obtained numerous kentrogonsattached to the edge of the ``paddle'' of the last, ®fth,thoracopod. The kentrogons ejected vermigons, whichwe observed in vivo and in situ with light microscopy inthe limbs of crabs immobilized with rubber bands.

Morphology of the vermigon

Just as in Loxothylacus panopaei, the L. texanusvermigon showed no signs of breaking up but remained

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intact during 4 h of observation following liberationfrom the kentrogon. The L. texanus vermigon is verysimilar to the one in L. panopaei. One distinct similarityis the presence of a central spiralling rod, which extendsthroughout the length of the body, except for the mostdistal regions at either end (Figs. 2C, 3A, D).

Immunostaining

Specimens stained for actin with phalloidin showed anintense positive reaction both peripherally in the ver-migon and in the central spiralling structure (Fig. 3A).Focussing at high magni®cation at the level of the epi-dermis revealed a dense layer of actin ®laments orientedparallel with the long axis of the vermigon (Fig. 3B).These peripheral actin ®laments are most likely locatedin the epidermal cells. Such a concentration of epidermalactin ®laments occurs quite commonly in larvae of ma-rine invertebrates, where they can play an important rolein rapid changes of form during metamorphic processes,e.g. in the retraction of the tail in ascidian tadpole larvae(Cloney 1982). The pronounced reaction in the centralspiralling structure is more elusive. Our LM and TEMmicrographs failed to reveal a single cell or any row ofcells that could correspond to the spiral structure, andwe remain con®dent that it is not a muscle cell. FurtherTEM observations are needed to elucidate the mor-phology of this structure.

Motility of the vermigon

We videotaped the movement of Loxothylacus texanusvermigons in the distal segment (``paddle'') of the ®fththoracopod of Callinectes sapidus and replayed the re-

cordings at normal and fast-forward speeds. Most, if notall, larvae were located in the tissues exterior to ratherthan inside blood spaces and left open ``channels'' be-hind them as they forced their way slowly through thedense tissue of the limbs. During these movements, thediameter of the vermigon body changed only slightly, i.e.there was no pronounced peristalsis as seen in L. pan-opaei by Glenner and Hùeg (1995). Just as portions ofthe rod performed irregular meandering movements ineither direction along the long axis, the vermigons alsorepeatedly reversed the direction of their movements. Ingeneral, a wave of movement would pass down the en-tire rod, but not in a very organized fashion, andmovement could stop and reverse direction at any time.

Discussion

Our study con®rms and extends the observations ofGlenner and Hùeg (1995) except for one crucial obser-vation. The injected vermigon does not break up andliberate its cellular contents following injection into thehost. Instead it stays intact and enveloped within itsoriginal sheath, now interpreted as a cuticle, and deve-lops directly into the internal parasite equipped with asystem of nutrient-absorbing rootlets. Since the injectedparasite stays intact and is never devoid of a cuticle, itcorresponds to a true instar following the kentrogon. Wetherefore name this internal parasitic stage ``the ver-migon''. The etymology derives from Greek ``vermes''(worm) and ``gonos'' (larva) and is given to comply withthe kentrogon stage in females and the trichogon stagein males. We emphasize, however, that none of thesethree ``-gons'' may necessarily represent larvae in thestrict sense.

The break up of the vermigon and the liberation of itscells observed by Glenner and Hùeg (1995) is now in-terpreted as an artifact probably caused by incubatingthe vermigons in sea water rather than in hemocoelic¯uid. Glenner and Hùeg (1995) chose this procedurebecause settlement occurred on gills that had earlierbeen dissected from the gill chambers of the crabs, inorder to facilitate videotaping emergence of the subse-quent stage. In the current study, cyprids were allowedto settle and infect megalopa larvae and juvenile crabs,and larvae were never exposed to arti®cial solutions.Consequently the migration and development of thevermigons was monitored in their natural environment.

It is surprising that Loxothylacus panopaei cypridswill settle on megalopae of Rhithropanopeus harrisii,since it is normally believed that infection ofrhizocephalan hosts occurs only in postlarval stages. InR. harrisii, however, the megalopae spend most of theirtime on the bottom and are morphologically very closeto the ®rst crab stage. Walker et al. (1992) observed thepresence of an L. panopaei externa on a small Stage 1crab, which can only mean that this host was infectedwhile still a megalopa. These authors also noted cyprids

Fig. 2 Loxothylacus panopaei. Early development of the vermigonwithin megalopae ofRhithropanopeus harrisii. Light micrographs from2-lm epoxy resin sections. A Cyprids (arrowheads) within the gillchamber and a vermigon (arrow) within a blood space. The vermigon isstillwithinagill ®lament.Bothcypridsandvermigons incross-section.BDetail of left side scene in A, but from an adjacent section. Cypris(arrowhead) in gill chamber and vermigon (arrow) within blood space ofa gill ®lament; both in cross-section. C Semilongitudinal view of arecently injected vermigon within a dorsal blood sinus. The body isprincipally ®lled with large-sized cells. The distinct globular body(arrowhead) of small-sized cells will develop into the visceral sac andovary of the mature parasite. An elusive spiralling structure (arrows)extends throughout the lengthof thevermigon.DCross-section throughthe heart of a megalopa. Several vermigons in cross-section within thepericardium (PE), two of these (arrows) penetrating into the lumen oftheheart (HE) throughostia.ELate ``vermigon'' developingadjacent tothe hepatopancreas. This internal parasite has grown signi®cantlycompared to the recently injectedones inAtoD.Theglobularbody inChas matured into the primordium of the visceral sac, surrounded by anincipient mantle cavity (arrows) and containing the anlage to the ovary(ov). Compared to C, the interior of the parasite has opened up into aloose system of lacunae (asterisks). FTEM section through integumentof an only minutes-old vermigon. Note that the body is enveloped by adelicate, but distinct cuticle (arrows); Scales in lm

b

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in the gill chambers, although settlement was notobserved. The internal anatomy of the R. harrisiimegalopa is similar to that of early instar juvenile crabs,and we are therefore con®dent that the development ofthe vermigons observed here would be the same as thatoccurring in later crab stages.

Motility of the vermigon

Our observations on Loxothylacus texanus vermigonsunder in vivo conditions con®rm that the motility of thevermigon observed in vitro by Glenner and Hùeg (1995)is real. These authors found that the vermigon lacked

Fig. 3 Loxothylacus texanus. A Vermigon in situ in tissue of ®fththoracopod (paddle) of Callinectes sapidus and stained with phalloi-din. Cuticle of limb has been opened to allow view of parasite andaccess of stain. Positive stain for actin ®laments occurs along surfaceof vermigon and in central spiralling structure. Inset shows wholevermigon. B Vermigon prepared and stained as in A, but picturefocussed on epidermis, showing numerous actin ®laments runningparallel to the longitudinal axis. C Vermigon dissected out of crabthoracopod and stained for nuclei (DNA) (stained areas correspondto nuclei of large cells that ®ll the vermigon in Fig. 2C). Specimen hasbeen punctured with a needle allowing the central spiralling structureto disintegrate or escape. D Unstained vermigon in situ in tissue of®fth thoracopod (paddle). Note that central spiralling structureterminates just short of distal end of vermigon (arrow). Videoobservations con®rm that vermigons can move proximally in thelimbs even through such dense tissue as seen here ; Scales in lm

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muscles and suggested that the motility was caused byactin ®laments inside some vermigon cells. The presenceof phalloidin-stained material within the L. texanus ver-migon strongly supports this conclusion. Glenner andHùeg (1995) observed that the vermigon remains im-motile until ca. 15 min after its expulsion from thekentrogon. Therefore it seems that the motility has nofunction during the injection process but serves to propelthe vermigon through the tissues and blood system ofthe host.

The sheer size of the Loxothylacus texanus vermigonimpedes its passage proximally in the limbs, whether ittravels through the muscle tissue or within one of thenarrow blood spaces (Fig. 3D). The L. panopaei ver-migon must likewise pass through the narrow bloodspaces of the gills and through a minute ostium before itreaches the heart and can enter one of the major vesselsleading to other parts of the body (Fig. 2D). We there-fore believe that the movements observed in L. panopaeiand L. texanus vermigons are crucial for reaching theirultimate destinations in their hosts. Although we did notobserve peristalsis in L. texanus vermigons we are un-certain whether this signi®es a true di�erence toL. panopaei.

The reaction with phalloidin in Loxothylacus texanusvermigons strongly suggests that actin ®bers located inthe epidermis play an important part in the observedmotility. The video recordings indicate that the centralspiralling structure also plays a part, but whether it is apassive or an active one remains obscure until new TEMstudies have clari®ed its nature.

Growth of the vermigon into an interna

It is now clear that the sheath enveloping the early ver-migon is a very thin cuticle that later develops into thecuticle covering the internal parasite and its rootletsystem. It therefore follows that kentrogonid rhizoce-phalans are never devoid of a cuticle at any stage of theirlife-cycle.

Growth of the internal parasite apparently starts al-most immediately after infection as many of the ver-migons studied here had increased substantially in sizeand reached a stage, at which they are more appro-priately called ``interna'' than vermigon.

The oldest stages studied by us, with clear signs offormation of the externa anlage and a developing rootletsystem, overlap with those that previously have beenobserved by other workers using both light and electronmicroscopy (Smith 1906; Brinkmann 1936; Rubilianiet al. 1982; Hùeg 1990). Rubiliani et al. (1982) exposedsmall Carcinus maenas to female cyprids of the ``classi-cal'' sacculinid Sacculina carcini and later producedhistological sections of the infected crabs. The earlieststages found were comparable to our oldest stages inLoxothylacus panopaei. In both species, the interna hada primordium, or ``nucleus'', beginning to di�erentiateinto the tissues and organs of the externa. In L. pan-

opaei, most if not all vermigon cell types, including thoseof the ovary, can be traced back to the kentrogon andeven to the cypris using TEM (Glenner 1998).

Vermigons in other Rhizocephala

Our study revealed the presence of almost identical ver-migons in Loxothylacus panopaei and L. texanus, bothbelonging to the Sacculinidae. Since L. panopaei cypridsattach on gills and those of L. texanus on the append-ages, we can infer that the presence of a vermigon stageis not coupled to any particular site of settlement. Theobservation by Walker et al. (1992) that L. panopaeicyprids can settle also externally on megalopae supportsthis conclusion.

Veillet (1963) was probably the ®rst who directlyobserved host infection in the Rhizocephala. He studiedthe process in the sacculinid Drepanorchis neglecta,which infects munidan crabs. His micrograph clearlyshows a kentrogon within a spent cypris and an elongatebody, somewhat more bulbous than Loxothylacus spp.,emanating from the injection stylet. In hindsight, there isno doubt that this depicts the injection of the vermigonof D. neglecta.

In the classical rhizocephalan Sacculina carcini, Hùeg(1992: Fig. 17D) observed and illustrated a bulbousbody emanating from the tip of a kentrogon. We have(Glenner and Hùeg unpublished) repeated this obser-vation numerous times in this species and always seenthe ejection of such bodies. From comparison withLoxothylacus spp. it is obvious that kentrogons of bothDrepanorchis neglecta and S. carcini expel vermigonstages, although of a somewhat more bulbous shapethan those found in L. panopaei and L. texanus.

In Lernaeodiscus porcellanae (Lernaeodiscidae), Hùeg(1985) used TEM to study the female cypris and thekentrogon in great detail. Hùeg did not actually observethe infection process but from a comparison of ken-trogons ®xed before and after infection he concluded thatonly a single, large cell was injected into the host. Glen-ner and Hùeg (1995) documented that the injection of thevermigons lasts but a couple of minutes, so ®xation forTEM of a specimen during this event is a daunting task.We still believe that Hùeg's single ``inoculation cell'' isinjected into the host of L. porcellanae, but we also pre-dict that a renewed study will show that it is part of a truevermigon, which could be exceedingly small in this spe-cies. If true, the presence of a vermigon would charac-terize all three families of the suborder Kentrogonida andperhaps also the groundplan of all Rhizocephala.

Closing the rhizocephalan life-cycle

The observation that the kentrogon injects a vermigon,which migrates through the crab tissues or blood spacesand grows directly into the internal parasite has closedthe life-cycle of the kentrogonid Rhizocephala (Fig. 1)

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after more than 100 years of study (Delage 1884).At present, all stages in the life-cycle have been subjectedto histological study on parasites reared under con-trolled laboratory conditions. Until now it was assumedthat the early rhizocephalan parasite developed de novofrom undi�erentiated cells injected by the kentrogon(Delage 1884; Smith 1907; Hùeg 1985; Glenner andHùeg 1994). The break up of the vermigon observed byGlenner and Hùeg (1995) initially seemed to con®rm thisview but was, as shown here, probably due to an artifactcaused by experimental conditions.

Our observations remove a crucial obstacle for un-derstanding the evolution of the rhizocephalan life-cycle,and future theories can now proceed from a solid ob-servational basis. There is no ``second embryogenesis''or ``naked, undi�erentiated stage'' in rhizocephalanontogeny, neither during host infection nor during theearly internal development. The demonstration that thecuticle-covered vermigon originates from the kentrogonand develops directly into the adult parasite allows amuch more conventional scenario for the evolution ofthe rhizocephalan life-cycle. The vermigon instar hasadmittedly an exceedingly simpli®ed structure, but thenow well-known trichogon instar formed by male cyp-rids is actually even less complex (Hùeg 1987). The ve-rmigon already includes the most important cellularelements for forming the adult parasite, including cuti-cle, epidermis and the body of cells that later mature intothe ovary.

Rhizocephala is the sister group of the cirripede orderThoracica (Spears et al. 1994; Hùeg 1995), and theurrhizocephalan was most likely a setose-feeding animalcomparable to pedunculated barnacles. Accordingly,Glenner and Hùeg (1994) have suggested that thekentrogon evolved as an extreme specialization of the®rst juvenile stage in setose-feeding barnacles. The on-togenetic continuity between the pelagic rhizocephalanlarvae and the internal parasitic phase has now beenlargely clari®ed, but we still need to understand the se-lective pressures that led to the evolution of an internalmigratory stage (the vermigon) and resulted in the de-velopment of the parasite far from the site of infection.

Acknowledgements J. Hùeg acknowledges the support of theDanish Natural Science Research Council (Grant Numbers 97-01589 and 96-01405) in ®nancing travels and computerized mi-croscopic facilities and is also indebted to the Carlsberg Founda-tion (Grant No. 97038/40-213) for advanced printer and imageprocessing equipment. HG was supported by The University ofCopenhagen and the Carlsberg Foundation (Grant No. 980076/20-1234). E. Hed was instrumental in ®nding and videotaping theL. texanus vermigons, and J. Freeman provided stains and the Leitzepi¯uorescence microscope. Experiments performed on the animalsconcerned comply with U.S. and Danish legislation.

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