CRUSTACEAN RESEARCH，NO .31: 39-46，2002

Interna structure and nucleus formation in Sacculinαpolygeneα( C r u s t a c e a : Cirripedia: : Sacculinidae)

Jorgen Lutzen

Abstract.-The root system and body and in sacculinids it is borne ven- nucleus formation in the colonial rhizo- trally on the abdomen of the host crab; cephalan Sα cculinαpolygeneαL u t z e n & it is solely concerned with sexual repro- Takahashi has been reinvestigated and duction.The externa (or externae in found to correspond to those of other re- asexually reproducing species) is/are lated species. A conflicting opinion of connected to the parasite's trophic org但し these subjects recently proposed by Rus- the interna or root system，which is 10- sian zoologists is due to the fact that cated within the host abdomen and these authors have mistaken anumber of cephalothorax.Primordial externae arise host structures for pa此 s of the parasite's as so-called nuclei that are part of the root system. root system and are located in the crab's abdomen，although they may start their 1ntroduction development in the cephalothorax. A nucleus is located within atumor ，an ex- 1saeva et al.(1999 ，2001) and Shukal戸lk pansion of the root system with an interior & 1saeva (2000) recently reported on the consisting of aloose tissue of widely organization of the root system and the separated stellate cells. mode of asexual reproduction in the north- ， west Pacific sacculinid Sα cculina pol)伊 nea Lutzen &Takahashi ，a parasite of the Materials and Methods littoral crab Hemigrapsus sα nguineus As new material was not available，1 (de Haan).Al though they described the reexamined part of myearlier section late development of the primordial series of Hemigrapsus sα nguineus para- externae correctly，their description of sitized by S .polygene α.The processing of the early development differs signific 田 lt- this material was described by Takahashi ly and fundamentally from results pre- & Lutzen (1998). It comprises 10μm thick sented earlier by Takahashi & Lutzen continuous Paraplast (R) serial sections of (1998)，組d this has prompted the present the abdomens and part of the cephalotho- reinvestigation of myearlier published rax of 10 sacculinized crabs and afew material. non-sacculinized crabs.Because of the The generallife cycle of rhizocepha- considerable thickness of the sections，the lans has been summarized by Hoeg & images are not optimal. 1n addition，1 Lutzen (1985，1995 ，1996) .The complete studied afew slides of cross-sections of life cycle is only known for asingle species ， abdomens of H .sanguineus parasitized by the sacculinid Loxothylαcus pαnopαei S .polygene αt h a t were kindly placed at (Gissler) (see Glenner，2001) .Terms used mydisposal by Dr.Valeria 1saeva，1nsti- in the following text are explained as fol- tute of Marine Biology，Russian Academy lows. The externa is the reproductive of Sciences，Vl adivostok，Russia. 40 J.LUTZEN

Results centrally in the visceral mass (Fig.1・ 8) Figure 1presents aseries ofnuclei and Figures 2-4 illustrate structures that tumors arranged to illustrate the growth are relevant for the Discussion. An expan- of the early nucleus from adiameter of cα. sion of the root system is always present 20μm to almost 50μm and the accompa- beneath the stalk of the externa (Fig. 2) . nying expansion and change in shape of Within the cephalothorax of parasitized the tumor.Th eillustrated stages are each crabs numerous rootlets lie between the chosen from among cα. 300 examined diverticula of the host's digestive gland stages of the same size class (仕 omten sec- (Fig. 3). Rootlets are particularly abun司 tioned crabs) .In the present study，which dant in the first segments of the abdomen aimed to document the development of and may even invade acluster of sub- the earliest nuclei，it was deemed un- gastrodermal glands，which occur in a necessary to present larger (and older) zone circumscribing the boundary be- tumors/nuclei. Subsequent development tween the crab's midgut and hindgut takes exactly the same course as in other (Fig. 4). The glands，which in the illus- species of three genera of sacculinids trated caSe are only super五cially invaded (Delage，1884: Sacculinα; Smith，1906: by rootlets ，consist of many small acini Drepαnorchis; George，1959: ). composed ofrelatively few large cells with The smallest recorded nuclei (15-25 darkly staining cytoplasm and peripher- μm ) and tumors always occurred at the ally placed nuclei . tips of rootlets，which as aresult had of- ten swollen into small buds (Fig.1 ・1，2，3). Discussion Accompanying bud-formation，the cuticu- The material used by the Russian au- larized epidermis surrounding these thors differs from myown only in that small tumors typically increased in theirs was collected in Vostok Bay，Siberia ， height and stainability，a fact which con- Sea of Japan，while my specimens are siderably helps in the detection of these from the Amakusa Islands，Kyushu ，Ja- small stages.In favorable sections ，the pan. In both cases the host crabs were H. nucleus' small prospective mantle cavity sα nguineus collected at the shore. was often seen to have fused with the in- side of the bud's epidermis (Fig.1・ 2) . The orgαnLZαtion of the root system In later stages the tumors expand pro- The generallayout of the root system portionally much more than the nuclei. in sacculinids is very simple (Delage， Nuclei ofcα.50μm in diameter often have 1884; Smith，1906; I仕uger，1940; Hoshino， tumors with dimensions of 250 x300μm 1965). Beneath the point where the stalk (Fig.1・8). In all growth stages，the nucle- of the externa penetrates the integument us is always positioned superficially of the host，the root system has asac- within the tumor. Opposite the nucleus， shaped enlargement，from which alarge larger tumors usually extend into many number of rootlets issue .By branching smaller or larger rootlets . profusely，the rootlets penetrate in be- Even the smallest buds contained a tween many organs of the host.An organ minute vesicle associated with asmall ， that is especially heavily infiltrated by compact body，which represent the earli- the rootlets is the digestive gland (hepato・ est rudiments of the mantle cavity and pancreas)，but the rootlets never enter the visceral mass，respectively .In nuclei of cα. tubular diverticula ofthe gland.The epi- 50μm in diameter or larger，it was pos- dermis ofthe rootlets consists of asimple ， sible to recognize acuticular lining in the cuticle-producing epithelium，which is of- mantle cavity and arudimentary ovary ten associated with asubjacent layer of INTERNA STRUCTURE IN POLYGENEA 41

日g. 1. Sacculinαpoか'genea.Eight successive stages (1-8) in the development and expansion of nuclei and tumors.ct ，contact between the epithelia of mantle ca吋ty and bud; ep，合 ontal epithe- lium ofbud，exhibiting increased height and"stainability; mc，prospective mantle cavity lined by cuticle (cu); nu，nucleus; tu，tumor; vm，prospective visceral mass. 10μm thick paraplast sections stained with hematoxylin and eosin. Scale bar 50μm. 42 J.LUTZEN

two distinct systems:n 田 TOWdistal rootlets and much larger transport trunks (or transport canals). They believed that the trunks connected the externae and/or their primordia with the rootlets. Although they did not present any histolo回cal sec- tions of rootlets issuing from the trunks， they did provide cross-sections of the lat- ter (Shukalyuk & Isaeva，2000 ，fig. 1d; Isaevaetαl. ，2001 ，fig .2E). In these figures the diameter of the trunks (cα.125-200 μm ) is seen to exceed that of the rootlets by several times. However，it is evident that the authors mistook the tubular diverticula of the host's digestive gland with what they construed to be transport trunks. According to their description，the lining of the purported trunks is made up of alongitudinally folded epithelium，but this is exactly characteristic of the diges- tive gland diverticula.The diameter and average wall thickness of the diverticula in H .sα nguineus (Fig. 3) also matches that ofthe “trunks". Furthermore，a careful examination by me of several sectioned externae in situ has shown that although the hemocoelic canal penetrating the stalk of the externa expands to abasin Fig. 2. Sαcculina polygeneα. Expansion of underneath the stalk basis，the rootlets the root system (ex) underneath the stalk of issue directly 企omthis expansion (Fig. 2) ， the externa and ramification of rootlets (r) as has been reported also in other species . 企omit; the expansion is continuous with the Even if it rnay give off afew larger roots as hemocoelic canal (hc) running through the well they are always short and their stalk. 10μm thick paraplast section stained ， with hematoxylin and eosin .Scale bar 100μm. walls are much thinner than the “trans- po吋 trunks"，and smooth，never folded.A single diverticulum and many rootlets were illustrated side by side in cross- axial cells (Bresciani & Hoeg，2001) .The section by Hoshino (1962:fig .2 ，p l. 1，fig.1; s仕ucture of the root sys旬 m and the histol- Pα chygrα:psus crαssipes Randall parasit- ogy of the roots of Sacculinαpolygeneαa s ized by S. confrαgosαB o s c h m a )and by reported by Takahashi &Lutzen (1998)， Takahashi & Lutzen (1998: 日g.5; present and those of S .plan αB o s c h m a reported species). by Liu & Lutzen (2000)，agree with this Figure 5(center and right frame) in description (Fig. 2). Isaeva et al .(2001) sums up their interpre- Shukalyuk & Isaeva (2000) and Isaeva tation of the root system in S .polygene α. et αl. (2001) arrived at quite another It falsely shows how the rootlets fuse with conclusion when investigating the mor- the “transport trunks"，which is ，of course， phology of the root system in S .polygenea . impossible.The error becomes all the They reported it to be differentiated into more incomprehensible in light ofthe fact INTERNA STRUCTURE IN SACCULINA POLYGENEA 43 that the digestive gland diverticula in H . sα nguineus never penetrate into the part of the abdomen where the externae are attached.

The origin of nuclei in αsexuαlly repro- ducing rhizocephαl α ns Until the study by Glenner (2001)，the exact origin of the earliest nuclei present in the root system was not known.By a patient and meticulous examination of the fine structure of the cyprids and later invasive larval stages of the sacculinid Loxothylαcus p αnopαei ，Glenner suc- ceeded to trace the origin of the earliest known nuclei of the root system-and at the same time the externae-back through the vermigon and the kentrogon to the female cyprid larva.Whi le L. panopaei is predominantly solitary on its host，many rhizocephalans are constantly colonial. It is generally recognized that all externae in these species arise by asexual reproduc- tion through abudding process from the interna which itselfhas originated oma Fig .3. Diverticula ofthe digestive gland (dg) ， 企 of the host (H. sαnguineus) and rootlets (r) of single female cyprid. The externae in S. polygeneα. 10μm thick paraplast section some colonial species may number several stained with hematoxylin and eosin .Scale hundreds or even thousands (Jespersen & bar 100μm. Lutzen，1992; Hoeg & Lutzen，1993); clearly，therefore ，the explanation ofhow the nucleus/externa originates in asoli- form ayoung tumor around the growing tary rhizocephalan fails to explain how nucleus (Potts ，1915; Lutzen，1992; Lutzen the many nucleilexternae arise in colonial & Jespersen 1992; Lutzen & Du，1999) . forms.If one does not resort to the unlikely As documented in the present study，and explanation that asingle introduced as has also been shown and illustrated nucleus splits or buds into many new earlier for this species (Takahashi & ones，one is left with the alternative that Lutzen，1998) and the allied S .plana (see the externae develop 企omnuclei that are Liu & Lutzen，2000) ，the earliest nuclei formed de novo within the root system. At are invariably localized within bud-like the moment，however ，this question has enlargements of the tips of the rootlets. not been settled. Therecorded minimum size ofthese small Asexual reproduction occurs in several nuclei is cα .15μm in both species .The families of Rhizocephala that do not col- smallness ofthe nuclei combined with the lectively form anatural taxon. The origin thickness ofthe sections (10μm) made it or the formation ofthe nuclei is not neces- impossible to study the details of their sarily the same in all of them，but in a structure; it was，however ，repeatedly number of species the smallest nuclei noted that the nuclei at one place adhered have been discovered in slightly swollen to the inside of the surrounding epidermis rootlet terminations，which by expansion (Fig.1・2). This may 0百er aclue as to how 44 J. LUTZEN

Fig. 4. Rhizocephalan rootlets (r) penetrating between the acini (ac) of the subg:a strodeEmli1 glands ofthe host gut. 10μm thick paraplast section stained with hematoxylin and eosin .Scale bar 50μm. they arise. Rubiliani et al. (1982) showed Isaeva et αl .(1999 ，2001) believe that that several nuclei 訂 e formed temporarily the nuclei of S .polygenea develop from in the early interna of totipotent embryonic stem cells ，which (Thompson)，arising by alocal invagina- are said to accumulate to form numerous tion of the epidermis; the invagination small nuclei consisting of compact clusters dedifferentiates and subsequently pro- of non-differentiated cells. They report duces the rudiments of the future mantle (p. 137) that these nuclei are located “in- cavity and ovary. The association of the side the major portion of the interna … smallest nuclei with the epidermis in S. close to the border between cephalothorax polygenea and S. plana might indicate and abdomen."Their fig. 3A shows a that they also arise by invagination. Deri- number of these so-called nuclei，which ， vation of the cuticularized epidermis of however，seem to be squeezed in between the mantle cavity from the equally cu- the elements of the root system rather ticle ・lined root epidermis could be easily than being part of it .1 have studied these explained，whereas formation of an ovary structures on slides borrowed 企omDr.V . 仕omcells of ectodermal origin is unortho- Isaeva，on which they are abundantly dox. If true，it has nevertheless aparallel present. Oncomparing them with myown in the morphogenesis as aresult of bud sections 1became certain that 1) they are formation exhibited by some colonial as- definitely not part of the root system，2) cidians (family Botryllidae).In the buds they are constantly present in unparasi- of these ascidians the ectodermally de- tized as well as parasitized host crabs， rived atrial epitheliallining is totipotent and 3) they are identical with the acini and gives rise to nearly all tissues of the of the subgastrodermal glands mentioned blastozooid，including such mesodermal in the Results section. The similarity in structures as the pericardium，epicar- structure and size of these so-called nu- dium，and gonads (Be町 ill ，1941，1947). clei and the glandular acini is apparent INTERNA STRUCTURE IN SACCULINA POLYGENEA 45 iffig.3B 企omIsaeva et al. (2001) is com- 249: 9-42. pared with Fig.4 in the present paper. Delage，Y .，1884. 邑volution de la sacculine Other bodies claimed to represent (8α cculinαc α rcini Thomps.) crustace endoparasitaire de l'ordre nouveau des stages in the formation of primordia of kentrogonides. Archives de Zoologie externae are shown by Shukalyuk & Experimentale et Generale，ser .2 ，2: 417- Isaeva (2000，fig. 1d: pe) and Isaeva et al. 736. (2001，fig .2E: PE).They resemble no George，A. 1.， 1959. Heterosαccus ruginosus other known stages in the development (Boschma)，a rhizocepha1an parasite ofthe of any sacculinid and do not fit into the crab Neptunus sanguinolentus (Herbst). Journal of the Zoological Society of India， series of developmental stages illustrated 11: 171-204. in the present paper. They are obviously Glenner，H. ，200 1. Cypris metamorphosis， part of the host's digestive gland diver- injection and earliest internal develop- ticula，and their unusual shape is possibly ment of the rhizocephalan Loxothylαcus aresult of poor histological preparation pα nopaei (Giss1er). Crustacea: Cirripedia: Rhizocepha1a: Sacculinidae. Journa1 of resulting in crumbling and partial de- Morpho1ogy，249: 43-75. tachment of the folded epithelial wall. Hoshino，K.， 1962.Morpho1ogy of 8α cculinα Equally baffling is an epithelium-lined conf同:g osαB o s c h m a ，arhizocephalan para- empty space claimed to be alater primor- site of P αchygrαpsus crαssipes Randall， and changes of the host induced by parasit- dium (I saeva et αl.，2001 ，fig. 3C ，D) ism.Contribution No. 5from the Science despite the absence in it of any landmarks Education Laboratory，Manazura Science such as arecognisable tumor or nucleus Education Station，Faculty of Artsand Sci- that could reveal its true nature. 1am ences，Yokohama National University，12 inclined to believe that the hollow struc- pp. (In Japanese) tures shown in fig. 3C are parts of the 一一一一， 1965. On the root system of 8α cculinα confragosαB o s c h m a arhizocephalan p訂 a- coiled tubular hindgut caecum and that ， site attached to P αchygrα.p sus crαssipes the structure labelled ICM(meaning in- Randal l. R氾searches on Crustacea，2: 3-9. ternal cell mass) in fig .3 D Is simply a Hoeg，J .T .，& L:u tzen，J. ，1985. Crustacea tangentially sectioned wall of one of the Rhizocepha1a. Marine Invertebrates of tubes，which bends at this place. Scandinavia 6.92 pp.Norwegian Univer- sity Press Oslo. The Russian authors have obviously ， 一一一一，& 一一一一， 1993.Comparative morpho1- mistaken anumber ofhost structures for ogy and phy10geny of the fami1y parts of the root system of S .polygene α Thompsoniidae (Cirripedia，Rhizocepha1a ， and have therefore reached some odd con- Akentrogonida)，with descriptions of three clusions，which are in conflict with all new genera and seven new species . Zoologica Scripta，22: 363-386. previous studies. Thepresent paper ce吋1- 一一一一一，& 一一一一一， 1995.Life cycle and repro- fies that the root system and nucleus for- duction in the Cirripedia Rhizocepha1a. mation in S. polygeneαa r e the same as in Oceanography and Marine Bio1ogy，an An- other species of sacculinids. nua1 Review，33: 427-485. 一一一一，& 一一一一， 1996. Super-ordre des Rhizocephales (Rhizocepha1a F .Muller ， Literature Cited 1862).In: Forest J，(ed .) ，Tr aite de Zoologie， Berrill ，N .J .，194 1. The development of the 7(2) Crustac岳s Generalites (suite) et bud in Botrッllus. Biological Bulletin，80: Systematique (Ire partie) .pp. 541-568. 169-184. Masson，Paris. 一一一一， 1947. The developmental cycle of Isaeva，V .V .，Rybakov ，A.V. ，& Kas'yanov， Botrylloides. Quarterly Journal of Micro- V .L.，1999 .In vitro visualization of co1onial scopical Science，88 :393 -407. organization in the interna ofrhizocephalan Bresciani，J. ，& Hoeg，J. T. ，200 1. Comparative barnac1es Peltogαsterella gracilis and ultrastructure of the root system in rhizo- Sα cculina polygenea.Doklady RAN，366: cephalan (Crustacea: Cirripedia 840-842. (In Russian) Rhizocephala). Journal of Morpho1ogy， Isaeva，V. V. ，Shuka1yuk ，A .I .， Trofimova， 46 J. LUTZEN

A.V.，Korn ，O.M. ，& Rybakov ，A.V. ，200 1. littorαlis (Crustacea: Cirripedia: The structure of colonial interna in Rhizocephala).Acta Zoologica，73 :1-23 . Sα cculinα polygenea (Crustacea: Potts，F. A.' 1915. Onthe rhizocephalan genus Cirripedia: Rhizocephala). Re- Thompsonia and its relation to the evolu- search， 133-146. tion of the group.Publications of the Jespersen，A.， & Lutzen，J. ，1992. Thompsonia Carnegie Institution，212 :1-32. dofleini Hafele，a colonial akentrogonid Rubiliani，C .，Turquier ，Y .，& Payen，G .G .， rhizocephalan with dimorphic，ova- or 1982.Recherche sur l'ontogen色se des sperm-producing，externae (Crustacea: rhizocephales.I. Les stades precoces de la Cirripedia). Zoomorphology，112: 10ι116. phase endoparasitaire chez Sα cculinα Kruger，P. ，1940 .Cirripedia. In: H.G.Bronn ， carcini Thompson. Cahiers de Biologie (ed.) ，Kl assen und Ordnungen des Tiereichs， Marine，23 :287-297 . Bd. 5，Abt. 1，Buch 3，Teil 3:1-560. Shukal抑止 ，A. I.， & Isaeva，V. V .，2000. Intra- Liu，H .-C. ，& Lutzen，J. ，2000. Asexual repro- vital and histological studies of the inter- duction in Sacculina plαna (Cirripedia: nal organization ofthe rhizocephalan bar- Rhizocephala)，a parasite of six species of nacle Sα cculinαpolygeneα. Russian Jour- grapsid crabs 仕omTaiwan. Zoologischer nal ofMarine Biology，26 :209-211 . Anzeiger，239 :277-287. Smith，G .，1906 .Rhizocephala .Fauna und Lutzen，J. ，1992 .Morphology of Thompsoniα Flora des Golfes von Neapel，29: 1- 129. reinhαrdi ，new species (Cirripedia: Takahashi，T .，& Lutzen，J. ，1998. Asexual re ・ Rhizocephala)，parasitic on the northeast production as part of the life cycle in Pacific hermit crab Discorsopαgurus Sα cculina polygeneα (Cirripedia: schmitti (Stevens). Journal of Crustacean Rhizocephala: Sacculinidae).Journal of Biology，12 :83-93 Crustacean Biology，18 ・ 321-331. 一一一一， & Du，P. T. ，1999 .Three colonial rhizocephalans 企ommantis shrimps and a crab in Vietnam，including Pottsiαserenei ， Address: Zoomorphology Department， new species (Cirripedia:Rhizocephala: Zoological Institute，Copenhagen University， Thompsoniidae).Journal of Crustacean Universitetsparken 15，DK ・2100 Copenhagen Biology，19: 902-90. 7. の， Denmark 一一一一， & Jespersen，A. ，1992 .A study of the E-mail:jlutzen@zi .ku.dk morphology and biology of Thompsonia