Reference: Biol. Bull. 201: 417-423. (December 2001) Shaping of Colony Elements in Laomedea flexuosa Hinks (Hydrozoa, Thecaphora) Includes a Temporal and Spatial Control of Skeleton Hardening IGOR A. KOSSEVITCH'. KLAUS HERRMANN2 *, AND STEFAN BERKING2 ^Department ofInvertebrate Zoology, Biology Faculty, Moscow State University, Moscow 119899. Russia; and 'Zoologisches Institut, Universitdt zu Koln, Weyertal 119, 50923 Koln. Germany Abstract. The colonies of thecate hydroids are covered Inside the perisarc is a hollow tube of soft tissue com- with a chitinous tubelike outer skeleton, the perisarc. The posed of two cell layers separated by an extracellular ma- perisarc shows a species-specific pattern of annuli, curva- trix, the mesogloea. This matrix is flexible. In general, a tures, and smooth parts. This pattern is exclusively formed colony comprises two pans: a net of tubes (stolons or at the growing tips at which the soft perisarc material is hydrorhiza) generally fixed to a substratum, and shoots expelled by the underlying epithelium. Just behind the apex (hydrocauli) emerging vertically from these stolons in a of the tip, this material hardens. We treated growing cul- more or less regular pattern. The shoots bear polyps (hy- tures of Laomedeaflexuosa with substances we suspected dranths) with which the animals catch their prey. All parts would interfere with the hardening of the perisarc (L-cys- ofthe colony are covered with the perisarc. In thecates, the teine, phenylthiourea) and those we expected would stimu- polyp expands out of the tubelike endings of the perisarc late it (dopamine. A'-acetyldopamine). We found that the covering (Fig. 1 ). formercaused a widening ofand the latterareduction in the The perisarc ofthe stolons is an almost uniform tube that diameter of the perisarc tube. At the same time, the length is flattened at the site oftight contact to the substratum. The of the structure elements changed so that the volume re- perisarc ofthe shoots in Laomedeaflexuosa Hinks, used in mained almost constant. We propose that normal develop- this research, forms a repetitive pattern (Fig. 1). One ele- ment involves a spatial and temporal regulation of the ment of the shoot the internode consists of two se- hardening process. When the hardening occurs close to the quences ofannuli separated by a smooth, slightly bent tube apex, the diameter of the tube decreases. When it takes and followed by the finely structured housing (hydrotheca) place farther from the apex, the innate tendency of the tip ofthe polyp. The sequence, the number, and the size ofthe tissue to expand causes a widening ofthe skeleton tube. An pattern elements are almost invariant and species specific oscillation of the position at which hardening takes place (Kosevich, 1990). The exact composition of the perisarc is causes the formation of annuli. unknown, but it appears to contain up to 30% of chitin (Jeuniaux, 1963: Holl et ai, 1992). Introduction Both the stolon and the shoot tubes increase in length The fragile, almost beautiful pattern ofhydrozoacolonies exclusivelyattheirtips (Kiihn, 1914:Hyman, 1940). Hence, attracts every observer's interest. There are many variations the pattern of the perisarc emerges exclusively at that site. reminiscent of plumes, plants, or minute trees. At closer Close to the apex of the elongating tube, this material is examination the pattern of the colonies is fixed by a rigid rather soft and flexible. Its shape is exactly that of the outer skeleton, the chinitous perisarc. We are interested in underlying tissue. The perisarc material "hardens" some how this perisarc is shaped. dozens of micrometers proximal to the apex, and from that time onward it has a fixed shape. The pattern ofthe perisarc R*eTcoeiwvehdom5 Fceobrrrueasrpyon2d0e0n1c;e aschcoeupltdedbe26adJdurleyss2ed0.01.E-mail: k.herrmannfs' is aOutrimientreerecsotrdiisngtoolfeatrhne ahcotwivitthyeopfertihseartcissisuesihnaptehde.tOipn.e uni-koeln.de possibility is that in the course ofgrowth, cell-cell interactions 417 418 I. A. KOSSEVITCH ET AL The treatment was performed in 4-ml petri dishes. Nor- hydranlh mally, the treatment lasted for 14 to 36 h. Under such shoot tip conditions the shoot tip completed the formation of the internode in about 20 to 24 h. The animals were not fed during the course of the experiment. However, the tips of fed and unfed specimens grew with the same speed (Ko- sevich, 1991). The medium was not changed. The results were scored at different times, starting 14 h after the begin- ning of the treatment. Measurements were made by means of an ocular micrometer. The proximal internodes of the hydrotheca isolated shoots that had completed theirdevelopmentbefore the start of the experiment served as the control, and were termed untreated. distal annulation zone regressed Chemicals usedfor treatments proximal hydranth The stock solutions of the following chemicals were prepared in distilled water: 10 mM/1 dopamine (Sigma). 10 mMl mM/1 W-acetyldopamine (Sigma). 5 phenylthiourea 1 mm (Sigma), 0.1% Calcofluor white (Fluorescent Brightener 28 Figure 1. AcolonyofLaomedeaflexuosa illustratingtheperisarc and [Sigma]). The following stock solutions were prepared in two hydramhs. seawater: 10 mM/1 L-cysteine (Sigma), adjusted to pH 8.2- 8.3, each time freshly prepared: staining solution for phenol compounds with fast red salt (Chroma, Stuttgart) according cause a differential curving of the tip surface, and that this to Romeis (Clara, after Romeis, 1968), treatment for 3 to 5 pattern is simply fixed by the perisarc. In this case the time at min under visual control; 0.001% Congo red (Merck) and whichtheperisarc hardenshasnoinfluenceonthe shapeofthe Evans blue (Merck), treatment for 5 to 15 min under visual perisarc. But shaping could also involve a differential pattern control; 4% formaldehyde (Merck), treatment for 24 h. in time and space of perisarc hardening. When the perisarc hardens closer to the apex, the diameter of the growing tube Statistics should decrease thereafter. But when hardening takes place mofortehedtiisstsaunetlyi,n tthhee daipaemxetteorexcpaanndi.ncrease due to the tendency folTlhoewinsgigtnhiefivcaarnicoeusoftredaitfmfeenrtesncweassbceatlwceuleanteddabtay moebatanisneodf Totestwhetheradifferential hardeningoftheperisarccould the F-test and the one-tailed t test. play a role in the process of shaping, we treated the colonies Results with substances that could be expected to either support or to antagonize the hardening process. The effects we observed Architecture ofthe stolon tip anil the shoot ti/> indicate that both a spatial and temporal pattern of perisarc hardening is involved in the shaping of the perisarc. The stolon and the shoot tip differ in size. During forma- tion of the smooth part of a shoot, the tissue tube in the tip Materials and Methods is about 160-250 ju.ni in diameterand is in tight contact with the perisarc over a length of 250-350 jam (the perisarc is Animals translucent). The tissue tube in the stolon tip is 200-300 ^m Colonies of Laomedea flexnosa Hincks (Thecaphora, in diameter and is in tight contact with the perisarc over a Campanulariidae) were cultured on glass microscope slides length of300-500 /urn. In both cases, adjacent to that region in artificial seawater (Tropic Marine, 1000 mOsmol, pH the tissue tube is much smaller in diameter and has tight 8.2-8.3) in a 5-1 aquarium at 18 C. The animals were fed contact with the perisarc at only a few positions. daily with Anemia sulinii nauplii. The mode ofperisarcformation Test system The composition of the perisarc is not well known but Shoots with newly emerged tips were used as test sys- includes chitin and proteins (Jeuniaux, 1963; Chapman, tems. Shoots including 4 to 6 distal internodes were isolated 1973). The proteins ofrelated species were found tocontain from the colony 2-4 h after feeding. The pieces were used a high concentration of disulfide bonds (Chapman, 1937; immediately. Bouillon and Levi, 1971). Phenol compounds are expected PERISARC FORMATION IN LAOMEDEA 419 Figure 2. Laomedeaflexuosa stained with various compounds, (a) A shoot stained with Calcofluor white. Notethat the new tip is strongly stained, butthe staining in the previously formed internode occurs in patches, (b) A stolon tip stained with formaldehyde. Note the decrease in the brightness of the fluorescence in the proximal direction, (c)Shoot perisarc stainedwith lm/M/1 ot dopamine. Theproximal partofthe intemodeand ofthe hydrotheca are well stained, but the distal annulation zone is almost unstained. The scale barrepresents 100 turn. to play a role in the hardening by causing a crosslinking also found in smaller numbers along the whole tissue prox- between the proteins and the chitin (Knight. 1970). imal to the tip (Fig. 2b). This result may indicate the Figure 2a shows the result of staining with Calcofluor presence of phenol compounds, which are known to play a white, which stains various carbohydrate fibrils, including role in the hardening process or sclerotization of the amorphous chitin (Compere, 1996). The treatment stains the chitin-containing exoskeletons of various animals, includ- perisarc of the tip and in particular the outer surface of all ing cnidarians (Knight, 1968. 1970; Holl et al. 1992). ectodermal cells in the tip, that is. in the region in which all Although the perisarc looks almost uniform within an ectodermal cellscontact the perisarc. Proximal tothe tip, the internode, it is not. Treatment with dyes including dopa- ectoderm is not in close contact with the perisarc. In this mine. fast red salt, Evans blue, and Congo red revealed a region, the surface ofthe ectoderm, staining is observed to distinct pattern ofstaining ofthe perisarc. The most intense be in the shape of patches. The diameter of such a patch and spatially different staining was obtained with dopamine corresponds to the diameter of one or several ectodermal (Fig. 2c). The staining intensity decreases gradually from cells. The perisarc in the proximal part shows very little the most proximal position to the distal end of the smooth staining. No correlation between the spatial pattern of part. The distal annulated zone is not stained, whereas in the stained cells and the perisarc pattern could be detected. It hydrotheca the staining is intense again. In elder internodes appears that perisarc material is almost continuously se- the pattern is identical, but the staining is deeper. Thus, the creted by the epithelial cells along the whole shoot, with the pattern of staining does not correspond simply to the thick- cells in the tip being the most active ones. That correlates ness ofthe perisarc wall. Because ofthe chemical nature of with the rinding that in old parts of the colony the perisarc the various agents and their binding specificity, we argue is thicker than in younger parts. For example, the thickness that these substances bind tophenol compounds, which may ofthe perisarc wall in the smooth part ofthe internodes was have played a role in cross-linking the proteins and the found to change from proximal (the eldest) towards distal chitin in the skeleton (cf. Holl et al., 1992). (the youngest) as follows (in /nm): 1 1.95-8.34-7.56-6.10- 4.39-3.17. Note that the distal part is stained but the prox- The influence ofL-cvsteine on shootpatterning imal is not. After formaldehyde was applied, a fluorescent stain ap- L-cysteine is able to interfere with the formation of di- peared in cells of the ectoderm or at their surface. The sulride bonds between and within proteins. Thus, the appli- stained cells were more numerous within the tip. but were cation of L-cysteine may antagonize perisarc hardening if 420 I. A. KOSSEVITCH ET AL. Figure 3. Alteration of the shoot perisarc shape due to treatment with L-cysteine and phenylthiourea. Treatment with 1 mAffl ofL-cysteine. (a); 2 mMI\ ofL-cysteine (b): 0.25 mM/1 ofphenylthiourea (c). Notethat the proximal annulation zone is smoothened, the smooth part is crumbled, and the distal annulation zone is smoothened and widened. Compare the normal pattern elements on the left side ofeach graph. The scale bar represents 100 ju.m. the formation ofdisulfide bonds is involved in this process. external and internal mechanical forces to produce the ob- In addition, L-cysteine impedes the formation of diphenols served malformations. (Horowitz et al., 1970). Diphenolic compounds including dopamine and A'-acetyldopamine were shown to be in- The influence ofphenylthiourea on stolon and shoot volved in the sclerotization ofthe cuticle ofinsects (Kramer patterning et ai, 1987; Sugumaran, 1987). Phenylthiourea, due to its sulfhydryl moiety, was also asnhadTpredeiaostfpmlteahnyetepdeorffiosslahdrsoco.tatTshuweniutpsehuraiLls-acrpycossttieutibineoenwsgird(eeFaintgel.dy,3aacl,treubrm)e.bdlTethdhe,e etwixaopsnecfotfoeuddnidpthofeoinrnotLle-srcfybesyrteeiinwntieet,rhapcththeienonyhlawtrihdtiehonutirhneegamohofinnotdhpeehrsepentrohielsafrmoco.rnmAoa-s- smooth part and the distal annulated zone were especially oxygenases (Lerch, 1983). Treatment of shoots by applica- affected. Most important, the annuli ofthe distal part, which tion of 0.25-0.5 mM/1 of phenylthiourea resulted in the form after the onset ofthe treatment, were not separated by the usual deep indentations, but displayed a much smoother fuloarrm,atthieondiosftablenatnnaunldatcerdumzpolneedapnadttetrhne eslmeomoetnths.paIrntpoafrttihce- mpaetnttersn.tha(tCfoomrpmaerdebeasforienttehrenasltacrtonotfrotlreathtemenotld[Fpiagt.t3e]r)n. Telhee- Ls-hcoyostteiwneer,ethaeffaencntueldi (wFeirg.e n3oct).seFpoalrlaotweidnbgyttrheeatumseunatl dweietph effect was observed following application of up to 1-2 indentations but displayed a much smoother pattern. The mA//l of L-cysteine. Concentrations ten times higher caused sequence of pattern elements was unchanged. the tissue to disintegrate. Although the shape ofthe perisarc was altered to a great Dopamine degree, the sequence of the pattern elements such as the proximal annulated zone, the smooth part, the distal annu- The diphenol dopamine is an intermediate on the way to lated zone, and the hydrotheca was laid down as usual. It those diphenols that are involved in cross-linking of com- appears that even the volume of these elements was not ponents of the cuticle in insects. In L. flexuosa Knight significantly changed. Thus, the applied concentrations of (1970) found dopamine and a phenoloxidase. He suggested L-cysteine did not strongly affect the pattern-forming pro- that both substances generate quinones that react to cross- cesses in the tissue, but rather adversely affected the normal link structural proteins. We found that 0.1 mM/1 of dopa- perisarc hardening. Due to the L-cysteine treatment, the mine reduced the maximal diameter ofboth the smooth part perisarc remained soft for a longer period oftime, allowing and the distal annulated zone. At the same time, the length PER1SARC FORMATION IN LAOMEDEA 421 Figure4. Alterationoftheshootpensarc shapeduetotreatmentwithdopamineand.V-ucetyldopamine. (aI Treatmentwith0.1 m/W/1 ofdopamine(b)Untreatedcontrol (c)Treatmentwith0.1 mM/1 ofiV-acetyldopamine. Notethatthesmoothpartandthedistalannulationzoneareincreasedinlength,andtheproximalannulationzone is irregular in shape due to treatment with W-acetyldopamine. Compare Fig. 4b as control. The scale bar represents 100 /xm. of these pattern elements increased (Fig. 4a, Fig. 5). In the proximal annulated zone and almost absent in the distal annulated zone, the ratio between the maximal outer diam- annulated zone (Fig. 2c). eter of the annuli and the diameter of the furrow between adjacent annuli remained almost unchanged (not shown). In N-acetyldopatnine the proximal annulated zone, the effect was less pro- nounced. One reason may be the short interval between the In insects, A/-acetyldopamine is thought to be an interme- onset oftreatment and the formation ofthe proximal annuli. diate between dopamine and the diphenols used for cross- Further, the composition of the perisarc may play a role. linking of the cuticle (Kramer et ni, 1987: Sugumaran. The resultant staining of the perisarc was strong in the 1987). Knight (1970). however, suggested that the mecha- S -2 0) re 0) 10 TO u c 422 I. A. KOSSEVITCH ET AL. nism of sclerotization of the hydroid perisarc differs from to support it. The putative "softeners" caused a crumbling thatofinsects, becausehe failed todetect/V-acetyldopamine and a widening of the perisarc. Of particular importance is and phenolic-/3-glucosides in hydroids. In L. flexuosa, a that the constrictions between the annuli were smoothed out concentration of 0.1 mA//l of/V-acetyldopamine caused the in the distal annulated zone. The putative "hardeners" smooth part and the annuli of the distal annulated zone to caused the perisarc tube in all internode parts to become become narrowerand longer(Figs. 4a, b; Fig. 5). Further, as narrower and longer. The applied concentration of the var- observed forthetreatment with dopamine, the ratiobetween ious chemicals was apparently not toxic to the animals: in the maximal outer diameter of the annuli and the diameter the presence of the chemicals the polyp and the hydrotheca of the furrow between adjacent annuli remained almost of the internode formed normally and the polyps behaved unchanged. Unlike dopamine, /V-acetyldopamine strongly normally forexample, in stretchingouttocatch theirprey. affected the proximal annulated zone, eliminating its regular We suggest that the composition of the soft perisarc annulation pattern (Fig. 4c). /V-acetyldopamine may act material surrounding the apex changes with time. The na- faster than dopamine. ture of the compounds is largely unknown. In insects, low- There is no indication of an unspecitic, cytotoxic action molecular-weight catechols such as /V-acetyldopamine and of the chemicals. One can see in the figures that the older /V-/3-alanyldopamine are involved in sclerotization. These colony elements are unaffected by the treatment: polyps are converted to quinones, which react in cross-linking stretch out of their hydrotheca, and they are able to catch proteins (for general review see Waite, 1990). Knight their prey. The hydrothecae formed during treatment with (1970) proposed a different mechanism of action for the chemicals are well shaped, and living polyps formed Laomedea flexuosa: failing to detect the mentioned sub- with tentacles. stances, he detected dopamine instead, and suggested that it was active in sclerotization. Waite (1990) stated that "this should be taken with caution since the entire animal was Discussion methanol-extracted." In organisms other than insects, dopa- Thedelicate species-specific pattern ofathecatecolony is containing proteins are thought to cause the sclerotization laid down exclusively at the growing tip. At this site, the through a process of "autotanning" (Smyth, 1954; Brown, tissue has permanent contact with the expelled soft material 1952; Pryor, 1962) in which thedopa moietiesare converted fromwhichthe outerskeleton, the perisarc, isformed. Some to quinones. Additional molecules of chitin, collagen, fi- dozen micrometers proximal to the apex of the tip. the broin, or cellulose, for example are necessary as "fillers." perisarc hardens, which fixes the pattern of the perisarc. This mode of sclerotization is well-distributed throughout It is obvious that the soft material is molded by the outer theanimal kingdom, and Waite andcoworkers (1990) found shape of the underlying tissue. This outer shape is deter- dopa-containing proteins in the cnidarian Pachycerianthus mined by the property and activity ofthe cells that built the fimbriatus. Our results do not help resolve the question of tissue tube, particularly those cells that produce the growing which mode of sclerotization acts in L.flexuosa. We know, tip. In the tip, the tissue moves back and forth rhythmically. however, that the concentration of one or several of the This phenomenon, termed growth pulsation, has been stud- components changes rhythmically during the growth of the ied extensively (Beloussov et ai, 1992). shoot internode. These rhythms are much slower than those The staining with Calcofluor white suggests that the of the aforementioned growth pulsations. If the hardening amorphous perisarc material that eventually forms fibrils, occurs closer to the apex, the diameter of the ring-shaped including chitin fibrils, is secreted by almost all the ecto- border between the hard and the soft perisarc decreases, dermal epithelial cells ofthe growingtip, as well as bysome forcing the tissue to squeeze through this opening. Under epithelial cells along the body axis. The phenolic com- these conditions, the perisarc tube elongates with a reduced pounds, which Knight contended to be involved in the diameter. A widening of the diameter needs at least two cross-linking of the perisarc, appear to be contained in prerequisites: the hardening has to happen more distally so-called tanning cells (Knight. 1970). These cells are con- from the apex than before, and the tissue of the apex must centrated in the tip and also exist in lower density in the form a bulb. Evidence for bulb formation may be that the proximal parts. They have no broad contact with the outer tissue tube in both the tip of the stolon and the tip of the surface of the epithelial sheet of the growing tip and are shoot has a tight contact to the perisarc, while in proximal embedded between the epithelial cells (Knight. 1970). regions the tissue tube is much smallerthan the inner lumen Our data suggest that a differential hardening of the of the perisarc tube. Further, the shoot and the stolon occa- perisarc is involved in the shaping of the perisarc tube. We sionally form a bulb at the wound after cutting (Kossevitch, treated a growing culture with substances that we expected, unpubl. obs.). from their chemical nature, to affect the hardening process. In the process of annulus formation, the zone of harden- Phenylthiourea and L-cysteine were expected to impede the ing may move rhythmically closer to and then farther away hardening; dopamine and /V-acetyldopamine were expected from the apex. This may occur in either a continuous or a PER1SARC FORMATION IN LAOMEDEA 423 stepwise manner. When a hydrotheca starts to form, the hydranthes-hydrothequeschezlespolypesThecata. Z. Zellforsch. 121: 218-231. zproonteruodfihngartdiesnsuien.gTlhaagts cbaeuhsiensdainwiredleantiionngtooftthheeatpiessxueoftutbhee BroMwinc,roCs.c.HS.ci.199532:.48S7-o5m0e2.structural proteins ofMylilus edulis. Q. J. and subsequently of the perisarc tube as well. Chapman. G. 1973. A note on the composition of some coelenterate In L. ficxHosa, the observed bending of the tube in the exoskeletal materials. Camp. Biochern. Physiol. 45B: 279-282. smooth part ofthe internode (cf. Fig. 1) may be the result of Chapman. S. S. 1937. Localization of-SH and-S-S- in Obeliagenicu- an asymmetry in the hardening of the perisarc along the lara. Growth 1: 299. catirtchuemfseirdeenctehaotfftahceestip.thIet msahoyotocacxuirs,cliosmeprostiontgheatisppaatpieaxl ComCpheirtein, iPn.L1if9e96.ScieCnycteos.cheMm.icaMl. lGaibrealuldi-nGguiolflec,hiteidn.. JPapc.qu6es6-8An7drien Publisher, Lyon, France. control of hardening in addition to the temporal control. Holl,S.M.,J.Schaefer,W.M.Goldberg.K.J.Kramer,T.D.Morgan, In other animals with an exoskeleton, such as arthropods, and T. L. Hopkins. 1992. Comparison ofblack coral skeleton and the integument may be shaped by changes in the hardening insect cuticle by a combination of carbon-13 NMR and chemical process together with changes in the pressure of the tissue analyses.Arch. Biochem. Biophys. 292: 107-111. against the forming integument. In arthropods other than Horowitz, N. H., M. Fling, and G. Horn. 1970. Tyrosinase (Neuro- thecate hydrozoa. the hardening can start at various posi- sphora crassa). Methods En-ymol. 17A: 615-620. Hyman, H. 1940. The Invertebrates: Protozoa through Ctenophom. tions and can spread at different speeds from those posi- McGraw-Hill. New York. Pp. 400-497. tions. The resultant shape of the integument can thus be Jeuniaux, C. 1963. Chitine et Chitinolyse. Un chapitre de la biologie more complex than in hydrozoa. moleculaire. P. Masson. Paris. The various treatments we applied caused the perisarc to Knight,D.P. 1968. Cellularbasisforquinonetanningoftheperisarcin bend, to fold, and to crumble. However, the sequence ofthe the thecate hydroid Campanularia (= Obelia)flexuosa Hinks. Nature pattern elements up to hydrotheca formation was as normal Knig2h1t8,: 5D.84P-.58169.70. Sclerotization of the perisarc of the caliptoblastic as possible. The volume of the tissue responsible for the hydroid, Laomedeaflexuosa. 1. The identification and localization of formation of the corresponding element was largely un- dopamine in the hydroid. Tissue Cell2: 467-477. changed. The decrease in the diameter of the perisarc tube Kosevich [Kossevitch], I. A. 1990. Developmentofstolon'sandstem's was compensated for by the elongation of the tube. This internodes in hydroid genera Obelia (Campanulariidae). Vestn. Mask. indicates thatthe very tipdetermines the sequence ofpattern Univ. Biol.. N 3: 26-32. [In Russian; English summary.] elements. The respective decisions of the tip were not in- Kosevich[Kossevitch].I.A. 1991. Comparisonofupright'sandstolon's fluenced by (1) the chemicals applied in the concentrations tiidpase).funVcetsitonn. iMnoshky.droUindivc.olBoionly.,ObNeli2:a l4o4v-e5n2i.(A[lIlnm.R)us(sCiaamnp;anEunlgalriis-h noted, (2) the disturbance of the shape and movements of summary.] the tissue in the tip, (3) the shape ofthe tissuetube inamore Kramer, K. J., T. L. Hopkins. J. Schaefer, T. D. Morgan, J. R. proximal region, nor (4) the altered tension and pressure of Garbow. G. S. Jacob, E. O. Stejskal, and R. D. Speirs. 1987. the proximal tissue on the tissue in the very tip. These four Mechanismsofinsectcuticlestabilization.Howdotobaccohornworms piomiennttsalalrye iinsoalgatreedemsehnototwittihp tchoentoibnsueerdvattiheonptahtattertnhienegxpperro-- KiilLdiinos.si.At.?NPe1p9w.143Y.3o1r-kE3.n5t5wiicnklMuonlgescguelsacrhiEcnhttoemoulnodgyV,erJw.anHd.tsLcahwa,ftesdb.eAzlieahnunR-. gram ofthe perisarc tube up to the formation ofthe polyp's genderHydrozoen. I.Teil; Die Hydroiden. ErgebnisseFortschr. Zool housing. The tissue itself was transformed into only the 4: 1-284. apical part of the polyp; the proximal part of the perisarc Lerch, K. 1983. Neurospora tyrosinase: structural, spectroscopic and tube was free of tissue (Kosevich, 1991). catalytic properties. Mol. Cell Biochem. 52: 125-138. Pryor, M. G. M. 1962. Sclerotization. Pp. 371-396 in Comparative Acknowledgments Biochemistry. Vol. IV, M. Florkin und H. S. Mason, eds. Academic Press, New York. This work was in part supported by the Deutscher Aka- Romeis, B. 1968. Mikroskopische Technik. R. Oldenbourg, Munich. demischer Austauschdienst (PKZ A/98/40374) for I.A.K. Germany. Smyth, J. D. 1954. A technique forthe histochemical demonstrationof polyphenoloxidase and its application to eggshell formation in hel- Literature Cited minths and byssus formation in Mytilus. O. J. Microsc. Sci. 95: 139- Beloussov, L. V., J. A. Labas, and N. I. Kazakova. 1992. Growth 152. poullosgayt.ioPnp.si1n8h3y-d1r9o3idinpoOlsycpisl:laktiinoenmsaatnicdsM,obripohlooggeicnaelsirsol.eLa.ndRecnystionpgh.yseid-. SugMuomlaercaunl.arME.nt1o9m8o7l.ogyQ.uiJ.noHn.eLamewt,hiedd.eASlclaenroRt.izaLtiisosn,.NPep.w3Y5o7r-k3.67 in Marcel Dekker, New York. Waite, J. H. 1990. The phylogeny and chemical diversity ofquinone- Bouillon. J., and C. Levi. 1971. Structure et ultrastructure des attaches tanned glues and varnishes. Camp. Biochem. Physiol. 97B: 19-29.