Marine Biology (2003) 143: 533–541 DOI 10.1007/s00227-003-1078-8

R. Martin Management of nematocysts in the alimentary tract and in of the aeolid gastropod peregrina

Received: 22 January 2003 / Accepted: 21 March 2003 / Published online: 27 May 2003 Ó Springer-Verlag 2003

Abstract The nudibranch gastropod cnidophages was not obtained. It is concluded that a (: Aeolidaceae), when feeding on polyps large proportion of the nematocysts ingested with the of the hydrozoan racemosum (: food are not digested, but are eliminated, structurally ), devours masses of small microbasic and functionally intact, via the alimentary canal and the eurytele and holotrichous isorhiza nematocysts. Large tips of the . proportions of these nematocysts were found undis- charged in the alimentary tracts of the snails. Feeding experiments in this study tracked the fate of nematocysts as they passed through the alimentary canal to the Introduction digestive gland in the dorsal appendages, the cerata, to the cnidosacs, and finally in the faeces. In digestive cells, Aeolidacean nudibranchs (: Opisthobran- many structurally intact nematocysts were present in chia), which feed on of the Cnidaria, devour large phagosomes that remained unaffected even after masses of nematocysts with their food. It has been 2 days fasting, and phagosomes containing nematocysts suggested that during ingestion the snails are able to were found in the faeces. Thus, it is inferred that fusion inhibit discharge of nematocysts, possibly by the secre- of nematocyst-containing phagosomes with lysosomes tion of mucus (Glaser 1910; Graham 1938; Rosin 1970; and subsequent digestion of nematocysts is blocked. Salvini-Plawen 1972; Conklin and Mariscal 1977; Masses of exposed, undigested and structurally intact Schmekel and Portmann 1982). However, we and others nematocysts were discarded in the faeces. In the tips of (Harris 1973; Greenwood and Mariscal 1984a; Martin the cerata, other nematocysts were phagocytosed by and Walther 2002a) found many discharged nemat- cnidophages and stored in the cnidosacs. After release, ocysts in a pharynx (63% empty capsules versus 37% in contact with seawater, nematocysts were able intact nematocysts) and in the stomachs of feeding snails to discharge. When cnidophore tentacles of E. racemo- (64, 33, 38, 64, 32, 7% empty capsules). An attractive sum with only holotrichous isorhizas were fed to the hypothesis is that the functionally mature nematocysts snails, the isorhizas arrived in the cnidosacs about 2 h discharge during ingestion, while those that are func- after feeding and mixed with existing small euryteles. tionally immature remain intact and undischarged Some cnidosacs also contained very large microbasic (Naville 1926; Greenwood and Mariscal 1984a, 1984b). eurytele or large macrobasic eurytele nematocysts, During evolution aeolid nudibranchs have acquired possibly from Eudendrium ramosum and Eudendrium protective mechanisms against injury from discharging glomeratum, respectively. This indicates that the various nematocysts (Edmunds 1966; Martin and Walther types of nematocysts from food were all incorporated 2002a, 2002b). into the cnidosacs. Evidence for a selection process An unresolved issue is whether snails are capable of or digestion of a certain nematocyst type in the digesting the intact nematocysts. Schmekel and Wechsler (1968a) reported evidence for the digestion of nemat- Communicated by O. Kinne, Oldendorf/Luhe ocysts in cells of the digestive gland. Kepner (1943) and Day and Harris (1978) assumed that nematocysts of a R. Martin certain type are digested in cells of the cnidosacs at the Zentrale Einrichtung Elektronenmikroskopie, Universita¨ t Ulm, tips of the dorsal appendages, the cerata. Moreteau Albert-Einstein-Allee 11, 89069 Ulm, Germany E-mail: [email protected] (1977) found both degraded and undegraded nemat- Tel.: +49-731-5023441 ocysts in the faeces. In any case, the nematocysts are Fax: +49-731-5023383 stored for long periods in cnidosacs (Wright 1863; 534

Grosvenor 1903; Kepner 1943; Conklin and Mariscal overnight were aspirated with a pipette and immersed in fixative. 1977; Day and Harris 1978), where they are thought to The fixative was 2% paraformaldehyde and 2% glutaraldehyde in seawater, which had been mixed 1:1 with H2O and to which 0.35 M mature (Naville 1926; Greenwood and Mariscal 1984a), sucrose, 0.17 M NaCl and 0.1 M sodium cacodylate (pH 7.4) were and, when ejected, many of them discharge when added. After a minimum of 7 days and up to many months in the coming in contact with either seawater (Conklin and fixative, the specimens were contrasted with 2% osmium tetroxide Mariscal 1977), the mouth of a fish (Herdman 1890), in water and 2% uranyl acetate in ethanol, then dehydrated, embedded in Epon and examined by light microscopy and trans- another aeolid nudibranch (Wolter 1967), human skin mission electron microscopy in the laboratory at Ulm. For phase (Thompson and Bennett 1969), or the tongue (Glaser contrast microscopy, 0.75 lm thick sections were mounted in 1910). Discharging cnidosac nematocysts may mechan- Depex. In digital images of these sections all nematocysts clearly ically attach the snail to the prey (Tardy 1964). The recognized to belong to a specific type were counted. cnidosacs with their discharging nematocysts, the In order to avoid artifacts due to chemical fixation, live snails were also brought to Ulm, and cerata were high-pressure frozen cleptocnidae, were thought to serve as a defense mech- and freeze-substituted as described in Martin and Walther (2002a). anism for the snails (Kepner 1943; Edmunds 1966); The method of Ericsson and Trump (1965) was applied for the however, others have suggested that cnidosacs fulfil a detection of acid phosphatase. Cerata were fixed as above for 1 h, function of storage and excretion for indigestible and then stored in 0.1 M cacodylate buffer with 1% gum arabic and 0.88 M sucrose. Cerata were opened by a longitudinal cut and nematocysts (Glaser 1910; Streble 1967; Miller and rinsed in 0.1 M cacodylate buffer with 10% sucrose, and then Byrne 2000). incubated for 45 min at 37°C in a freshly prepared mixture of A question raised by several authors is whether the 1 part of 3% Na-ß-glycerophosphate and 10 parts of 0.6 g lead cnidosac cells of the snails are able to recognize and select nitrate in 500 ml of 0.05 M acetate buffer (pH 5.6). They were distinct types of nematocysts. The proportions of nem- subsequently rinsed in 0.1 M acetate buffer (pH 5.6), treated for 30 s with 2% acetic acid, rinsed again in the acetate buffer, stained atocyst types in the cnidosacs were at variance with the with 2% OsO4 in 0.1 M cacodylate buffer and embedded in Epon. proportions of nematocyst types in the food organisms Enzymatic activity of control specimens was blocked by 10% glu- (Grosvenor 1903; Kepner 1943; Moreteau 1977; Day and taraldehyde before addition of the substrate. Harris 1978; O¨ stman 1997). It was suggested that snails are able to select and store in the cnidosacs the types of nematocysts that are most effective against a specific Results enemy in a given habitat, such as penetrating nemat- ocysts where fish predators dominate and entangling Are nematocysts digested in the digestive glands? nematocysts where crustacean predators prevail (Edm- unds 1966; Day and Harris 1978). This recognition pro- Many intact nematocysts as well as components of dis- cess is thought to occur at the cell membrane, or in the charged nematocysts were transported by ciliary action cytoplasm of cnidophages, which has been suggested to and/or peristaltic contractions of the ceratal muscles selectively digest specific nematocyst types (Kepner 1943; from the stomach into the lumen of the cerata with li- Day and Harris 1978). Receptors or ligand molecules for quid and solid food (Fig. 1). Different cell types of the the numerous different nematocyst types are not known. digestive gland line the ceratal lumen (Schmekel and In the present study, intact nematocysts have been Wechsler 1968a, 1968b; Griebel 1993; Kress et al. 1994). tracked through the alimentary canal to the faeces, and Digestive cells of cerata of four Cratena peregrina, fixed through the cerata to the digestive gland and into the after feeding, included many nematocysts (Fig. 2a, b). cnidosacs of the common aeolid nudibranch Cratena There were up to five nematocysts per cell in sections. peregrina (Gmelin, 1791) (Gastropoda: Opisthobran- Most of them were lying in large phagocytic vacuoles, chia). These usually feed on colonies of the the phagosomes (Fig. 2a, b); others were closely sur- hydrozoan Eudendrium racemosum (Gmelin, 1791) rounded by the phagosome membrane (Fig. 2a). In (Cnidaria: Anthoathecata). Questions addressed were: electron micrographs of conventionally fixed specimens whether nematocysts are digested and whether cnidosac the enveloping host cell membrane often was difficult to cells are able to select specific nematocyst types. detect, but it was clearly seen in high-pressure frozen cerata (Fig. 2e). This was examined in more detail, be- cause Greenwood and Mariscal (1984b) suggested that a Materials and methods direct contact of the nematocysts with the cytoplasm of the host cell is responsible for the absence of enzymatic Specimens of Cratena peregrina, Eudendrium racemosum and Eu- digestion in lysosomes. Phagosomes with nematocysts dendrium armatum were collected in May and October 2000, 2001 were clearly separated from heterolysosomes. Hetero- and 2002 by SCUBA diving near the island of Giglio (Italy, Tus- lysosomes, but not the phagosomes with nematocysts, cany). The animals were kept in the laboratory at the Istituto di Biologia Marina, Campese, 58012 Isola del Giglio, Italy, at about exhibited acid phosphatase reaction product (Fig. 2d). I 18°C. The snails were usually fed with colonies of E. racemosum conclude that digestive cell phagosomes with nemat- and fixed under varying conditions (see ‘‘Results’’). In a series of ocysts usually do not fuse with lysosomes. experiments, two snails, fasted overnight, were each fed 10 or 12 In snails feeding on Eudendrium racemosum, the cnidophore tentacles of E. racemosum that had been collected. Three or four cerata were removed and fixed during feeding, 2 and structurally intact nematocysts in digestive cells were of 4 h after one snail started feeding, and at feeding and 3.5 h, after both types, small microbasic euryteles and holotrichous the other snail started feeding. Faecal pellets of four snails fasted isorhizas. A very large microbasic eurytele of the 535

of nematocysts among masses of faecal pellets, bacteria and undefined material (Fig. 3a, b). Most of them were undischarged and appeared structurally intact (Fig. 3b). Internally degraded nematocysts, or empty capsules and profiles of nematocyst threads were also found. Both types of nematocysts from E. racemosum (small micro- basic euryteles and holotrichous isorhizas) were present. While most of the well-preserved, exposed, intact cysts were very likely transported through the alimentary ca- nal directly to the anus, without passing through ceratal digestive glands, there were also nematocysts within phagosomes (Fig. 3c) that presumably were extruded from digestive cells. Figure 3c also shows a large het- erolysosome with a degrading nematocyst. Fragments of digestive cells were regularly found in the faeces. This indicates a process of disintegration and turnover of digestive gland cells, the fragments of which are dis- carded in the faeces. By this process, apparently, phagosomes with nematocysts, as well as heterolyso- somes and their contents appear in the faeces. A large quantity of intact nematocysts was not digested, either in the alimentary tract or in digestive cells, but were eliminated with the faeces in a structurally intact state.

Do cnidosac cnidophages select specific nematocyst types? Fig. 1 Cratena peregrina. A schematic horizontal cross section through the anterior part showing anus (an), cerata (cr), cnidosacs I observed specimens of C. peregrina feeding on speci- (cs), digestive gland (dg), epithelium with cuticula (ec), epithelium mens of E. racemosum (Gmelin, 1791) and E. armatum with special vacuoles (ev), lips (lp), labial tentacle (lt), pharynx (ph), Tichomiroff, 1887. E. racemosum has 7–10 lm long, (rd) and stomach (st) small microbasic eurytele nematocysts on the tentacles. When longitudinally cut they are oval with a Eudendrium ramosum type was found in a digestive cell. pointed end and exhibit a distinct shaft or rhabdome Degraded nematocysts with indistinct interior structures, (Fig. 4a). At the base of the polyps there are rings with empty and collapsed capsules (Fig. 2b; Table 1) and larger holotrichous isorhiza nematocysts, 10–14 lm thread and barb components of discharged cysts, as well long. They are oval in a longitudinal plane and do not as remnants of diatoms, and even spindles, the secretion exhibit a rhabdome (Fig. 4a). Identical isorhizas occur products of the epithelial cells lining the snail’s stomach on the cnidophores, very long defense tentacles origi- (Martin and Walther 2002b), were also contained in nating at the base of about one of five polyps in a colony phagosomes. Thus, phagocytosis of nematocysts and (Martin and Walther 2002a). E. armatum has small food particles by digestive cells is rather non-selective. microbasic euryteles only, both on the tentacles and on These findings were not much different in snails that cnidophores, which totally replace some of the polyps. had fasted 2 days after an abundant meal. The digestive The nematocysts of this species are indistinguishable cells in the cerata still exhibited phagosomes with from the small euryteles of E. racemosum. structurally intact nematocysts, which were separated The cnidosacs of snails feeding on E. racemosum in- from heterolysosomes (Fig. 2c; Table 1). Nonetheless, a cluded masses of small microbasic eurytele nematocysts, few structurally intact and degraded nematocysts and smaller numbers of holotrichous isorhizas (Fig. 4a; occurred in heterolysosomes (Table 1). Thus, the block- Table 2). In some C. peregrina there were cnidosacs with ade of fusion of phagosomes with lysosomes is not an very large nematocysts (25–28 lm long) with a distinct all-or-nothing process. I conclude that many nematocysts shaft or rhabdome, possibly from E. ramosum (Linna- in the phagosomes of digestive cells remain structurally eus, 1758), in addition to small microbasic euryteles intact for at least 2 days and are not digested. (Fig. 5). In cnidosacs of other snails there were different large nematocysts (25–28 lm long), with a spirally wound shaft, macrobasic euryteles, possibly of E. glom- Nematocysts are eliminated with faeces eratum Picard, 1952, besides small microbasic euryteles. However, because I was unable to collect these two Faeces from snails that had eaten and were isolated hydrozoan species, it was not possible to compare the overnight from food organisms contained large numbers two large nematocyst types in the cnidosacs with those 536

Fig. 2a–e Cratena peregrina. Electron micrographs of digestive cells four nematocyst types, which are known to occur in the in the cerata fixed after feeding on Eudendrium racemosum. a, b four hydrozoan species (Schuchert 2003), were found in Many structurally intact (stars) and degraded (triangles) nemat- ocysts are included in phagosomes of digestive cells bordering the cnidosacs of C. peregrina. There was clearly no evidence lumen (lu) of the ceras. c A digestive cell of an after 2 days of selective exclusion of specific nematocyst types by starvation, with a structurally intact nematocyst in a phagosome cnidophagous cells. (asterisk) next to a heterolysosome (hl). d A digestive cell with acid The time course of fresh nematocysts incorporated phosphatase reaction product in heterolysosomes (hl, arrows). e A into cnidosacs was evaluated by feeding cnidophores of high-pressure frozen digestive cell with a nematocyst (star) showing the host cell membrane (arrow) closely surrounding the nematocyst E. racemosum containing only holotrichous isorhiza capsule nematocysts to starved specimens of C. peregrina. Three cerata were removed during feeding, at intervals of 2, 3.5 of E. ramosum or E. glomeratum, nor was it possible to and 4 h, and the nematocysts were counted in light observe the snails feeding on them. In the course of the microscopical sections of cnidosacs. In all cnidosacs over 2 years of this study, in spring and in October, all there was a majority of small euryteles and a lower 537

Table 1 Cratena peregrina. Nematocysts in digestive cells Nematocysts in lysosomes, Nematocysts in phagosomes Number of of cerata degraded structure cerata/animals Intact structure Degraded structure Empty capsules

After feeding – 30 9 10 1/1 – 57 12 28 1/1 – 10 1 – 1/1 After 2 days starvation 5 30 2 – 6/2

Fig. 3a–c Cratena peregrina. Light (a) and electron micrographs 11.7%, 7.0% and 5.5% in a second snail). This indicates (b, c) of the faeces, fixed after feeding on Eudendrium racemosum. more of a random than of a selective handling of ne- a The arrows point to some of the numerous structurally intact, exposed, microbasic euryteles. b Euryteles, bacteria (arrowheads) matocysts during ingestion, transport, phagocytosis, and and faecal pellets (stars). c A phagosome with a nematocyst (large storage in the cnidosacs. arrow), a heterolysosome with a degraded nematocyst (small arrow) At the ultrastructural level, evidence for digestion of and faecal pellets (stars) nematocysts in cnidosac cells, as suggested by Day and Harris (1978), has not been observed. On the contrary, it was the nematocyst-filled cnidophage cells, not nemat- number of isorhizas. During feeding the relative num- ocysts per se that showed signs of disintegration towards bers of isorhizas were about equal in the proximal and the distal end of the cnidosacs. Cnidophage cells in the distal halves of the cnidosacs and in groups of extruded proximal end were compact and appeared undifferenti- cysts (Fig. 6). There were relatively more isorhizas in ated, while in distal ends the nematocyst-filled cells proximal halves 2, 3.5 and 4 h after feeding as compared exhibited large vacuoles. In the packages of expulsed to distal halves and ejected nematocysts (Fig. 7). This nematocysts (Fig. 4b) the enveloping cellular mem- indicates that the fresh isorhizas from the food had ar- branes had ruptured, and many nematocysts at the rived in the cnidosacs at least 2 h later. In the cnido- surface of the packages, which were exposed to seawater, phage cells of the cnidosacs, the phagocytosed isorhizas had discharged. had mixed with already present small euryteles. Because of this mixing with present nematocysts, the quality and proportions of the nematocysts in the cnidosacs were different as compared to those in the stomach, in which Discussion only cnidophore isorhizas had been ingested in these experiments. When the aeolid nudibranch Cratena peregrina feeds on Considering snails feeding on entire E. racemosum the hydrozoan Eudendrium racemosum, the stomach of polyps, the percentages of isorhizas varied significantly the snail contains a large proportion of intact nemat- in stomachs and cnidosacs (P<0.01) (Table 2). Also in ocysts, besides a significant number of discharged different cnidosacs of the same snail the proportions of nematocysts (Martin and Walther 2002a). When undis- isorhizas varied (7.7%, 4.2% and 3.3% in one snail; charged, structurally intact nematocysts were tracked 538 b Fig. 4 Cratena peregrina. Light micrographs of cnidosacs 3.5 h (upper) and 4 h (lower) after feeding cnidophores of Eudendrium racemosum with isorhizas only to C. peregrina. Upper: the arrowheads point to holotrichous isorhizas, and the arrows to small microbasic euryteles, in the proximal (p) and distal (d) cnidosac. Lower: during fixation the cnidosac released masses of nematocysts at the tip (star)

they were found: (1) in digestive gland cells of cerata, (2) in the cnidosacs at the tips of the cerata and (3) in the faeces, at various times after feeding. The intact nematocysts in the stomach may be immature, but when extruded from the cnidosacs many of them dis- charged in contact with seawater. A maturation process in the cnidosacs has been postulated (Naville 1926; Greenwood and Mariscal 1984a).

Fig. 5 Cratena peregrina. A cnidosac with small (arrows) and large microbasic euryteles (arrowheads), the latter possibly from Eudend- rium ramosum 539

also detected in the heterolysosomes of the digestive cells, structurally intact nematocysts usually were not included in heterolysosomes. After starving for 2 days intact ne- matocysts still were contained in large phagosomes or tightly surrounded by membranes of the host cells, sep- arated from heterolysosomes. Apparently, the properties of nematocysts are such as to interfere or block fusion of the phagosome with a lysosome. This is the case with chloroplasts in digestive gland cells of herbivorous saccoglossan species (Taylor 1968; Trench 1969). Fusion- inhibiting mechanisms have been examined when pathogenic mycobacteria were taken up in phagosomes of mammalian macrophages (Ku¨ hnel et al. 2001). In Fig. 6 Cratena peregrina. The number of isorhizas per 100 previous studies of nudibranchs the occurrence of empty nematocysts (±standard deviation) in three cnidosacs of snail I (squares) and three cnidosacs of snail II (triangles), in the proximal capsules in lysosomes of digestive cells was taken to (p) and distal (d) halves, and in ejected nematocysts (e), during indicate intracellular digestion of nematocysts (Schmekel feeding on cnidophores of Eudendrium racemosum with isorhizas and Wechsler 1968a), and Moreteau (1977) found spe- only. The counts in the different areas are not significantly different cific nematocyst types in degraded state in the faeces of (P>0.05); for each point >850 nematocysts were counted. There is . However, these degraded components no evidence for a higher proportion of isorhizas in the proximal part may have been derived from the discharged nematocysts in the stomachs, which were transported to and absorbed by digestive cells, and/or delivered directly to the faeces. However, in the present study a few nematocysts with decomposing internal structures were also found in heterolysosomes, especially after 2 days starvation (Table 1). Thus, some nematocysts, possibly damaged cysts, were passed from phagosomes to lysosomes. If large numbers of intact nematocysts are not effi- ciently processed for digestion in lysosomes, how do the digestive cells eliminate the nematocyst loads? Signs of decomposition of nematocyst-laden digestive cells and fragments of digestive cells, as well as phagosomes with nematocysts in the faeces of the snails, indicated a fast turnover of the digestive cells. This was found also by Fig. 7 Cratena peregrina. As in Fig. 6, but 2 h after feeding (three cnidosacs of snail I, inverted triangles), 3.5 h after feeding (four Schmekel and Wechsler (1968a, 1968b). cnidosacs of snail II, circles) and 4 h after feeding (four cnidosacs The very large number of structurally intact, exposed of snail I, triangles). The counts in the proximal halves (p) are nematocysts in the faeces of C. peregrina, one night after significantly different from the counts in the distal halves (d) feeding, suggests that many of the nematocysts derived (P<0.01); for each point >750 nematocysts were counted. There are more isorhizas in the proximal halves, as compared to the distal from food pass through the alimentary tract without halves and the ejected nematocysts (e) digestion (for experiments with fibrin-coated colloidal gold, see Ho¨ rstadius 1933). While masses of nematocysts Nematocysts in the digestive cells of the cerata were are discarded directly or via the digestive gland, some contained in large phagosomes, as were other food are transported to the tips of the cerata, enter the particles and also empty capsules of discharged nemat- cnidosacs and are phagocytosed by cnidophages (Wright ocysts. While food particles as well as the capsules were 1863; Grosvenor 1903). They are then stored for many

Table 2 Cratena peregrina. Proportional content of intact nematocyst types in sections through stomachs and cnidosacs

Small microbasic Holotrichous Very large Large macrobasic Number of euryteles isorhizas microbasic euryteles with stomachs or (see Fig. 4) (see Fig. 4) euryteles spiral shaft cnidosacs/number (see Fig. 5) of animals

Stomachs of snails fed on 66.0% 34.0%±13.5 SD – – 6/3 Eudendrium racemosum Cnidosacs of snails fed on E. racemosum 92.1% 7.9%±5.1 SD – – 8/4 Cnidosacs collected in May (>150) 2 53 – 5/1 (>150) 1 7 – 5/1 92.0% – 8.0% – 2/2 Cnidosacs collected in October 99.0% – – 1.0% 2/2 540 days in the cnidosacs (Kepner 1943; Day and Harris proportion is discarded intact and undigested with the 1978; Conklin and Mariscal 1977), and extruded when faeces, while others are incorporated and stored in the the animal is disturbed. Many of these nematocysts are tips of the cerata, and are eventually ejected. They may functionally mature, since they discharge in contact with serve for defense (Herdman 1890; Grosvenor 1903; seawater. Kepner 1943; Edmunds 1966). Others suggested that the A selection process in the cnidosacs has been postu- cnidosacs are organs for the storage and disposal of lated (Kepner 1943; Edmunds 1966; Moreteau 1977; nematocysts (Glaser 1910; Streble 1967; Miller and Day and Harris 1978; O¨ stman 1997). Specific penetrat- Byrne 2000), which do not serve a defense function. The ing nematocysts where fish predators dominated and two functions would not be mutually exclusive. entangling nematocysts where crustacean predators prevailed were thought to be selectively stored in cni- Acknowledgements Support by the Deutsche Forschungsgemeins- dosacs of certain snails (Edmunds 1966). The selection chaft (Ma 259/16-3) is gratefully acknowledged. I would like mechanism was predicted to be in the plasma membrane to thank P. Walther for hospitality in the laboratory at Ulm and of the cnidophages and/or in the cytoplasm of the cni- help with high pressure freezing, E. Koenig (University at Buffalo) for revision of the text, two anonymous and a non-anonymous dophages, where it was suggested that specific nemato- colleague for criticisms of the manuscript, and M. Hagedorn cyst types were digested (Kepner 1943; Day and Harris (Ulm) for help with the computer work. C. Valentin and his staff 1978). A membrane-bound recognition mechanism for provided excellent working conditions at the field station at Giglio. the many different nematocyst types expressed in The experiments comply with the current laws of Italy and cnidarians seems difficult to conceive. Intracellular Germany. digestion of nematocysts could not be confirmed, either in the digestive cells or in the cnidophages of C. pere- References grina. 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