Management of Nematocysts in the Alimentary Tract and in Cnidosacs of the Aeolid Nudibranch Gastropod Cratena Peregrina
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Marine Biology (2003) 143: 533–541 DOI 10.1007/s00227-003-1078-8 R. Martin Management of nematocysts in the alimentary tract and in cnidosacs of the aeolid nudibranch gastropod Cratena peregrina Received: 22 January 2003 / Accepted: 21 March 2003 / Published online: 27 May 2003 Ó Springer-Verlag 2003 Abstract The nudibranch gastropod Cratena peregrina cnidophages was not obtained. It is concluded that a (Opisthobranchia: Aeolidaceae), when feeding on polyps large proportion of the nematocysts ingested with the of the hydrozoan Eudendrium racemosum (Cnidaria: food are not digested, but are eliminated, structurally Anthoathecata), devours masses of small microbasic and functionally intact, via the alimentary canal and the eurytele and holotrichous isorhiza nematocysts. Large tips of the cerata. 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 (Gastropoda: Opisthobran- many structurally intact nematocysts were present in chia), which feed on species 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, cnidosac 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 animals 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.