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A Modified View on Octocorals: Heteroxenia fuscescens Nematocysts Are Diverse, Featuring Both an Ancestral and a Novel Type

  • Chen Yoffe,

    Affiliation Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel

  • Tamar Lotan,

    Affiliation Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Haifa, Israel

  • Yehuda Benayhau

    YehudaB@tauex.tau.ac.il

    Affiliation Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel

A Modified View on Octocorals: Heteroxenia fuscescens Nematocysts Are Diverse, Featuring Both an Ancestral and a Novel Type

  • Chen Yoffe, 
  • Tamar Lotan, 
  • Yehuda Benayhau
PLOS
x

Abstract

Cnidarians are characterized by the presence of stinging cells containing nematocysts, a sophisticated injection system targeted mainly at prey-capture and defense. In the anthozoan subclass Octocorallia nematocytes have been considered to exist only in low numbers, to be small, and all of the ancestral atrichous-isorhiza type. This study, in contrast, revealed numerous nematocytes in the octocoral Heteroxenia fuscescens. The study demonstrates the applicability of cresyl-violet dye for differential staining and stimulating discharge of the nematocysts. In addition to the atrichous isorhiza-type of nematocysts, a novel type of macrobasic-mastigophore nematocysts was found, featuring a shaft, uniquely comprised of three loops and densely packed arrow-like spines. In contrast to the view that octocorals possess a single type of nematocyst, Heteroxenia fuscescens features two distinct types, indicating for the first time the diversification and complexity of nematocysts for Octocorallia.

Introduction

Within certain epithelial cells of members of the phylum Cnidaria there are distinct intracellular secretion products, known as nematocysts [1], [2]. They are commonly found in the body, and especially in the tentacles, of jellyfish, hydroids, sea anemones, and corals, and are used for capturing food, defense against predation and, in certain cases, for adherence to a substrate [1], [2]. In response to appropriate stimuli, the nematocysts discharge, releasing long, thin, folded tubules, commonly onto a prey where, depending upon the nematocyst type, they either entwine, pierce, or adhere to it [2], [3], [4], [5].

Although nematocysts have been long studied, little is known on those of the anthozoan subclass Octocorallia, most probably due to the generally held view that octocorals possess only a few small nematocysts [6], [7]. To date, most studies report atrichous isorhiza as the sole nematocyst type found in octocorals, as opposed to all other cnidarians, which typically feature several types [8], [9]. Several studies have referred to Mariscal's (1974) review on octocoral nematocysts, and further argued that octocorals generally lack stinging nematocysts, or possess only the atrichous isorhiza type [5], [7]. David et al. [9] indicated that the latter are the ancestral-type for all cnidarians, and the only type present in Octocorallia. A study on Leptogorgia virgulata (Alcyonacea) and Renilla mülleri (Pennatulacea) indicated the presence of only a very few nematocysts of a single type [6]. Subsequently, Schmidt [10] studied nematocysts in Alcyonium digitatum, Cornularia cornucopiae, Parerythropodium coralloides, Pseudopterogorgia aerosa and Paralcyonium elegans (Alcyonacea), as well as in Pteroeides spinosum and Veretillum cynomorium (Pennatulacea), all of which featured solely the atrichous isorhiza type. Schmidt and Moraw [11] examined the nematocysts of A. digitatum, P. coralloides, P. aerosa and V. cynomorium, concluding that their structure (and possibly also their function) is much simpler than that in the hydrozoans. Sebens and Miles [12] revealed hundreds of nematocysts in the sweeper tentacles of Erythropodium caribaeorum (Alcyonacea), characterized as the holotrichous type. Piraino et al. [13] were the only ones to describe two nematocyst types- atrichous and holotrichous isorhiza in Corallium rubrum. Jimbo [14] revealed on both the nematocysts and on the zooxanthellae surface in Sinularia lochmodes (Alcyonacea), a D-galactose binding lectin (SLL-2), indicating the possibility of a lectin-mediated host-symbiont interaction.

The octocoral Heteroxenia fuscescens (Ehrenberg, 1834) is a shallow-water reef-dweller on Eilat's reefs (northern Gulf of Aqaba, Red Sea), where it has been extensively studied, including aspects of its sexual reproduction [15], planula-metamorphosis [16], and acquisition of zooxanthellae [17]. The colonies neither feed on prey nor ingest particulate food, but utilize dissolved organic material and the photosynthates provided by their zooxanthellae [18]. The current study reports for the first time the finding of abundant nematocysts in H. fuscescens, including a novel type. These findings shed new light on octocoral nematocysts and challenge the commonly held perception on stinging cells in this group.

Results

Cresyl violet stained nematocysts

Examination of cresyl-violet stained histological sections of H. fuscescens polyps revealed numerous nematocytes (i.e., stinging cells containing the sub-cellular nematocyst), differentially stained red, and thus distinct from the violet-stained epidermis and gastrodermis, pinkish mesoglea, or dark violet zooxanthellae (Fig. 1). The nematocytes were mostly found on the surface of the gastrodermis of the gastrovascular cavity of the polyps (Fig. 1a) and on the tentacular pinnules (Fig. 1b). Notably, the epidermis rarely featured nematocytes (Fig. 1a). In the tentacles the nematocytes mainly appeared within the cavity of the pinnules adjacent to the gastrodermis and less in the epidermis (Fig. 1b). SEM micrographs revealed numerous nematocytes exposed on the gastrodermis, some entangled with the cilia (Fig. 2a). The nematocytes are elongated, about 9 µm in length, have a round and a pointed end, and a laterally-positioned cnidocil (Fig. 2b), which is the mechanosensory cilium receptor for nematocyst discharge [19]. Interestingly, the cresyl-violet- treated wet preparations of the polyp content and tentacular pinnules stimulated massive discharge of nematocyst, which otherwise remained intact. Two types of nematocysts were observed, both red-stained, well differentiated in shape and location. The dominant nematocysts in the gastrovascular cavities feature a capsule, oriented at ∼90° to a uniform long thread tubule (Fig. 3a, c), known to be characteristic to the atrichous isorhiza type [20]. A second, but less common, type was primarily discharged from the tentacular-pinnules. It has a unique thread tubule, with three loops, which narrows toward its distal end (Fig. 3b, d).

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Figure 1. Light microscopy of cresyl-violet stained micrographs of Heteroxenia fuscescens.

a. Cross-section of polyp at pharyngeal level. b. Cross-section of tentacular pinnules. Nematocytes stained red (white arrows), pharynx (ph); epidermis (ep); mesoglea (me), gastodermis (ga), mesenterial filament (mf); gastrovascular cavity (gvc), zooxanthellae (white arrow heads).

https://doi.org/10.1371/journal.pone.0031902.g001

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Figure 2. Scanning-electron micrographs of nematocytes within the gastrovascular cavity of Heteroxenia fuscescens.

a. Nematocytes on the gastrodermis (white arrows); insert: cross-section of exposed gastovascular polyp cavity with mesenteries (mf), enlarged area of a indicated by rectangle. b. Enlarged view of a nematocyte, cnidocil (cn).

https://doi.org/10.1371/journal.pone.0031902.g002

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Figure 3. Light microscopy of wet mounts of discharged nematocysts of Heteroxenia fuscescens stained by cresyl violet.

a. Atrichous isorhiza nematocysts isolated from polyp cavity, zooxanthellae indicated by arrow. b. Novel type of nematocysts adjacent to surface of tentacular pinnules, zooxanthellae indicated by arrow. c. Enlarged view of a discharged atrichous isorhiza nematocyst-type from the gastrovascolar cavity. d. Novel macrobasic-mastigophore nematocyst-type from pinnules.

https://doi.org/10.1371/journal.pone.0031902.g003

Nematocyst characterization

The thread tubule of the atrichous isorhiza type, isolated from the gastrovascular cavity, has a uniform diameter along its entire length (Fig. 4a) and an open tip (Fig. 4b). Fine conical spines emerge along the thread tubule, each about 0.5 µm long (Fig. 4b, c). Notably, the spines are indistinct under light microscopy (Fig. 3c), thus corresponding to the definition of atrichous isorhiza nematocysts [8], [21]. The capsule is 9.7±0.75 µm long and 4.3±0.39 µm wide, and the thread tubule is 273.32±55.74 µm long (Table 1).

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Figure 4. Scanning-electron micrographs of discharged atrichous isorhiza nematocyst of Heteroxenia fuscescens.

a. Whole nematocyst with capsule (cp) and elongated thread tubule. b. Enlarged view of the tip (arrow) of the thread tubule with spines (s). c. Spines along the thread tubule (s).

https://doi.org/10.1371/journal.pone.0031902.g004

SEM provided further structural details on the novel type of nematocyst (Fig. 3b, d). It features a basal portion, termed shaft [8], which is spiral and comprises three loops (Fig. 5a). The surface of the shaft features densely packed spines, each ∼1 µm long. These arrow-like spines are each composed of a pair of side-pointed hooks (Fig. 5b). The shaft gradually tapers to a narrower plain thread-tubule without spines (Fig. 5c). The thread-tubule is 136±19.69 µm long (n = 15 nematocysts) and significantly shorter than the atrichous isorhiza-type. Its capsule is 7.77±0.90 µm long, also significantly shorter than the latter, but 4.62±0.56 µm wide, like the atrichous isorhiza (Table 1). To the best of our knowledge this type of nematocyst (Fig. 5) has not been recorded to date in the literature. Since the shaft length is more than four-fold that of the capsule (4.9±0.61, n = 15; Table 1), we have defined the type of nematocyst as macrobasic-mastigophore following [21], [22].

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Figure 5. Scanning-electron micrographs of novel macrobasic-mastigophore nematocyst type of Heteroxenia fuscescens.

a. Whole nematocyst with capsule (cp), spiral shaft and elongated thread. b. Enlarged view of spiral shaft covered with arrowhead-shaped spines (white arrow). c. View of the plain proximal thread tubule. Scale at b also applies to c.

https://doi.org/10.1371/journal.pone.0031902.g005

Discussion

Our preliminary work has indicated that nematocysts of H. fuscescens are not stained by the commonly used hematoxylin and eosin dye. Safranin O and methylene blue, known stains of nematocysts, yield non differential results and thus do not distinguish between the different tissue cell-types. Our study indicates for the first time that cresyl violet offers a straightforward staining of nematocysts (“drip and wash”), applicable both to histological sections and live nematocytes. The cresyl violet differentially stained the nematocysts red, in contrast to the rest of the cells (Fig. 1). Moreover, the current study shows for the first time that this dye successfully stimulates discharge of nematocysts both in vivo and ex vivo (Fig. 3). Cresyl violet is commonly used for neuronal tissue studies, and due to its cationic characteristics it binds readily to anionic macromolecules of the neurons [23]. Thus, we assume that the basic cresyl-violet dye binds to the anionic poly γ glutamic homopolymer inside the nematocyst, releasing cations from the capsule matrix and consequently driving its discharge [24].

The current study in H. fuscescens has revealed numerous nematocysts of at least two distinct types: atrichous isorhiza and a novel macrobasic-mastigophore. The former type was mainly found in the gastrovascular cavity, adjacent to the gastrodermis, while the novel one was prevalent in the tentacles. We suggest that the atrichous isorhiza nematocysts play a role in H. fuscescens in management of microorganisms introduced with the seawater into the gastrovascular cavity. Whether this is a defense mechanism against parasites and pathogens, as often performed by nematocysts [4], [25], remains to be studied. Interestingly Cheng and Wong [26] described a number of nematocysts of gastrodermal origin that had been discharged into the gastrovascular cavity of the stony coral Porites spp., in the vicinity of parasite cysts; and the subsequently found abundance of nematocysts on the surface of the gastrodermis may further support this view. The microstructure of the novel macrobasic-mastigophore-type, and in particular its spiral shaft bearing arrow-like spines (Fig. 4), suggests a specialized protective adaptation whose nature awaits future studies.

The present study has revealed that nematocysts in octocorals are more diversified than previously considered, featuring side-by-side the ancestral atrichous isorhiza and a novel macrobasic-mastigophore. Mastigophore-type nematocysts are found in the most related Hexacorallia sister subclass of Octocorallia, as well as in some other cnidarian groups. It was suggested that the mastigophore-type is in fact an ancestral hexacorallian cnidom and that nematocysts development preceded the diversification of Hexacorallia [27]. The Octocorallia phylogeny places H. fuscescens in one of their two major clades, but distant from its suggested root [28], [29]. The Octocorallia subclass is highly diverse, containing over 3,000 species with a remarkably limited nematocyst data-record. It is consequently evident that a thorough study of octocoral nematocytes is still needed. Such a study should also include analysis of the minicollagen proteins, which were shown to vary between simple and more sophisticated nematocysts [9], [30]. This may reveal whether H. fuscescens is unique in its subclass, suggesting an independent evolvement of the mastigophores in this species; or, perhaps, that other octocorals also contain this nematocyst-type, thereby not only indicating the existence of a wider array of nematocysts among Octocorallia, but also placing the mastigophore together with the atrichous-isorhiza as an ancestral type of nematocyst [9].

Materials and Methods

Specimen collections and maintenance

Colonies of H. fuscescens were collected by SCUBA diving in Eilat, shipped to Tel Aviv University, and maintained there in a closed artificial seawater system: seawater temperature ∼24°C, salinity 35–38 ppm, and natural sunlight (2009–2011).

Light and scanning-electron microscopy

For light and scanning-electron microscopy (SEM) studies, polyps were either removed and preserved in 4% glutaraldehyde in seawater or examined alive. The preserved material was rinsed in double-distilled water (DDW) and decalcified in a mixture of equal volumes of formic acid (50%) and sodium citrate (15%) for 20 min [16]. The polyps then underwent dehydration through a graded series of ethanol and xylene, were embedded in paraffin, and serial cross-sections (5–6 µm thick) were obtained by Shandon MIR microtome. Removal of the paraffin was followed by two short xylene immersions, rehydration with a decreased graded series of ethanol, and a rinse in DDW. Finally, the slides were dried at room temperature, cover slips were glued with Eokkit (03989 Fluka), and the slides were examined under a Nikon OPTIPHOT light microscope.

For SEM studies the decalcified polyps underwent dehydration (see above) and were critically point-dried. The gastrovascular cavity was then exposed with a scalpel blade under a dissecting microscope (Nikon SMZ800), mounted on stubs, and gold-coated. Live nematocysts that were initially stained with cresyl violet (see ahead) were dry-smeared on microscope slides, rinsed with running tap water, dried at room temperature, and then gold-coated. The material was examined under a JEOL JSM 840A SEM (25 kV) and under a Quanta 200 FEG Environmental Scanning Electron Microscope (ESEM) (200 V–30 kV).

Isolation of live nematocysts

In order to isolate live nematocysts from the gastrovascular cavity of the H. fuscescens polyps, a sterile pipette tip (100 µm) was inserted through the mouth opening and the cavity content was removed into Eppendorf tubes. These were centrifuged for five min (600 g),

Cresyl violet staining

Sections were stained with 0.1% cresyl violet (Sigma C- 1791). The stain was applied by dripping onto the sections, and after two min rinsed under running tap water.

Live nematocysts and wet mounts of tentacular pinnules were rinsed in cresyl-violet before further observation.

Acknowledgments

We are grateful to A. Colorni and W. Litaker for advice and fruitful discussions during the early stages of the study, S. Dowd for professional help and kind assistance, C. Mcfadden, R. Mariscal, A. Diamant and P. Keeling for valuable guidance. M. Weis and N. Shalev are acknowledged for help in the field, and the staff of the Interuniversity Institute for Marine Science at Eilat for kind hospitality and facilities. We wish to thank Z. Barkay and Y. Delaria for valuable assistance with electron microscopy, V. Wexsler for digital editing, and N. Paz for skillful editorial assistance. Collection of animals complied with a permit issued by the Israel Nature and National Parks Protection Authority

Author Contributions

Conceived and designed the experiments: CY TL YB. Performed the experiments: CY. Analyzed the data: CY. Contributed reagents/materials/analysis tools: YB TL. Wrote the paper: CY.

References

  1. 1. Mariscal RN, Bigger CH, Mclean RB (1976) Form and function of cnidarian spirocysts .1. Ultrastructure of capsule exterior and relationship to tentacle sensory surface. Cell and Tissue Research 168: 465–474.RN MariscalCH BiggerRB Mclean1976Form and function of cnidarian spirocysts .1. Ultrastructure of capsule exterior and relationship to tentacle sensory surface.Cell and Tissue Research168465474
  2. 2. Tardent P (1995) The cnidarian cnidocyte, a high-tech cellular weaponry. Bioessays 17: 351–362.P. Tardent1995The cnidarian cnidocyte, a high-tech cellular weaponry.Bioessays17351362
  3. 3. Holstein TW, Benoit M, Herder GV, David CN, Wanner G, et al. (1994) Fibrous mini-collagens in hydra nematocysts. Science 265: 402–404.TW HolsteinM. BenoitGV HerderCN DavidG. Wanner1994Fibrous mini-collagens in hydra nematocysts.Science265402404
  4. 4. Kass-Simon G, Scappaticci AA (2002) The behavioral and developmental physiology of nematocysts. Canadian Journal of Zoology-Revue Canadienne De Zoologie 80: 1772–1794.G. Kass-SimonAA Scappaticci2002The behavioral and developmental physiology of nematocysts.Canadian Journal of Zoology-Revue Canadienne De Zoologie8017721794
  5. 5. Fautin DG (2009) Structural diversity, systematics, and evolution of cnidae. Toxicon 54: 1054–1064.DG Fautin2009Structural diversity, systematics, and evolution of cnidae.Toxicon5410541064
  6. 6. Mariscal RN, Bigger CH (1977) Possible ecological significance of octocoral epithelial ultrastructure. In: Taylor DL, editor. Third International Coral Reef Symposium. Miami, Florida: pp. 127–134.RN MariscalCH Bigger1977Possible ecological significance of octocoral epithelial ultrastructure.DL TaylorThird International Coral Reef SymposiumMiami, Florida127134
  7. 7. Sammarco PW, Coll JC (1992) Chemical adaptations in the Octocorallia - evolutionary considerations. Marine Ecology-Progress Series 88: 93–104.PW SammarcoJC Coll1992Chemical adaptations in the Octocorallia - evolutionary considerations.Marine Ecology-Progress Series8893104
  8. 8. Mariscal RN (1974) Nematocysts. In: Muscatine L, Lenhoff HA, editors. Coelenterate Biology Reviews and New Perspectives. New York: London: Academic Press. pp. 129–178.RN Mariscal1974Nematocysts.L. MuscatineHA LenhoffCoelenterate Biology Reviews and New PerspectivesNew YorkLondon: Academic Press129178
  9. 9. David CN, Ozbek S, Adamczyk P, Meier S, Pauly B, et al. (2008) Evolution of complex structures: minicollagens shape the cnidarian nematocyst. Trends in Genetics 24: 431–438.CN DavidS. OzbekP. AdamczykS. MeierB. Pauly2008Evolution of complex structures: minicollagens shape the cnidarian nematocyst.Trends in Genetics24431438
  10. 10. Schmidt H (1981) The cnidogenesis of the Octocorallia (Anthozoa, Cnidaria) .1. secretion and differentiation of capsule and tube. Helgolander Meeresuntersuchungen 34: 463–484.H. Schmidt1981The cnidogenesis of the Octocorallia (Anthozoa, Cnidaria) .1. secretion and differentiation of capsule and tube.Helgolander Meeresuntersuchungen34463484
  11. 11. Schmidt H, Moraw B (1982) The cnidogenesis of the Octocorallia (Anthozoa, Cnidaria) .2. maturation, migration and degeneration of cnidoblast and nematocyst. Helgolander Meeresuntersuchungen 35: 97–118.H. SchmidtB. Moraw1982The cnidogenesis of the Octocorallia (Anthozoa, Cnidaria) .2. maturation, migration and degeneration of cnidoblast and nematocyst.Helgolander Meeresuntersuchungen3597118
  12. 12. Sebens KP, Miles JS (1988) Sweeper tentacles in a gorgonian octocoral - morphological modifications for interference competition. Biological Bulletin 175: 378–387.KP SebensJS Miles1988Sweeper tentacles in a gorgonian octocoral - morphological modifications for interference competition.Biological Bulletin175378387
  13. 13. Piraino S, Ulianich L, Zupo V, Russo GF (1993) Cnidocyst morphology and distribution in Corallium rubrum (L.) (Cnidaria, Anthozoa). Oebalia XIX: 67–78.S. PirainoL. UlianichV. ZupoGF Russo1993Cnidocyst morphology and distribution in Corallium rubrum (L.) (Cnidaria, Anthozoa).OebaliaXIX6778
  14. 14. Jimbo M, Yanohara T, Koike K, Sakai R, Muramoto K, et al. (2000) The D-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates. Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology 125: 227–236.M. JimboT. YanoharaK. KoikeR. SakaiK. Muramoto2000The D-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates.Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology125227236
  15. 15. Ben-David-Zaslow R, Henning G, Hofmann DK, Benayahu Y (1999) Reproduction in the Red Sea soft coral Heteroxenia fuscescens: seasonality and long-term record (1991 to 1997). Marine Biology 133: 553–559.R. Ben-David-ZaslowG. HenningDK HofmannY. Benayahu1999Reproduction in the Red Sea soft coral Heteroxenia fuscescens: seasonality and long-term record (1991 to 1997).Marine Biology133553559
  16. 16. Yacobovitch T, Weis VM, Benayahu Y (2003) Development and survivorship of zooxanthellate and azooxanthellate primary polyps of the soft coral Heteroxenia fuscescens: laboratory and field comparisons. Marine Biology 142: 1055–1063.T. YacobovitchVM WeisY. Benayahu2003Development and survivorship of zooxanthellate and azooxanthellate primary polyps of the soft coral Heteroxenia fuscescens: laboratory and field comparisons.Marine Biology14210551063
  17. 17. Barneah O, Weis VM, Perez S, Benayahu Y (2004) Diversity of dinoflagellate symbionts in Red Sea soft corals: mode of symbiont acquisition matters. Marine Ecology-Progress Series 275: 89–95.O. BarneahVM WeisS. PerezY. Benayahu2004Diversity of dinoflagellate symbionts in Red Sea soft corals: mode of symbiont acquisition matters.Marine Ecology-Progress Series2758995
  18. 18. Schlichter D (1982) Epidermal nutrition of the alcyonarian Heteroxenia-Fuscescens (Ehrb) - absorption of dissolved organic material and lost endogenous photosynthates. Oecologia 53: 40–49.D. Schlichter1982Epidermal nutrition of the alcyonarian Heteroxenia-Fuscescens (Ehrb) - absorption of dissolved organic material and lost endogenous photosynthates.Oecologia534049
  19. 19. Hwang JS, Takaku Y, Chapman J, Ikeo K, David CN, et al. (2008) Cilium evolution: Identification of a novel protein, nematocilin, in the mechanosensory cilium of Hydra nematocytes. Molecular Biology and Evolution 25: 2009–2017.JS HwangY. TakakuJ. ChapmanK. IkeoCN David2008Cilium evolution: Identification of a novel protein, nematocilin, in the mechanosensory cilium of Hydra nematocytes.Molecular Biology and Evolution2520092017
  20. 20. Weill R (1930) Essai d'une classification des nematocystes des cnidaires. The Biological Bulletin 141–153.R. Weill1930Essai d'une classification des nematocystes des cnidaires.The Biological Bulletin141153
  21. 21. Weill R (1934) Contribution a l'étude des cnidaires et de leurs nématocystes. Travaux de la station zoologique de Wimereux Wimereux 10–11: 1–701.R. Weill1934Contribution a l'étude des cnidaires et de leurs nématocystes.Travaux de la station zoologique de Wimereux Wimereux10–111701
  22. 22. Ostman C (2000) A guideline to nematocyst nomenclature and classification, and some notes on the systematic value of nematocysts. Scientia Marina 64: 31–46.C. Ostman2000A guideline to nematocyst nomenclature and classification, and some notes on the systematic value of nematocysts.Scientia Marina643146
  23. 23. Tureyen K, Vemuganti R, Sailor KA, Dempsey RJ (2004) Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques. Journal of Neuroscience Methods 139: 203–207.K. TureyenR. VemugantiKA SailorRJ Dempsey2004Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques.Journal of Neuroscience Methods139203207
  24. 24. Ozbek S, Balasubramanian PG, Holstein TW (2009) Cnidocyst structure and the biomechanics of discharge. Toxicon 54: 1038–1045.S. OzbekPG BalasubramanianTW Holstein2009Cnidocyst structure and the biomechanics of discharge.Toxicon5410381045
  25. 25. Aeby GS, Santavy DL (2006) Factors affecting susceptibility of the coral Montastraea faveolata to black-band disease. Marine Ecology-Progress Series 318: 103–110.GS AebyDL Santavy2006Factors affecting susceptibility of the coral Montastraea faveolata to black-band disease.Marine Ecology-Progress Series318103110
  26. 26. Cheng TC, Wong AKL (1974) Chemical, Histochemical, and histopathological studies on corals, Porites Spp., parasitized by trematode metacercariae. Journal of Invertebrate Pathology 23: 303–317.TC ChengAKL Wong1974Chemical, Histochemical, and histopathological studies on corals, Porites Spp., parasitized by trematode metacercariae.Journal of Invertebrate Pathology23303317
  27. 27. Daly M, Fautin DG, Cappola VA (2003) Systematics of the Hexacorallia (Cnidaria: Anthozoa). Zoological Journal of the Linnean Society 139: 419–437.M. DalyDG FautinVA Cappola2003Systematics of the Hexacorallia (Cnidaria: Anthozoa).Zoological Journal of the Linnean Society139419437
  28. 28. McFadden CS, France SC, Sanchez JA, Alderslade P (2006) A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Molecular Phylogenetics and Evolution 41: 513–527.CS McFaddenSC FranceJA SanchezP. Alderslade2006A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences.Molecular Phylogenetics and Evolution41513527
  29. 29. Mcfadden CS, Brisson V, France SC (2010) Molecular Phylogenetic Insights into Octocoral Evolution. Integrative and Comparative Biology 50: E113–E113.CS McfaddenV. BrissonSC France2010Molecular Phylogenetic Insights into Octocoral Evolution.Integrative and Comparative Biology50E113E113
  30. 30. Adamczyk P, Meier S, Gross T, Hobmayer B, Grzesiek S, et al. (2008) Minicoliagen-15, a novel minicollagen isolated from hydra, forms tubule structures in nematocysts. Journal of Molecular Biology 376: 1008–1020.P. AdamczykS. MeierT. GrossB. HobmayerS. Grzesiek2008Minicoliagen-15, a novel minicollagen isolated from hydra, forms tubule structures in nematocysts.Journal of Molecular Biology37610081020