Different nematocytes have different synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa)

Sea anemones feed by discharging nematocysts into their prey, but the pathway for control of nematocyst discharge is unknown. The purpose of this study was to investigate the ultrastructural evidence of neuro-nematocyte synapses and to determine the types of synaptic vesicles present at different kinds of nematocyst-containing cells. The tip and middle of tentacles from small specimens of Aiptasia pallida were prepared for electron microscopy and serial micrographs were examined. We found clear vesicles in synapses on mastigophore-containing nematocytes and dense-cored vesicles in synapses on basitrich-containing nematocytes and on one cnidoblast with a developing nematocyst. In addition, we found reciprocal neuro-neuronal and sequential neuro-neuro-nematocyte synapses in which dense-cored vesicles were present. It was concluded that : (1) neuro-nematocyte synapses are present in sea anemones, (2) different kinds of synaptic vesicles are present at cells containing different types of nematocysts, (3) synapses are present on cnidoblasts before the developing nematocyst can be identified and these synapses may have a trophic influence on nematocyst differentiation, and (4) both reciprocal and sequential synapses are present at the nematocyte, suggesting a complex pathway for neural control of nematocyst discharge. J. Morphol. 238:53–62, 1998. © 1998 Wiley-Liss, Inc.     

Adaptable defense: a nudibranch mucus inhibits nematocyst discharge and changes with prey type

Nudibranchs that feed on cnidarians must defend themselves from the prey's nematocysts or risk their own injury or death. While a nudibranch's mucus has been thought to protect the animal from nematocyst discharge, an inhibition of discharge by nudibranch mucus has never been shown. The current study investigated whether mucus from the aeolid nudibranch Aeolidia papillosa would inhibit nematocyst discharge from four species of sea anemone prey. Sea anemone tentacles were contacted with mucus-coated gelatin probes, and nematocyst discharge was quantified and compared with control probes of gelatin only. Mucus from A. papillosa inhibited the discharge of nematocysts from sea anemone tentacles. This inhibition was specifically limited to the anemone species on which the nudibranch had been feeding. When the prey species was changed, the mucus changed within 2 weeks to inhibit the nematocyst discharge of the new prey species. The nudibranchs apparently produce the inhibitory mucus rather than simply becoming coated in anemone mucus during feeding. Because of the intimate association between most aeolid nudibranchs and their prey, an adaptable mucus protection could have a significant impact on the behavior, distribution, and life history of the nudibranchs.     

A sea anemone's environment affects discharge of its isolated nematocysts

Nematocysts were isolated from individuals of Calliactis tricolor maintained under different feeding schedules or in different salinities in an attempt to determine how these culture conditions influence the discharge of isolated nematocysts. In addition, the discharge frequencies of nematocysts isolated from two different populations of sea anemones found in two different environments were also compared. Undischarged acontial nematocysts were isolated by extrusion into 1 M sodium citrate and were then treated with 5 mM EGTA to initiate discharge. Nematocysts isolated from anemones maintained under three different feeding schedules showed significantly different responses to the test solution. Nematocysts isolated from anemones maintained in two different salinities did not differ significantly in discharge frequency. Nematocysts isolated from individuals from two separate populations of C. tricolor responded significantly differently to 5 mM EGTA and to deionized water, and these responses also depended upon the isolation solution used. Environmental conditions are known to have an impact on the physiological state of most organisms, but this is the first study providing evidence that the environment or feeding state of an anemone affects discharge of isolated nematocysts. Inherent differences in ionic and osmotic characteristics among nematocysts could explain some of the ambiguities when comparing past studies of isolated nematocyst discharge.     

The cnidarian nematocyst: a miniature extracellular matrix within a secretory vesicle

Nematocysts are the taxon-defining features of all cnidarians including jellyfish, sea anemones, and corals. They are highly sophisticated organelles used for the capture of prey and defense. The nematocyst capsule is produced within a giant post-Golgi vesicle, which is continuously fed by proteins from the secretory pathway. Mature nematocysts consist of a hollow capsule body in which a long tubule is coiled up that, upon discharge, is expelled in a harpoon-like fashion. This is accompanied by the release of a toxin cocktail stored in the capsule matrix. Nematocyst discharge, which is one of the fastest processes in biology, is driven by an extreme osmotic pressure of about 150 bar. The molecular analysis of the nematocyst has from the beginning indicated a collagenous nature of the capsule structure. In particular, a large family of unusual minicollagens has been demonstrated to form the highly resistant scaffold of the capsule. Recent findings on the molecular composition of Hydra nematocysts have confirmed the notion of a specialized extracellular matrix, which is assembled during an intracellular secretion process to form the most complex predatory apparatus at the cellular level.     

Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.     

Predation resistance and nematocyst scaling for Metridium senile and M. farcimen

Previous studies suggest that large body size reduces the risk of predation for acontiate sea anemones. For two species of Metridium, we found significant increases in the length of the acontial threads and in the mean lengths of the unfired acontial nematocyst capsules, with increasing body size. This supports the hypothesis that more damaging acontial defenses protect larger acontiate anemones from their predators. Metridium is planktivorous, and food size does not increase substantially with body size; so we expected smaller increases in nematocyst size for the feeding tentacles. In fact, scaling exponents were significantly smaller for the tentacle nematocysts than for acontial nematocysts of the same types in 3 out of 4 cases. This suggests that nematocyst scaling responds predictably to selection pressure. When specimens of the same size were compared, the non-clonal, subtidal species, M. farcimen, had significantly larger acontial nematocysts than did its clonal congener, M. senile, which lives at the upper tidal limits for major subtidal predators in the northeastern Pacific. Therefore, larger acontial nematocysts may be particularly advantageous where predation levels are high. These data demonstrate that closely related anemone species can be distinguished on the basis of ecologically and functionally relevant differences in nematocyst scaling.     

Receptors for N-acetylated sugars may stimulate adenylate cyclase to sensitize and tune mechanoreceptors involved in triggering nematocyst discharge.

In fishing tentacles of sea anemones, cnidocyte/supporting cell complexes (CSCCs) trigger the discharge of nematocysts following stimulation by swimming prey of specific mechanoreceptors and chemoreceptors located on the supporting cells. Two types of mechanoreceptors have been identified: a contact-sensitive mechanoreceptor (CSM), and a vibration-sensitive mechanoreceptor (VSM). The CSMs become predisposed to initiate nematocyst discharge into static (i.e., nonvibrating) test probes in the presence of submicromolar free and conjugated N-acetylated sugars, a process referred to as sensitization. In seawater, the VSMs cause maximal discharge in response to test probes vibrating at 30, 50-55, and 75 Hz, whereas in the presence of submicromolar N-acetylated sugars the VSMs cause maximal discharge into test probes vibrating at 5, 15, 30, and 40 Hz, a process referred to as tuning. Tuning of the VSMs is accompanied by elongation of the stereocilium bundles comprising the VSMs. We report that dibutyryl cyclic-AMP sensitizes CSMs and tunes VSMs to the lower frequencies of 5, 15, 30, and 40 Hz, while cyclic-AMP has no such effects. Endogenous adenylate cyclase activity at the apical plasma membrane of the supporting cells is detectable by cytochemical methods in the presence of N-acetylated sugars but not in seawater alone. By activating adenylate cyclase with L858051, an analogue of forskolin, or by activating the stimulatory form of G proteins (Gs) with cholera toxin, CSCCs are induced to sensitize CSMs and to tune VSMs to the lower frequencies of 5, 15, 30, and 40 Hz. Caged GTP-gamma S also sensitizes CSMs but tunes VSMs to 5, 15, 30, 40, 55, 65, and 75 Hz, suggesting that VSM tuning may be regulated both by Gs and inhibitory G-proteins. Together, these results implicate cAMP as the second messenger for activated supporting cell chemoreceptors involved in sensitizing the CSMs and tuning the VSMs to lower frequencies.     

Active Nematocyst Isolation Via Nudibranchs

Cnidarian venoms are potentially valuable tools for biomedical research and drug development. They are contained within nematocysts, the stinging organelles of cnidarians. Several methods exist for the isolation of nematocysts from cnidarian tissues; most are tedious and target nematocysts from specific tissues. We have discovered that the isolated active nematocyst complement (cnidome) of several sea anemone (Cnidaria: Anthozoa) species is readily accessible. These nematocysts are isolated, concentrated, and released to the aqueous environment as a by-product of aeolid nudibranch Spurilla neapolitana cultures. S. neapolitana feed on venomous sea anemones laden with stinging nematocysts. The ingested stinging organelles of several sea anemone species are effectively excreted in the nudibranch feces. We succeeded in purifying the active organelles and inducing their discharge. Thus, our current study presents the attractive possibility of using nudibranchs to produce nematocysts for the investigation of novel marine compounds.     

Rhythmic sensitization of nematocyst discharge in response to vibrational stimuli

Sea anemones capture prey by discharging nematocysts and other cnidae. Discharge of microbasic p-mastigophore (mpm) nematocysts is regulated in part by hair bundle mechanoreceptors on tentacles arising from multicellular complexes consisting of supporting cells and a sensory neuron. Anemone hair bundles detect movements of prey and then sensitize cnidocytes (cnida-containing cells) to discharge mpm nematocysts in response to contact between the prey and tentacle. Data from a simple bioassay based on counting nematocysts discharged into test probes, indicate that approximately twice as many nematocysts discharge into test probes touched to tentacles after sensitization than before sensitization. We here report that sub-second bursts of vibrational stimuli at key frequencies (51, 55, 65, or 74 Hz; Watson GM, Mire P, Hudson RR. 1998. J Exp Zool 281:582-593) sensitize discharge for at least 90 sec. Very few complete cycles of vibration are sufficient to sensitize discharge. However, as the number of cycles of vibration is increased, discharge is sensitized in rhythmic patterns. Computer analysis of the data by fast Fourier transforms indicates discharge to vibrations at 65 Hz is sensitized every 6.75 cycles. At 51 Hz discharge is sensitized every 2.00 cycles. At 74 Hz, discharge is sensitized in a polyrhythm occurring every 4.26, 3.76, 2.46, and 2. 10 cycles, respectively. At 55 Hz, discharge is sensitized in a polyrhythm occurring every 6.09, 3.20, 2.91, and 2.0 cycles, respectively. Apparently, cells in the neuronal pathway interconnecting anemone hair bundles with cnidocytes count cycles of vibration and then sensitize discharge or not according to the tally. J. Exp. Zool. 286:262-269, 2000.     

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa,Cnidaria)

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.     

Characterization of nematocyst proteins from the sea anemones Aiptasia pallida and Pachycerianthus torreyi (Cnidaria: Anthozoa)

Nematocysts from the anemones Aiptasia pallida and Pachycerianthus torreyi were investigated. SDS-polyacrylamide electrophoresis of solubilized Aiptasia nematocysts revealed one major protein band (mol. wt 31,800) and several minor components. Coelectrophoresed whole venom contained numerous protein components, of which a major one appeared to be identical to the major nematocyst protein. Nematocyst capsules and everted threads from both species contained levels of glycine and proline-hydroxyproline characteristic of vertebrate collagens. Cysteine was present in significant amounts. Aiptasia whole venom contained high levels of glutamic acid and/or glutamine (71%) with no detectable cysteine or proline-hydroxyproline. The 31,800-dalton venom protein possessed only glycine (80%) and glutamyl and/or glutaminyl (20%) residues.     

Inorganic Nutrient Concentrations and Chlorophyll in the Euphotic Zone of Lake Tanganyika

The taxonomic value of nematocyst size in sea anemones is still being assessed. We evaluate size distribution of nematocysts of one type in a single individual anemone. Length of unfired nematocysts was measured along the column, tentacles, and actinopharynx of a preserved specimen of Actinodendron arboreum (Quoy & Gaimard, 1833). Mean, range, minimum, and maximum length of nematocysts vary along the column, those in the middle region being least variable. The length of nematocysts in mature (split) acrospheres is less variable than in immature (unsplit) acrospheres. There is significant variability between nematocysts in tentacles of the primary and quaternary cycles, and along a tentacle, the middle being least variable. Size distribution of actinopharynx nematocysts is complex. The results of this study suggest that assembling data on nematocysts from multiple individuals for taxonomic purposes should be used with an awareness that sampling site can be an important variable. Ideally, the position of tissue sampled should be documented, an attempt should be made to be consistent in sampling from the same position in individuals being compared, and the variability of nematocyst length at each sampled site should be assessed. Inferences can also be made on ontogeny from these data; we conclude that an actinodendrid tentacle grows from the base and at the tips of its branches.     

Inotropic response elicited by nematocyst contents of Hydra oligactis (Coelenterata: Hydrozoa)

1. Intact, isolated nematocysts from the non-toxic, freshwater coelenterate Hydra oligactis contain soluble material(s) capable of producing a sustained increase in the rate of developed force in the vertebrate myocardium. 2. The positive inotropic effects of this material(s) appear grossly comparable to those described for Anthopleurin-A (AP-A) and Toxin II (ATX-II) from sea anemones. 3. The effects of the nematocyst material are distinct from those of known vasoactive peptides reported to occur in Hydra.     

The Effects of Satiation on Strike Mode and Prey Capture Kinematics in the Largemouth bass, Micropterus salmoides

The feeding mechanism and kinematics of prey capture have been studied in many fishes. However, the effects of satiation on the strike mode and prey capture kinematics have never been directly measured. We analyze 12 kinematic variables to determine the effects of satiation on prey capture in five largemouth bass, Micropterus salmoides, by using high speed videography. We also present the first experimental test for modulatory capabilities in response to satiation, by using the ram-suction index. Significant changes in the kinematic variables of maximum lower jaw depression, maximum gape distance, maximum hyoid depression, time to maximum hyoid depression, and time from maximum hyoid depression to recovery were seen with the effects of satiation. Change in the kinematic variables imply a decrease in jaw opening velocity and the magnitude of suction velocity created during repetitive strikes by M. salmoides with increasing satiation. The bass primarily uses a ram strike mode, with some suction bites occasionally. Ram-suction index analyses suggests that M. salmoides does not modulate strike mode in response to satiation. However, the bass modulate prey capture kinematics without altering strike mode with the effects of satiation. Prey capture success decreases in each bass, as the probability of a successful prey capture event becomes lower, with increasing satiation. These findings demonstrate that satiation can have major effects on prey capture kinematics and future studies of feeding kinematics should account for satiation in their analyses.     

Evolution of complex structures: minicollagens shape the cnidarian nematocyst

The generation of biological complexity by the acquisition of novel modular units is an emerging concept in evolutionary dynamics. Here, we review the coordinate evolution of cnidarian nematocysts, secretory organelles used for capture of prey, and of minicollagens, proteins constituting the nematocyst capsule. Within the Cnidaria there is an increase in nematocyst complexity from Anthozoa to Medusozoa and a parallel increase in the number and complexity of minicollagen proteins. This complexity is primarily manifest in a diversification of N- and C-terminal cysteine-rich domains (CRDs) involved in minicollagen polymerization. We hypothesize that novel CRD motifs alter minicollagen networks, leading to novel capsule structures and nematocyst types.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

The influence of feeding on behavior and nematocyst discharge of the sea anemone Calliactis tricolor

A decrease in basitrichous isorhiza and spirocyst nematocysts is observed in a fully fed sea anemone, Calliactis tricolor, as compared to unfed animals. Discharge of tentacle nematocysts of an intact living animal is inhibited by anesthetics and a decrease in temperature. The chemical (food origin) threshold for discharge of basitrichs is higher than for spirocysts and the implications of these results are discussed. The most probable cause for the observed decrease in nematocyst discharge is a combination of food present in the gastrovascular cavity and a physical stretching of the cavity.     

Response in nematocyst uptake by the nudibranch Flabellina verrucosa to the presence of various predators in the Southern Gulf of Maine

Aeolid nudibranchs maintain nematocysts sequestered from their cnidarian prey for protection against predators. Selection for nematocyst incorporation is a function of diet and prey choice, but ratios vary among nudibranchs feeding on a given diet, indicating that other factors may be involved. It is proposed that the presence of predators influences nematocyst incorporation. Nematocyst uptake in the nudibranch Flabellina verrucosa collected from the southern Gulf of Maine was examined in response to various potential predators, including Crossaster papposus, Tautogolabrus adspersus, and Carcinus maenas. Nudibranchs in individual flow-through containers feeding on a diet of the hydroids Tubularia spp. and Obelia geniculata were subjected to tanks containing a predator, then their nematocyst distribution was examined. Although most of the changes over the experimental period were attributable to diet, F. verrucosa responded to both T. adspersus and C. papposus by significantly increasing microbasic mastigophore incorporation. No differential uptake was seen with C. maenas. Response was evident in the nudibranchs both for predators present in the collection area and for those with which they had no previous exposure, indicating that F. verrucosa modulates nematocyst incorporation in response to the presence of predators as well as to diet. A coevolution of nudibranchs and potential predators may govern changes in nematocyst uptake.     

The utilization of cnidarian nematocysts by aeolid nudibranchs: Nematocyst maintenance and release in spurilla

Some nudibranchs that feed on cnidarians are known to store nematocysts within cnidophage cells and use them for their own defense. Most of the nematocysts are in direct contact with the cytoplasm of the cnidophage. Nematocysts are not subjected to lysosomal enzymes because any phagocytic membrane that surrounded the nematocyst after engulfment does not persist. Cnidophage organelles are restricted to regions surrounding the nematocysts and may aid in the maintenance and development of the nematocysts. The release of cnidophages is initiated by a contraction of a dense muscle complex surrounding the cnidosac. Nematocysts do not discharge if the cnidophage membrane does not rupture upon release. A comparison of nematocyst maintenance in Spurilla neapolitana and nematocyst retention in other organisms is presented.