Immunochemical and electrophysiological analyses of magnetically responsive neurons in the mollusc Tritonia diomedea

Tritonia diomedea uses the Earth’s magnetic field as an orientation cue, but little is known about the neural mechanisms that underlie magnetic orientation behavior in this or other animals. Six large, individually identifiable neurons in the brain of Tritonia (left and right Pd5, Pd6, Pd7) are known to respond with altered electrical activity to changes in earth-strength magnetic fields. In this study we used immunochemical, electrophysiological, and neuroanatomical techniques to investigate the function of the Pd5 neurons, the largest magnetically responsive cells. Immunocytochemical studies localized TPeps, neuropeptides isolated from Pd5, to dense-cored vesicles within the Pd5 somata and within neurites adjacent to ciliated foot epithelial cells. Anatomical analyses revealed that neurites from Pd5 are located within nerves innervating the ipsilateral foot and body wall. These results imply that Pd5 project to the foot and regulate ciliary beating through paracrine release. Electrophysiological recordings indicated that, although both LPd5 and RPd5 responded to the same magnetic stimuli, the pattern of spiking in the two cells differed. Given that TPeps increase ciliary beating and Tritonia locomotes using pedal cilia, our results are consistent with the hypothesis that Pd5 neurons control or modulate the ciliary activity involved in crawling during orientation behavior.     

Pedal neuron 3 serves a significant role in effecting turning during crawling by the marine slug <Emphasis Type="Italic">Tritonia diomedea</Emphasis> (Bergh)

The marine nudibranch Tritonia diomedea crawls using its ciliated foot surface as the sole means of propulsion. Turning while crawling involves raising a small portion of the lateral foot margin on the side of the turn. The cilia in the lifted area no longer contribute to propulsion, and this asymmetry in thrust turns the animal towards the lifted side. Neurons located in the pedal ganglia of the brain contribute to these foot margin contractions. T. diomedea has a natural tendency to turn upstream (rheotaxis), and pedal flexion neuron Pedal 3 elicits foot margin lift and receives modulatory input from flow receptors. To assess the contribution of this single cell in turning behavior, two fine wires were glued to the surface of the brain over left and right Pedal 3. We determined that Pedal 3 activity is correlated with subsequent ipsilateral turns, preceding the lift of the foot margin and the change in orientation by a consistent interval. Both Pedal 3 cells show synchronous bursts of activity, and the firing frequency of the ipsilateral Pedal 3 increased before turns were observed to that side. Stimulation of the electrode over Pedal 3 proved sufficient to elicit an ipsilateral turn in Tritonia.     

Neuropeptide TPep action on salivary duct ciliary beating rate in the nudibranch molluscTritonia diomedea

Recently, in the marine molluscTritonia, a family of three peptides (TPep-NLS,-PLS,-PAR) from identified pedal ganglion neurons has been characterized and shown to regulate ciliary beat frequency in epithelia and isolated cells of the molluscan foot. In this study, using an antiserum raised against TPep-NLS, immunofluorescent labelling was observed in specific nerve cell bodies and axons in the buccal ganglia ofTritonia, as well as in axons leading to and innervating the salivary ducts, salivary glands, oesophagus and foregut. This pattern of innervation suggests that buccal ganglion neurons containing TPep control the beating rate of ciliated cells in feeding organs. Accordingly, TPeps were introduced to isolated ciliated salivary ducts. It was found that TPeps and serotonin increased the ciliary beat frequency of cells of the salivary duct similarly; other peptides (such as APep fromAplysia) had no such effect. Threshold sensitivity both for TPeps and serotonin was approximately 10⁻⁸ M, with maximal response occurring above 10⁻⁵ M. Taken together, these structural and physiological results suggest that TPep-like peptides are present in the salivary and other feeding organs ofTritonia and are involved in the regulation of salivary transport.     

Water-flow sensitive pedal neurons in Tritonia: role in rheotaxis

Summary We have identified 13 pairs of neurons in the pedal ganglia of the marine nudibranch slug Tritonia diomedea that responded tonically and/or phasically to water-flow directed at the rhinophore sheaths and oral veil tips. Most of the neurons responded equally to inputs from either side of the body, but 6 pairs responded with greater intensity to ipsilateral water-flow stimuli. When stimulated intracellularly in a semi-intact, whole-animal preparation, 4 of these 6 pairs of neurons caused ipsilateral movements that may turn the animal towards that side. These observations suggest a role for these current-sensitive neurons in the previously described orientation to water-currents in Tritonia diomedea.     

The Mitochondrial Genomes of the Nudibranch Mollusks,Melibe leonina and Tritonia diomedea,and Their Impact on Gastropod Phylogeny

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.     

Shoreward orientation involving geomagnetic cues in the nudibranch mollusc Tritonia diomedea

The marine nudibranch mollusc Tritonia diotnedea orients to the geomagnetic field in the laboratory, and has identifiable brain cells (Pd5, 6) which respond electrically when the ambient magnetic field is rotated artificially. Field studies reported here seek to determine if and why Tritonia diomedea uses geomagnetic cues to orient in the field. Animals were collected in their natural habitat using SCUBA, and placed on lines with magnetic headings parallel to the shore, at different locations with respect to their site of origin. Observations made at two or more tidal cycles later indicate that most animals move from the line in a direction corresponding to the original shoreward direction, regardless of the actual shoreward direction at the site of release, suggesting guidance by geomagnetic cues. Tritonia diomedea are close to neutrally buoyant, and subject to transport over great distances when dislodged by tidal currents or during escape swimming behavior. Since the natural distribution of food and mates is along the shoreline, shoreward orientation using geomagnetic cues, particularly when other cues are weak or ambiguous, may have adaptive value.     

About possible functional interaction between serotonin and neuropeptides in control processes of embryogenesis (Experiments on embryos ofTritonia diomedea)

Ritanserin and inmecarb hydrochloride, antagonists of serotonin, act cytostatically and teratogenically on early embryos ofTritonia diomedea, a nudibranch mollusk. On the basis of a pharmacological analysis and the type of developmental abnormalities observed, this action appears to be due to disturbances in the functional activity of endogenous serotonin and is associated with damage to the cytoskeleton. The effects of ritanserin and inmecarb are prevented or attenuated by lipophilic serotonin analogs (serotoninamides of polyenoic fatty acids), as well as by polypeptides isolated from neurons Pd5 and Pd6 of the pedal ganglia of the adultTritonia. In late embryos (stage of veligers), serotonin and to a lesser extent its lipophilic analogs strongly increase embryonic motility. This effect of serotonin is potentiated by some neuropeptides and inhibited by others. These results provide evidence for functional interaction between serotonin and neuropeptides in the control processes of embryogenesis.     

Development in Berthella californica (Gastropoda: Opisthobranchia) with comparative observations on phylogenetically relevant larval characters among nudipleuran opisthobranchs

Abstract. Morphological criteria defining the Nudipleura and sister group relationships among the three nudipleuran subgroups (pleurobranchoideans, anthobranch nudibranchs, and cladobranch nudibranchs) have been controversial. Analysis of larval stages may help resolve these uncertainties by identifying additional phylogenetically informative characters, but existing information on pleurobranchoidean larvae is meager. We studied larval development and metamorphosis of the pleurobranchoidean Berthella californica using histological sections, scanning and transmission electron microscopy, and immunolabeling of neurons within the larval apical ganglion. We also provide comparative data on other nudipleuran larvae that may be useful for phylogenetic reconstruction. Berthella californica fills a previously unoccupied place within an evolutionary scenario that derives nudibranchs from pleurobranchoideans, two groups in which the larval mantle fold forms the post‐metamorphic notum (dorsal epidermis). In B. californica, reflection of the mantle fold epithelium to form the notum begins at metamorphosis, as also occurs in nudibranchs, whereas mantle reflection in other pleurobranchoideans begins well before metamorphosis. Dissolution of overgrown shell walls inside the protoconch and formation of the post‐metamorphic notum from the inner epithelium of the larval mantle fold may be synapomorphies of the Nudipleura. The larval shell in B. californica is additionally noteworthy because it acquires bilateral symmetry later in development, which is very unusual among larval opisthobranchs. We demonstrate an osphradium in the larvae of two pleurobranchoideans and one anthobranch nudibranch, although adults lack this trait. We also identified an autapomorphy of cladobranch nudibranchs in the form of five ampullary neurons within the larval apical ganglion, whereas other planktotrophic opisthobranch larvae have only four of these neurons. Although our data provide morphological criteria defining both the Nudipleura and the cladobranch nudibranchs, they are insufficient to resolve sister group relationships within the Nudipleura.     

Nudibranch predation and dietary preference for the polyps of <Emphasis Type="Italic">Aurelia</Emphasis><Emphasis Type="Italic">labiata</Emphasis> (Cnidaria: Scyphozoa)

There is concern that jellyfish blooms may be increasing worldwide. Some factors controlling population size, such as temperature and food, often have been studied; however, the importance of predators is poorly known. Aeolid nudibranchs feed on cnidarians, but their predation on the benthic polyps of scyphozoan rarely has been documented. To understand the potential of nudibranchs to consume polyps, we tested several predation preference hypotheses with the generalist feeding nudibranch, Hermissenda crassicornis, and polyps of the common moon jellyfish, Aurelia labiata. Of the six prey species tested during feeding experiments, A. labiata polyps and the tunicate Distaplia occidentalis were significantly preferred. Nudibranch size, diurnal cycle, and ingestive conditioning did not significantly influence prey choice. Nudibranchs showed significant positive chemotaxis toward living polyps, hydroids, and tunicates, but not to sea anemones. Nudibranch chemotaxis was significantly more positive to polar extract of A. labiata than of D. occidentalis. Consumption of polyps was correlated with nudibranch size, with mean consumption by large nudibranchs (>0.92 g) of about 31 polyps h⁻¹. Three other nudibranch species also ate A. labiata polyps. Our results emphasize the potential importance of predation for controlling jellyfish benthic polyp populations and consequent jellyfish blooms.     

Homologues of serotonergic central pattern generator neurons in related nudibranch molluscs with divergent behaviors

Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal–ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia. This material is based upon work supported by the National Science Foundation, under Grant No. 0445768, and a GSU Research Program Enhancement grant to PSK.     

The effect of density and mutual interference by a Predator: A laboratory study of predation by the Nudibranch Coryphella rufibranchialis on the hydroid Tubularia larynx

The nudibranch Coryphella rufibranchialis (JOHNSTON) feeds on a variety of hydroids, including Tubularia larynx Ellis & Solander. Experiments in which density of prey and predators were altered showed that more prey were eaten as prey density increased. However, more prey were consumed at low predator densities, presumably because of mutual interference among nudibranchs at the higher predator densities. The number of prey consumed per nudibranch was maximal with low predator densities and a ratio of 25–50 polyps per predator. Coryphella seems to show an opportunistic feeding strategy involving solitary predators rapidly depleting hydroid colonies and moving on to new colonies.     

Chemistry of Two Distinct Aeolid Spurilla Species: Ecological Implications

The lipophilic extracts of two marine aeolid nudibranch molluscs of the genus Spurilla collected in distinct geographical areas have been chemically analyzed. The Et₂O extracts of the nudibranchs were dominated by the presence of usual fatty acids and sterols and contained terpenoid compounds 1  –  3 as minor metabolites. Spurillin A ( 1 ) and spurillin B ( 3 ) were new molecules whereas cis‐γ‐monocyclofarnesol ( 2 ) was already reported in the literature as a synthesis product. Interestingly, bursatellin ( 4 ), previously isolated from anaspidean molluscs of the genus Bursatella, was found in the butanol extract of both Spurilla species. Compounds 1  –  4 were not detected in the extracts of the sea‐anemone preys collected together with the molluscs.     

Charismatic microfauna alter cyanobacterial production through a trophic cascade

The trophic ecology of cyanobacterial blooms is poorly understood on coral reefs. Blooms of toxic cyanobacteria, Lyngbya majuscula, can quickly form large mats. The herbivorous sea hare, Stylocheilus striatus, and the predatory nudibranch, Gymnodoris ceylonica, often associate with these blooms, forming a linear food chain: nudibranch—sea hare—cyanobacteria. Using laboratory studies, this study quantified (1) the functional response of nudibranchs, (2) the effect of sea hare size on predation rates, and (3) the strength of the indirect effect of sea hare predation on cyanobacteria (i.e., a trophic cascade). Nudibranchs consumed on average 2.4 sea hares d^?1, with the consumption of small sea hares 22 times greater than the consumption of large sea hares. Predation of sea hares reduced herbivory. Cyanobacterial biomass was 1.5 times greater when nudibranchs were present relative to when nudibranchs were absent. Although sea hare grazing can substantially reduce cyanobacterial biomass, predation of sea hares may mitigate grazing pressure, and therefore increase the abundance of cyanobacteria.     

Thermal stress response in a dinoflagellate-bearing nudibranch and the octocoral on which it feeds

In this study, we examined two non-scleractinian taxa, the rare nudibranch Phyllodesmium lizardensis and Bayerxenia sp., the octocoral on which the nudibranch lives and feeds, to investigate the effect of experimental heat stress on their symbioses with Symbiodinium. Bleaching has not been studied in nudibranchs. Bayerxenia sp. belongs to the alcyonacea family Xeniidae, members of which are known to be heat sensitive, but the genus has never been subject to heat stress experiments or bleaching observations. While qPCR did not reveal any changes to the symbiont community composition, the two host species responded differently to increased temperature. There were changes in the relative proportion of tissue types in Bayerxenia sp., but these were not attributable to the temperature treatment. Bayerxenia sp. exhibited no changes in cellular structure (apoptosis or cell necrosis), or symbiont functioning, cell size, density, or cladal community structure. On the other hand, the host, P. lizardensis, experienced tissue loss and symbiont densities decreased significantly with the majority of the remaining symbiont cells significantly degenerated after the heat stress. This decrease did not influence symbiont community composition, symbiont cell size, or photosynthetic efficiency. While the bleaching process in nudibranchs was demonstrated for the first time, the physiological and molecular pathways leading to this response still require attention.     

Development,metamorphosis, and natural history of the nudibranch Doridella obscura Verrill (Corambidae: Opisthobranchia)

The doridacean nudibranch Doridella obscura Verrill was raised through one complete generation in laboratory culture, and spawning behavior monitored for a year at monthly intervals in Barnegat Bay, New Jersey.The nudibranch deposited egg masses throughout the year in Barnegat Bay, and the larvae remained viable at temperatures ranging from 1.5 to 28 °C. At 25 °C the eggs hatch 4 days after oviposition, and the planktotrophic veliger larvae swim and feed for 9 days before they metamorphose. Settlement occurs specifically on the bryozoan Electro crustulenta (Pallas). The spirally coiled larval shell grows rapidly until the dorsal mantle fold is retracted from the aperture 5–6 days after hatching. Although starved larvae grow only slightly and do not metamorphose, they resume normal development on introduction of suitable food. Newly metamorphosed juveniles consume algae and debris on the surface of the bryozoan until they grow large enough to attack the living zooids of E. crustulenta.The life cycle of Doridella obscura is short (26 days at 25 °C), allowing the nudibranchs to take advantage of short-lived Electra crustulenta colonies in unstable habitats in bays and estuaries.     

A light and electron microscope study of some opisthobranch eyes

Summary The retina of nudibranch eyes contains two types of large cells; pigment cells which comprise about two-thirds of the total, with unpigmented sensory cells making up the remainder. Both pigment and receptor cells carry microvilli on their distal borders, but no traces of cilia were observed among them. The cornea of the eyes of aeolid and dendronotid nudibranchs is composed of a single layer of small cells, unlike the dorids where the cornea is made up of one of more large cells. The latter contain nuclei comparable in size with those of the pigment cells in the retina, but are themselves unpigmented.The elliptical eyes ofAplysia contain three types of retinal cell; the pigment cells and two kinds of receptor cells. The ciliary receptor cells bear equal numbers of cilia (9+2) and microvilli, while the microvillous receptor cells carry long tufts of microvilli with only an occasional cilium among them. The proximal cytoplasm of the receptor cells inAplysia and the nudibranchs contains large quantities of the small spherical vesicles (averaging 660 Å in diameter) which appear to be characteristic of gastropod eyes.     

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.     

FEEDING AND GROWTH OF THE AEOLID NUDIBRANCH CUTHONA NANA (ALDER AND HANCOCK, 1842)

Feeding and growth of the aeolid nudibranch Cuthona nana wasdocumented. Cuthona nana feeds specifically on the colonialhydroid Hydractinia echinata. Nudi-branchs were observed onhermit crab shells in Gos-port Harbor at the Isles of Shoals,Maine and on old bridge pilings near Gerrish Island, Maine. Grazing rates of nudibranchs (3–25 mm) were quantifiedover 24 hour periods in the laboratory. Larger nudibranchs consumedmore hydroid tissue feeding mostly on polyps, whereas smallerindividuals consumed less and fed on mat tissue. Grazing intensityin the field at Gosport Harbor was determined by estimatingthe area of grazed patches of hydroid colonies attached to hermitcrab shells. The average patch size was 5.75% of the total colonyarea. Growth rates for nudibranchs were determined in the laboratory.Growth rates were greatest prior to maturation and oviposition.Animals from Gerrish Island and Gosport Harbor decreased insize after oviposition due to senescence. Nudibranchs from GerrishIsland reached a maximum size of 12 mm, compared to 23 mm foranimals at Gosport Harbor. ^*Present address: Division of Science and Mathematics. Universityof Minnesota, Morris. Minnesota 56267, USA     

Properties of central motor neurons exciting locomotory cilia inTritonia diomedea

Summary A pair of large, identifiable neurons (Pd 21), one in each pedal ganglion, can excite previously inactive locomotory cilia on the sole of the foot ofTritonia diomedea (Audesirk, 1978; Fig. 3). These neurons exert their effect via axons which innervate the foot and are probably central motor neurons for pedal cilia. IntactTritonia are stimulated to crawl by the application of 1.5 M NaCl to the tail, and conversely usually stop crawling when the chemosensitive oral veil is touched with food (sea whip,Virgularia sp.). The Pd 21 neurons are excited by 1.5 M NaCl applied externally to the tail, and are inhibited by sea whip touch to the oral veil (Figs. 4 and 5). When aTritonia performs its escape swim, the cilia move strongly, and the Pd 21 neurons fire bursts of spikes in phase with dorsal flexions (Figs. 6 and 7). After a swim, aTritonia rapidly crawls along the substrate; during this time the spiking rate of the Pd 21s is greatly accelerated. Interneurons thought to drive swim bursts produce monosynaptic EPSPs in the Pd 21s (Fig. 8). The Pd 21s are coordinated in their spike activity by synaptic activity which is synchronous in the two neurons regardless of the site of external stimulation, and by electrical coupling between the two cells via axons in a pedal commissure (Figs. 9 and 10). The coupling coefficient for passively conducted potentials is quite high, about 0.15, despite an axon 8 to 12 mm long separating the two cells.Abbreviations BPSP biphasic postsynaptic potential - SW sea water