Chemical ecology of the Antarctic nudibranch Bathydoris hodgsoni Eliot, 1907: defensive role and origin of its natural products

A variety of Antarctic marine invertebrates contains secondary metabolites that may provide defense against potential predators. However, only in a few cases have tissues, extracts or isolated compounds of these invertebrates been tested against sympatric predators. The Antarctic nudibranch Bathydoris hodgsoni Eliot, 1907 contains hodgsonal, a compound only present in the external body (mantle tissues), which may protect the slugs from predators. To test this defensive hypothesis for hodgsonal, we carried out a series of experiments using the sympatric omnivorous seastar Odontaster validus Koehler, 1906 as a potential predator. Our experiments revealed that natural concentrations of hodgsonal elicit significant feeding deterrent responses in O. validus. Furthermore, hodgsonal is probably biosynthesized de novo by the nudibranch, since it was not detected in the viscera (as it should be in the case of a dietary compound), its concentration in the mantle (0.05-0.15% dry mass) is quite constant in individuals from different localities and depths, and its sequestration from a particular dietary source is unlikely because B. hodgsoni is an omnivorous feeder.     

Packaging and Delivery of Chemical Weapons: A Defensive Trojan Horse Stratagem in Chromodorid Nudibranchs

Background

Storage of secondary metabolites with a putative defensive role occurs in the so-called mantle dermal formations (MDFs) that are located in the more exposed parts of the body of most and very likely all members of an entire family of marine mollusks, the chromodorid nudibranchs (Gastropoda: Opisthobranchia). Given that these structures usually lack a duct system, the mechanism for exudation of their contents remains unclear, as does their adaptive significance. One possible explanation could be that they are adapted so as to be preferentially attacked by predators. The nudibranchs might offer packages containing highly repugnant chemicals along with parts of their bodies to the predators, as a defensive variant of the strategic theme of the Trojan horse.

Methodology and Principal Findings

We detected, by quantitative ¹H-NMR, extremely high local concentrations of secondary metabolites in the MDFs of six species belonging to five chromodorid genera. The compounds were purified by chromatographic methods and subsequently evaluated for their feeding deterrent properties, obtaining dose-response curves. We found that only distasteful compounds are accumulated in the reservoirs at concentrations that far exceed the values corresponding to maximum deterrent activity in the feeding assays. Other basic evidence, both field and experimental, has been acquired to elucidate the kind of damage that the predators can produce on both the nudibranchs'' mantles and the MDFs.

Significance

As a result of a long evolutionary process that has progressively led to the accumulation of defensive chemical weapons in localized anatomical structures, the extant chromodorid nudibranchs remain in place when molested, retracting respiratory and chemosensory organs, but offering readily accessible parts of their body to predators. When these parts are masticated or wounded by predators, breakage of the MDFs results in the release of distasteful compounds at extremely high concentration in a way that maximizes their repugnant impact.     

Geographical variation in defensive chemicals from pacific coast dorid nudibranchs and some related marine molluscs

1. Comparison of the chemical constituents of geographically diverse collections of six dorid nudibranchs has revealed that those species that are capable of de novo biosynthesis of defensive metabolites always produce the same array of chemicals.2. Conversely, those species that acquire chemicals from their sponge diet have a variable chemical arsenal.3. It is proposed that chemical consistency be used as an indicator of the origin of chemicals in the defensive arsenal of dorid nudibranchs.     

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.     

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.     

Predator-induced polymorphism in the bryozoan Membranipora membranacea (L.)

Three described species of the bryozoan genus Membranipora, M. membranacea (L.), M. serrilamella Osburn, and M. villosa Hincks, occurring together on brown macroalgae off the west coast of North America are eco-phenotypic variants. These forms should be called M. membranacea on the basis of priority.The presence of spines, an important diagnostic feature separating these Membranipora forms, is induced by two nudibranch predators, Corambe pacifica MacFarland & O'Donoghue and Doridella steinbergae Lance. Spines protect Membranipora zooids against predation by these nudibranchs. The population costs of spine production include lower growth and reproductive rates.     

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.     

Morphology and biological roles of spicule networks in Cadlina luteomarginata (Nudibranchia, Doridina)

Abstract. Many cryptobranch dorid nudibranchs contain innumerable calcareous spicules, yet the function of these elements is unknown. Two possible roles are defense against predators and structural support. In one dorid, Cadlina luteomarginata , whole-mount and thin-section staining revealed an intricate network of spicule tracts and connective tissue ramifying throughout the body, with muscle fibers associated with this spicule/connective tissue matrix and inserting into it. Spicules were present in high concentrations in all areas of the body, but highest in exterior mantle tissue. Relative investment increased isometrically with body size for most body regions, in contrast to the positively allometric investment seen in prosobranch shells. Bioassays with artificial food indicated that spicules alone did not deter generalist crabs and anemones, and only weakly increased the deterrence of secondary chemicals to anemones. Thus, while nudibranch spicules may serve as a defense against other predators, their primary role may be in body support.     

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri as predators of glass sponges

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri were found on glass sponges (Porifera, Hexactinellida) during remotely operated vehicle surveys of three reefs in the Strait of Georgia, British Columbia, Canada. Eight nudibranchs were sampled from 2009 to 2011. Identification of sponge spicules found in their gut and fecal contents confirmed the nudibranchs to be predators of the reef‐forming hexactinellids Aphrocallistes vastus and Heterochone calyx, as well as of the demosponge Desmacella austini, which encrusts skeletons of the glass sponges. Four of five nudibranchs dissected for gut content analysis had stomachs containing sponge spicules. Counts from high‐definition video footage taken during systematic surveys done in 2009 showed that nudibranchs were found in only two of the three glass sponge reefs. These data provide the first quantitative evidence of a molluscan predator on glass sponges found outside of Antarctica, and establish the first trophic link between glass sponges and their associated community of animals in a sponge reef ecosystem on the western Canadian continental shelf.     

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.     

Inflated protoconchs and internally dissolved, coiled protoconchs of nudibranch larvae: different developmental trajectories achieve the same morphological result

Abstract. Light and scanning electron microscopy were used to examine protoconch form in eight species of planktotrophic heterobranch larvae, including four nudibranch species with a coiled (type 1) protoconch, two nudibranch species with an inflated (type 2) protoconch, and two cephalaspid species with a coiled protoconch. The coiled protoconchs of the cephalaspids and nudibranchs have a similar form at hatching, and shell growth up to metamorphic competence is hyperstrophic. Shell added to coiled protoconchs during the larval stage overgrows all but the left wall of the initial protoconch that exists at hatching. The entire protoconch of cephalaspids, including overgrown areas, is retained through metamorphosis. However, during later larval development in nudibranchs with a coiled protoconch, overgrown shell is completely removed by dissolution. As a result, regardless of whether nudibranch larvae hatch with an inflated or coiled protoconch type, the protoconch is a large, hollow cup at metamorphic competence. The protoconch of nudibranchs is shed at metamorphosis and absence of a post-metamorphic shell is correlated with absence of visceral coiling in this gastropod group. Internal dissolution of the coiled protoconch in nudibranchs allows the left digestive gland to uncoil prior to metamorphic shell loss. Retention of overgrown protoconch whorls in cephalaspids allows the attachment plaque of the pedal muscle to migrate onto the parietal lip of the post-metamorphic shell. Release from this constraint in nudibranchs, in which the larval pedal muscles and the entire protoconch are lost at metamorphosis, may have permitted internal protoconch dissolution and precocious uncoiling of the visceral mass, as well as evolutionary emergence of the inflated larval shell type.     

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.     

MORPHOLOGICAL AND CHEMICAL CAMOUFLAGE OF THE MEDITERRANEAN NUDIBRANCH DISCODORIS INDECORA ON THE SPONGES IRCINIA VARIABILIS AND IRCINIA FASCICULATA

The nudibranch Discodoris indecora shows a perfect camouflageon its prey, the dictyoceratid sponge Ircinia variabilis. Shapeand colour of the nudibranch are remarkably similar to thatof the sponge which is widespread in the shallow waters of theMediterranean Sea. The metabolite pattern of I. variabilis isdominated by unusual molecules, the sesterter-penoids palinurinand variabilin, containing 25 carbons and displaying at theends a ß-substituted furan ring and a tetronic acid.However, a chemical polymorphism has been recently describedfor I. variabilis. Some specimens contain either palinurin orvariabilin whereas others possess a mixture of both sestertepenoids.During the study of Mediterranean sponges, a linear sesterterpenoidrelated to palinurin and variabilin was found in Ircinia fasciculata,that from a taxonomic point of view is closely relatedto Irciniavariabilis. Discodoris indecora shows a typical defence behaviourwhen it is disturbed, secreting a copious white slime that containslarge amounts of palinurin and variabilin. Most likely the animalis able to transfer the sponge metabolites from the digestiveglands to mantle glands. Electron micrographs of the nudibranchmantle showed the presence of unicellular glands characterizedby a cylindrical vacuole with a big nucleus on the basis. Theglands were concentrated in the most conspicuous tubercles ofthe dorsal mantle. Probably, the sponge metabolites are storedin these glands. Comparative chemical analysis of mucous secretions, dorsal tubercles,mantle, foot, digestive glands and hepatopancreas of D. indecoraconfirmed this hypothesis. Palinurin and fasciculation were,of course, present in the digestive glands but they were alsofound in the mucus and in the mantle sections enriched by theabove described unicellular glands. The defensive role of palinurinand variabilin, strongly suggested by their anatomical location,was supported by recording the induction of feeding-deterrencein the fresh-water fish Carassius auratus and in the marinefish Chromis chromis and Sparus auratus. (Received 15 March 1996; accepted 27 January 1997)     

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.     

Chemical defense of Mediterranean sponges Aplysina cavernicola and Aplysina aerophoba

The Mediterranean sponges Aplysina aerophoba and A. cavernicola accumulate brominated isoxazoline alkaloids including aplysinamisin-1 (1), aerophobin-2 (2), isofistularin-3 (3) or aerothionin (4) at concentrations up to 10% of their respective dry weights. In laboratory feeding experiments employing the polyphagous Mediterranean fish Blennius sphinx crude extracts of both Aplysina sponges were incorporated into artificial fish food at their physiological concentrations (based on volume) and offered to B. sphinx in choice feeding experiments against untreated control food. In addition to the Aplysina sponges, extracts from nine other frequently occurring Mediterranean sponges were likewise included into the experiments. Both Aplysina species elicited strong feeding deterrence compared to the other sponges tested. Bioassay-guided fractionation of A. cavernicola yielded the isoxazoline alkaloids aerothionin (4) and aplysinamisin-1 (1) as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) as major deterrent constituents when tested at their physiological concentrations as present in sponges. Aeroplysinin-1 (5) and dienone (6), however, which are formed in A. aerophoba and A. cavernicola from isoxazoline precursors through bioconversion reactions upon tissue injury showed no or only little deterrent activity. Fractionation of a crude extract of A. aerophoba yielded aerophobin-2 (2) and isofistularin-3 (3) as major deterrent constituents against B. sphinx. We propose that the isoxazoline alkaloids 1-4 of Mediterranean Aplysina sponges as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) (in the case of A. cavernicola) act as defensive metabolites against B. sphinx and possibly also against other predators while the antibiotically active bioconversion products aeroplysinin-1 (5) and dienone (6) may protect sponges from invasion of bacterial pathogens.     

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.     

GC-MS and NMR analysis of Phyllidiella pustulosa and one of its dietary sources,the sponge Phakellia carduus

Gas-chromatographic and mass-spectrometric and NMR analyses of the lipophilic extract of the nudibranch Phyllidiella pustulosa and the sponge Phakellia carduus clearly showed that the sponge formed a major part of the nudibranchs diet. The analyses also indicated that the nudibranch accumulates some of the sponge metabolites in preference to others. The main components of the extracts were identified as the new natural product 10-isothiocyano-4-cadinene (1), axisonitrile-3 (2), and a number of other sesquiterpenes similar to 1 and 2. Also positively identified, in the extract of the sponge were the two sterols (3beta,22E)-ergosta-5,8,22-trien-3-ol (4) and (3beta,22E)-stigmasta-5,7,22-trien-3-ol (5), and in the nudibranch extract caryophyllene (3). The new natural product 10-isothiocyano-4-cadinene (1) was shown to have moderate antiplasmodial activity (IC(50) 1.5 microgram/ml towards Plasmodium falciparum clones K1, and NF54).     

Does concentrating chemical defenses within specific regions of marine sponges result in enhanced protection from predators?

Chemical defenses are an effective mode of predator deterrence across benthic marine organisms, but their production may come with associated costs to the organism as limited resources are diverted away from primary processes like growth and reproduction. Organisms concentrating ecologically relevant levels of these defenses in tissues most at risk to predator attack may alleviate this cost while deterring predators. We addressed this hypothesis by investigating the deterrence of chemical extracts from the inner and outer regions of the sponges Aplysina fulva, Ircinia felix, and I. campana from a temperate hard-bottom reef in the South Atlantic Bight. Assays were conducted using natural fish assemblages and sea urchins. Although, A. fulva and I. felix have higher concentrations of defensive metabolites in the outer and inner regions, respectively, extracts from these regions did not display enhanced deterrency against fish or mobile invertebrate predators. Likewise, extracts from both regions of the sponge Ircinia campana, which has a uniform distribution of defensive chemicals throughout, did not differ in their ability to deter either group of predators. Since chemical defenses were effective deterrents at lower concentrations, secondary metabolite allocation patterns observed among these sponges are likely not driven by predation pressure from generalist fish and mobile invertebrate predators on these reefs. Alternatively, these patterns may be driven by other ecological stressors, another suite of predators, or may be more effective at deterring predators when combined with structural defenses.     

Discharge of nematocysts isolated from aeolid nudibranchs

Nematocysts were extracted from 3 nudibranch species and one sea anemone species, and the ability of several test fluids to promote discharge was examined. Except when isolated in sodium citrate, nudibranch nematocysts did not discharge in response to any test fluids. Nudibranch nematocysts isolated in sodium citrate discharged when tested with EGTA, distilled water, and calcium-free artificial seawater, but there were differences among the 3 nudibranch species. Nematocysts isolated from one nudibranch species and nematocysts isolated from that nudibranch's sea anemone prey differed in the percentage that discharged in response to EGTA and distilled water. These results suggest that nematocysts stored by nudibranchs are altered in some way, resulting in the different discharge responses.     

Conspicuous visual signals do not coevolve with increased body size in marine sea slugs

Many taxa use conspicuous colouration to attract mates, signal chemical defences (aposematism) or for thermoregulation. Conspicuousness is a key feature of aposematic signals, and experimental evidence suggests that predators avoid conspicuous prey more readily when they exhibit larger body size and/or pattern elements. Aposematic prey species may therefore evolve a larger body size due to predatory selection pressures, or alternatively, larger prey species may be more likely to evolve aposematic colouration. Therefore, a positive correlation between conspicuousness and body size should exist. Here, we investigated whether there was a phylogenetic correlation between the conspicuousness of animal patterns and body size using an intriguing, understudied model system to examine questions on the evolution of animal signals, namely nudibranchs (opisthobranch molluscs). We also used new ways to compare animal patterns quantitatively with their background habitat in terms of intensity variance and spatial frequency power spectra. In studies of aposematism, conspicuousness is usually quantified using the spectral contrast of animal colour patches against its background; however, other components of visual signals, such as pattern, luminance and spectral sensitivities of potential observers, are largely ignored. Contrary to our prediction, we found that the conspicuousness of body patterns in over 70 nudibranch species decreased as body size increased, indicating that crypsis was not limited to a smaller body size. Therefore, alternative selective pressures on body size and development of colour patterns, other than those inflicted by visual hunting predators, may act more strongly on the evolution of aposematism in nudibranch molluscs.