Defenses of nudibranchs and their relatives include secondary metabolites, considered in this section, and
CAMOUFLAGE (CRYPSIS),
FAST CRAWLING & SWIMMING,
MUCOUS COATINGS,
CERATAL AUTOTOMY,
NUTRITIONAL CONTENT,
SPICULES,
NEMATOCYSTS,
VACUOLATED SKIN WITH PROTECTIVE SPINDLES,
ACID SECRETIONS,
INK & OPALINE SECRETIONS,
ALARM PHEROMONES,
APOSEMATIC (WARNING) COLORATION & BATESIAN MIMICRY, and
NAVANAX: A SPECIAL CASE STUDY, considered in other sections. 

Over the last few decades a rich literature¹ on the chemistry of potentially defensive secondary metabolites² in nudibranchs has emerged, and many reports pertain to west-coast species, mainly owing to a strong research focus in labs in California and British Columbia.  Much of this information is just a record of “interesting chemistry”, but there are a number of publications with toxicity bioassays or environmental connotations worth considering here. 

"Skin chemistry" is a feature of dorids, mostly relating to diets of sponges with their inherent toxicity. No aeolids³ contain such secondary metabolites. Thus far, about 30 species of dorids are known to possess secondary metabolites, but few experiments have been done on possible defensive functions. More dorid species would likely be identified, were it not for the problem of obtaining enough specimens for extractions and analyses. Unfortunately, there are no reliable data on the number of dorids without skin chemistry. Clearly, such data would be of inestimable interest, especially if the chemistry of the diets were also known. The entries below are listed alphabetically by genus.

NOTE¹  for more information readers are directed to reviews by Proksch 1994 Toxicon 32: 639; Cimino & Ghiselin 1998 Chemoecology 8: 51; Cimino & Ghiselin 1999 Chemoecology 9: 187; Andersen et al. 2006 p. 277 In, Progress in molecular and subcellular biology Subseries: Marine molecular biotechnology (Cimino & Gavagnin, eds.): Molluscs Springer-Verlag, Berlin; and Cimino & Ghiselin 2009 Proc Calif Acad Sci 60: 175 (this last is a useful compendium of current knowledge of chemical defenses in world opisthobranch species.  It deals not just with details of defensive chemistry, but also with how the chemistry has influenced opisthobranch evolution)

NOTE² these are organic compounds of restricted distribution among certain taxa, with no obvious metabolic function and, for this reason, with an often presumed defensive role

NOTE³  Melibe leonina, an aeolid, has repugnatorial glands that release a noxious secretion, but skin chemistry as such is lacking

The dorid nudibranch Achanthodoris nanaimoensis primarily eats bryozoans, and has a characteristic fragrant odour.  Analysis of specimens collected in Barkley Sound, British Columbia reveals the presence of 3 related sesquiterpenoid aldehydes, at least one of which, nanaimoal, imparts the species’ characteristic odour.  The other 2 metabolites are acanthodoral  and isoacanthodoral.  The consistency of presence of these 3 terpenoids in skin extracts of A. nanaimoensis collected at many sites along the coast of British Columbia suggests that they are biosynthesised de novo, rather than simply sequestered from their foods.  Ayer et al. 1984 Tetrahedron Letters 25: 141; Ayer et al. 1984 J Org Chem 49: 2654; Graziani & Andersen 1996 J Am Chem Soc 118: 4701. Photograph courtesy N. Elder and Linda Schroeder, Pacific Northwest Shell Club, Seattle, Washington PNWSC.

Analysis of the sponge-eating dorid Aldisa sanguinea from Barkley Sound, British Columbia reveals the presence of 2 related steroidal ketones that may have anti-feedant properties.  Ayer & Andersen 1982 Tetrahedron Letters 23: 1039.

A known predator of juvenile sea hares Aplysia californica is the green sea anemone Anthopleura xanthogrammica.  Field observations in the Palos Verdes area of California show that young sea hares are commonly caught and ingested, but that the digestive glands are usually regurgitated before digestion. Apparently, most of what is visible in the photograph is digestive-gland material. The authors conjecture that on the first breach of the membrane surrounding the gland, toxins may be released into the gastrovascular cavity that initiate regurgitation.  The toxin, referred to as aplysin by the authors, is noted to be a powerful cholinergic agent that, when injected intraperitoneally into mice, frogs, and chicks, has neuromuscular effects leading in some cases to death. Its effect on A. xanthogrammica, however, appears to be minimal.  Winkler & Tilton 1962 Pac Sci 16: 286; Winkler 1961 Pac Sci 15: 211. Photograph courtesy Kevin Lee, Fullerton, California diverKevin.

NOTE  in the case of 10 sea hares removed from anemone digestive tracts, digestion amounts to about 70% of body mass, but in no cases are the digestive glands attacked

NOTE  obtained by water- and acetone-extraction of the digestive gland.  Apparently, a similar type of toxin is present in both A. californica and A. vaccaria

In a later study related to toxins in sea hares, the senior author of the foregoing Research Study analyses various body parts of Aplysia californica for presence of brominated and lipid compounds.  Tissues of a single specimen from La Jolla, California are first minced, extracted with acetone, and then chromatographically separated.  Results of this preliminary study reveal that most bromine is stored in the digestive gland and little in the other body parts examined. Bromine compounds are absent in the opaline-gland secretions. Winkler 1968/69 Veliger 11: 268.

NOTE  bromine was earlier implicated in the defenses of sea hares through work by Japanese scientists on Aplysia kurodai. It was these researchers who coined the term aplysin, but apparently not for the cholinergic agent described in Research Study 1 above.  A previous search for the source of the organic bromides in the red-algal food of A. californica, namely, Plocamium pacificum, was unsuccessful.  Darling & Cosgrove 1965/66 Veliger 8: 178.

NOTE  the data for each body part are presented as % bromine and % lipid of the total found in that body part. As not all body parts are included, the totals may fall short of 100%

Analyses of digestive-gland constituents of Aplysia californica at the Scripps Institution of Oceanography, La Jolla, California reveal the presence of several halogenated natural products derived from metabolites of the red alga Laurencia pacifica, one of the preferred foodstuffs of the sea hare.  Additionally, the researchers identify 2 new halogenated monoterpenes derived from another preferred food of A. californica, the red alga Plocamium coccineum var. pacificum.  The authors speculate that the digestive gland may be used to store algal metabolites used in a chemical defense system, but how the substances are mobilised is, at the time, unknown.  Stallard & Faulkner 1974 Comp Biochem Physiol 49B: 25.

NOTE  these are aplysin, debromoaplysin, laurinterol, pacifenol, johnstonol, and pacifidiene, all previously isolated and named from studies on other Aplysia species and from analyses of seaweed foods of these particular sea hares

A companion study at the Scripps Institution of Oceanography, La Jolla by these same researchers on digestive-gland constituents in Aplysia californica starts with the questions: 1) is the digestive gland used solely for storage of toxic chemicals to be employed in defense, and 2) does the digestive gland chemically transform these toxic substances?  The answer turns out to be a little of both, as chemicals are stored there and they also undergo transformation.  The authors report that 2 algal metabolites derived from the preferred diets of red algae Laurencia pacifica and Plocamium coccineum var. pacificum, namely, laurinterol and pacifenol, undergo transformations to aplysin and pacifidiene, respectively, in the digestive gland.  Stallard & Faulkner 1974 Comp Biochem Physiol 49B: 37.

A comparative study on the location of sequestered secondary metabolites in 3 anaspid opisthobranchs, including the sea hare Aplysia californica, shows that the compounds are almost exclusively stored in the digestive glands, and not in skin, ink, or eggs.  The authors remark on the counterintuitive nature of the results, in that skin and eggs would be ones first encountered by a potential predator, and ink would allow predators to taste and reject the prey without causing it mortal damage.  The metabolites are found in and sequestered from the Aplysia’s natural diet of red algae.  The principal metabolite identified in the ink of Aplysia is the same monoterpene as found in the digestive gland.  Overall, the authors conclude that the sequestered compounds in A. californica and in the other anaspids studied are not located primarily for defense.  This is an interesting idea and certainly warrents further research. Pennings & Paul 1993 Mar Biol 117: 535.

NOTE  2 of the species Stylocheilus longicauda and Dolabella auriularia are tropical, and will not be dealt with here.  The sea hare species A. californica is collected at Santa Catalina Island, California

NOTE  the primary metabolite found in the preferred algal food Plocamium spp. is a halogenated monoterpene which, at the time of the study, had not yet been specifically identified

In San Diego, California, Cadlina luteomarginata eats at least 10 species of sponges, with Myxilla incrustans and Axinella sp. being the most common.  Chemical analysis reveals several secondary metabolites in Cadlina, and these are only present in the dorsal mantle, the part exposed to potential predators.  Preliminary tests show some antifeedant activity of 5 of these metabolites against fishes.  Thompson et al. 1982 Tetrahedron 38: 1865. Photo courtesy Jeff Goddard, UC Santa Barbara, California.

NOTE  the characteristic fruity odour of C. luteomarginata has been identified in British Columbia specimens as luteone, a 23-carbon terpenoid.  Hellou et al. 1981 Tetrahedron Letters 22: 4173

Several terpenoids, including cadlinolide A, and glaciolide (a degraded diterpenoid), have been isolated from skin extracts of Cadlina luteomarginata feeding on the sponge Aplysilla glacialis, and are identified as being the same as, or derived from related compounds, in the sponge.  Tischler & Andersen 1989 Tetrahedron Letters 30: 5717; Tischler et al. 1991 J Org Chem 56: 42.

NOTE  another terpenoid, marginatafuran, has additionally been isolated from specimens of C. luteomarginata collected in Haida Gwaii, British Columbia, also possibly derived from sponge food.  Gustafson et al. 1985 Tetrahedron Letters 26: 2521

Two new sesquiterpenoids, acanthene A and acanthene B have been isolated from the marine sponge Acanthella collected at Graham Island, British Columbia.  Further, an analogue of these 2 substances, acanthene C, has been identified in Cadlina luteomarginata, which feeds on Acanthella.  Burgoyne et al. 1993 Tetrahedron 49: 4503.

Other studies on distributions of terpenoids in Cadlina luteomarginata from populations in British Columbia and California show that endogenous concentrations vary inversely with the availability of structurally similar compounds in their foods.  This suggests that Cadlina regulates its de novo biosynthesis of defensive compounds according to need.  Based on the assumption that sequestration of terpenoids and other (defensive?) compounds from the diet is more primitive than de novo biosynthesis of the same compounds, the authors place C. luteomarginata, which does both, in an intermediate position in the evolution of nudibranch chemical defenses.  Kubanek et al. 2000 J Chem Ecol 26: 377; for an overview of de novo synthesis of various terpenoids in C. luteomarginata see Kubanek et al. 1997 J Org Chem 62: 7239.

NOTE  of the 100 or so compounds isolated from nudibranchs to date, most are terpenoids. Andersen et al. 2006 p. 277, In Progress in molecular and subcellular biology Subseries: Marine molecular biotechnology (Cimino & Gavagnin, eds.): Molluscs Springer-Verlag, Berlin

Several novel terpenoids, including cadlinaldehyde, spongian, seco-spongian, 20-acetoxy-12- marginatone, and lutenolide have been identified in skin extracts of Cadlina luteomarginata, and a new drimane sesquiterpenoid 1alpha, 2alpha-diacetoxyalbicanyl acetate in its egg masses.  Several new sesquiterpenoids, as well as 4 other terpenoids previously isolated from C. luteomarginata skin extracts, have been identified for the first time in the sponges Pleraplysilla sp. and Aplysilla sp., both species eaten by the nudibranch. The authors are interested in collecting and analysing specimens of Cadlina from as many sites in British Columbia as possible, in part, to attempt to answer the question as to the origin of the terpenoids (see map). There is indirect evidence that the nudibranch may have the capability for de novo synthesis or even for modifying some of the sequestered metabolites. The authors provide a good overview of the research that they have done to date on the subject. Dumdei et al. 1997 Can J Chem 75: 773.

Test of function of supposed chemical defenses in an animal, whether vertebrate or invertebrate, marine or terrestrial, is difficult, mainly owing to lack of knowledge of natural predators and to lack of suitable controls.  If a predator is confronted with a nudibranch and doesn’t attack, or attacks and doesn’t eat, the reason may be unrelated to presence of purported chemical defenses.  There could be physical reasons for unpalatability such as spines or thick integument, or a failure to recognise the nudibranch is a potential prey, or satiation of the predator from an earlier meal.  The difficulty of interpreting results of such predator/prey experiments is exemplified in a study of Cadlina luteomarginalis at Bamfield Marine Sciences Centre, British Columbia.  As we have seen, Cadlina has a well-characterised “secondary chemistry” in the form of various terpenoids, thought to be defensive but never rigorously tested.  Predators used in the experiment include crabs Cancer spp. that cut and crush their prey, and kelp-greenling fishes Hexagrammos decagrammus, sea stars Pycnopodia helianthoides, and sea anemones Anthopleura elegantissima, that mouth and swallow their prey.  The predators are kept under laboratory conditions for 1wk and fed bits of squid at regular intervals to accustom them to accepting food, but are not given enough to satiate them.  Half the trials involve presenting squid bits first, followed by Cadlina individuals, with the order reversed in the other half of the trials.  Predators that do not eat the control bits of squid are discarded.  Out of approximately 50 trials, only 1 Cadlina is eaten.  Many of the test nudibranchs are wounded during the feeding trials, but recovery (except when they are partially digested) is good over a subsequent week’s recuperation time.  The author concludes that C. luteomarginata is effectively defended against a range of predators with different feeding modes.  Penney 2004 J Moll Stud 70: 399.

NOTE  48 predators (approximately 10 of each species) are each tested once with a piece of squid (known to be edible) and once with Cadlina (hypothesised to be inedible)

Whole-body extracts of specimens of Diaulula sandiegensis from British Columbia contain steroids, diaulusterol A and diaulusterol B.  This differs from 9 chlorinated acetylenes previously reported for another dorid species D. sandiegensis collected at Point Loma, California.  In both localities sponges comprise the main dietary component and the authors conclude that the difference in skin chemistry is a consequence of eating different sponge diets at the 2 sites.  At the time the work was published the actual sponges being eaten by Discodoris had not been identified.  Williams et al. 1986 Can J Chem 64: 1527.

NOTE  extraction procedure involves immediately immersing freshly collected specimens in methanol for several days.  Speed is essential because many nudibranchs lose their chemicals quickly, perhaps from handling which may mimic attack from a predator or other animall.  No mention is made in these Research Studies on Diaulula of cleaning out gut contents (including digestive glands), or in other similar studies on skin chemistry of nudibranchs, for that matter, and one wonders if there is any significant contribution from residual sponge food in the gut system to the overall results

NOTE  a later study by the same research group shows that the polyketide fragment of diaulusterol are actually synthesised de novo in Diaulula sandiegensis collected at various sites in British Columbia.  The authors note that the ability to make these potentially defensive chemicals, known as allomones, should increase the range of habitats available to a species by eliminating its dietary dependence on prey rich in the required secondary metabolite(s).  Kubanek & Andersen 1999 J Nat Prod 62: 777

in an early study on toxicity of dorid nudibranchs in Monterey Bay, California, aqueous extracts of digestive glands of several species, including Doris montereyensis, Cadlina flavomaculata, Doriopsilla albopuntata, Doris odhneri, and Peltodoris nobilis are shown to lethal when injected into shore crabs (Pachygrapsus or Hemigrapsus) and mice.  The authors designate the unidentified pharmacologically active substances collectively as “dorid toxin”.  Fuhrman et al. 1979 Biol Bull 156: 289.

Doris montereyensis (?) 1X

Later studies show that extracts of the sponge-eating A. montereyensis and D. odhneri contain different forms of acid glycerides.  Extracts of D. montereyensis incorporated into food pellets significantly reduce feeding by tidepool sculpins Oligocottus maculosus, suggesting a defensive function.  Andersen & Fuk Wah Sum 1980 Tetrahedron Letters 21: 797; Gustafson et al 1984 Tetrahedron Letters 25: 11; Gustafson & Andersen 1985 Tetrahedron 41: 1101.

NOTE  these contain drimenoic acid glyceride and glyceryl ether

Doris odhneri 0.5X

Nudibranchs with potentially defensive metabolites either get them from their foods or synthesise them.  Determination of the latter is often a complex business, requiring that a labelled precursor substance be injected in order to obtain an identifiable secondary metabolite.  A simpler method, as proposed by the authors of a study using nudibranchs collected at several west-coast sites, is to see if a nudibranch in question exhibits considerable variation in their chemical constituents.  If so, they undoubtedly obtain these chemicals from dietary sources.  Conversely, if the chemicals are the same wherever they are collected, the species is most likely capable of de novo synthesis.  Let’s see how this might work with 2 common sponge-eating species, selected from several examples cited by the authors.  Doris montereyensis at La Jolla, California contain as their major component agathanori acid glycerides, identical to what is contained in specimens from Barkley Sound, British Columbia and elsewhere.  Individuals of this species always contain the same specific array of metabolites no matter where or when they are collected, suggesting identical biosynthetic pathways. 

In comparison, Cadlina luteomarginata has the following array of metabolites geographically:

Barkley Sound, British Columbia: 5 types of sesquiterpenes
Sandford Island, British Columbia: 3 types of diterpenes
Howe Sound, British Columbia: one norsesterpene and one sesquiterpene (but different from the ones in Barkley Sound)
Point Loma, California: 1 sesquiterpene isonitrile (different again from the others)

As to whether one strategy has an advantage over the other, the authors simply note that any advantage of not being dependent on specific food sources for defensive metabolites is balanced against the need to maintain a complex biosynthetic mechanism.  Faulkner et al. 1990 Comp Biochem Physiol 97C: 233; see also Hellou et al. 1982 Tetrahedron 38: 1875. Photo courtesy Jeff Goddard, Santa Barbara.

Cadlina luteomarginata 1.3X

Tests of possible anti-feedant activity of extracts of Doris montereyensis have also been done at Bamfield Marine Sciences Centre, British Columbia.  Crabs Cancer productus presented with alginate-gel pellets containing different amounts of powdered squid¹, with and without the addition of A. montereyensis extracts at physiological levels², eat significantly less pellets containing extract³ than pellets containing no extract (by about 50%), supporting an anti-feedant role for acid glyceride and glyceryl ether present in the nudibranch's tissues (see accompanying histogram). 

In considering the results of the chemical-deterrent part of the study, the author notes the potential pitfalls in using 1-factor studies of chemical³ defense.  For example, if a chemical is added to a high-quality food (e.g., one that is more nutritious or even more tasty) it may have less apparent deterrence than if it were added to a low-quality food.  Additionally and obviously, chemicals do not act in isolation, and this must be factored into deterrence-assessment experiments.  The literature on the subject of marine chemical defense is voluminous, especially relating to seaweeds, sponges, gorgonians and, especially, nudibranchs. Penney 2002 Oecologia 132: 411.

NOTE¹  two artificial foods are created, one with 12% dry mass organic content to mimic the organic content of spiculated dorid nudibranchs (intended as a "low" quality food), the other with 22% dry mass organic content (a "high" quality food).  One-half the pellets is treated with methanol extract of dried A. montereyensis before being set in alginate gel (WITH EXTRACT); the other half is treated only with methanol solvent as a WITHOUT EXTRACT control.  Low nutritional content of an opisthobranch as a "defense" is considered elsewhere in the ODYSSEY: DEFENSES AGAINST PREDATORS: NUTRITIONAL CONTENT

NOTE²  at concentrations matching those found naturally in the bodies of the nudibranch

NOTE³ the author does not consider possible diffusion or degradative losses of the extracted chemicals during the trials (lasting up to a day), perhaps because these are likely to be small owing to low water-solubility of the chemicals. Even if losses do occur, strong significance is shown in any case in the statistical tests

Melibe

The hooded nudibranch Melibe leonina responds to touch by releasing secretions from ectodermal glands distributed widely over the body.  The secretion, described as smelling like oil of bergamot, may be defensive, although the author does not test this.  Kjerschow Agersborg 1921 The Amer Nat 55: 222. Photograph courtesy Charles Seaborn, Malibu, California.

NOTE  a small pear-shaped citrus fruit Citrus aurantium from Italy whose peel yields an aromatic oil used in perfumes, tea, and aromatherapy

Direct observations of predation on Melibe leonina in the field are rare.  In 5 such instances, kelp crabs Pugettia producta are observed to catch and eat Melibe that they pluck from their perches in Macrocystis kelp plants. 

Tests in which glass rods are stroked over the bodies and cerata of Melibe and then touched to arms of sunflower stars Pycnopodia helianthoides result in the sea stars curling the arms upwards and towards the central disc. Ajeska & Nybakken 1976 Veliger 19: 19.

NOTE  clean (control) rods also elicit arm responses, but are much weaker in intensity

Investigations of the gland secretions of Melibe leonina in Victoria, British Columbia show that the glands are, indeed, repugnatorial.  Tests with sea stars Pycnopodia helianthoides, Leptasterias hexactis, Dermasterias imbricata, and Pisaster ochraceus show that tube-foot contact with live Melibe leonina causes arm withdrawal and retreat of the test subjects. Small specimens of P. helianthoides also respond similarly to contact with filter paper saturated with the secretion. 

Application of seawater filtrate of ground-up tube feet of Pycnopodia to test Melibe, however, fails to elicit discharge of the substance.  Feeding tests with cottid and gobiid fishes, more likely to be predators of Melibe than sea stars (owing to their habitat of living in kelp plants) result in rejection of test pieces of Melibe cerata.

Each gland comprises several secretory cells connected to a duct opening via a pore on the skin surface.  The glands are readily visible as tiny opaque spots (about 100µm diameter) in the translucent body wall, and are most abundant on the cerata and dorsal surface of the oral hood. The secretory cells contain large vacuoles that function as reservoirs.  A complex array of striated muscles squeezes the cells to force out their contents.  The gland is supplied with nerves leading to the muscles and to mechanoreceptor-type ciliated sensory cells surrounding the pore.  The glands discharge a ropy, sticky secretion when the overlying epidermis is touched.  On filter paper the secretion has the fruity odour typical of a physically disturbed Melibe.  Two types of cells comprise the main secretory component of the gland, and these are thought by the author to release defensive chemical and mucin, respectively.  Bickell-Page 1991 Zoomorph 110: 281.

Experiments on Melibe leonina collected from Bowen Island, British Columbia show that the characteristic fruity odour originates from the epidermal repugnatorial glands.  The substance is identified as a degraded terpenoid 2,6-dimethyl-5-heptenal, and is made by the nudibranch via de novo biosynthesis.   Barsby et al. 2002 Chemoecology 12: 199.

Peltodoris

In a study on Peltodoris nobilis from Monterey Bay, California, an active substance in the digestive glands is characterised as a new N-methylpurine riboside which the authors call doridosine.  The substance causes several related effects on heart function in rodents, including reduction of heart rate in anaesthetised mice by 50% for several hours, after which the mice recover completely.  Fuhrman et al. 1980 Science 207: 193.

Polycera

Extracts of Polycera tricolor contain triophamine. Reviewed in Gustafson & Andersen 1985 Tetrahedron 41: 1101.  Photo courtesy Kevin Lee, Fullerton, California diverkevin.

Shown here: the related Polycera atra, which
consumes bryozoans Membranipora spp.

Tochuina

The dendronotid nudibranch Tochuina tetraquetra in Port Hardy, British Columbia has at least 6 different, but related, terpenoids identifiable in skin extracts.  All but one of these metabolites can be directly traced to the soft coral Gersemia rubiformis, which is one of the main dietary items of Tochuina in this area.  Williams & Andersen 1987 Can J Chem 65: 2244.

Tochuina tetraquetra eating the
alcyonarian Gersemia rubiformis 0.6X

Although most secondary metabolites reported from skin extracts of dorid nudibranchs are sequestered unchanged from their diets of sponges, soft corals, tunicates, and bryozoans, a few species are capable of de novo biosynthesis of these chemicals. In the former category are nudibranch species whose skin chemistry varies with the different food-species eaten in their diverse habitats, while those in the second category have constant metabolic compositions over their entire geographic range.  The bryozoan-eating Triopha catalinae is an example of the latter, and its skin chemistry is identical at all collecting sites from Alaska to southern California.  Its principal skin metabolite triophamine, a diacylguanidine comprised of several acetate units, is found in studies at Bamfield, British Columbia to be synthesised de novo.  Graziani & Andersen 1996 Chem Commun 2377.