Palatability of Macroalgae that Use Different Types of Chemical Defenses

This study compared algal palatability and chemical defenses from subtropical green algae that may use different types of defense systems that deter feeding by the rock-boring sea urchin Echinometra lucunter. The potential defense systems present include (1) the terpenoid caulerpenyne and its activated products from Caulerpa spp., and (2) dimethylsulfoniopropionate (DMSP)-related defenses in Ulva spp. Secondary metabolites from these chemical groups have been shown to deter feeding by various marine herbivores, including tropical and temperate sea urchins. Live algal multiple-choice feeding assays and assays incorporating algal extracts or isolated metabolites into an artificial diet were conducted. Several green algae, including Ulva lactuca, Caulerpa prolifera, and Cladophora sp., were unpalatable. Nonpolar extracts from U. lactuca deterred feeding, whereas nonpolar extracts from C. prolifera had no effect on feeding. Polar extracts from both species stimulated feeding. Caulerpenyne deterred feeding at approximately 4% dry mass; however, dimethyl sulfide and acrylic acid had no effect at natural and elevated concentrations. E. lucunter is more tolerant than other sea urchins to DMSP-related defenses and less tolerant to caulerpenyne than many reef fish. Understanding the chemical defenses of the algae tested in this study is important because they, and related species, frequently are invasive or form blooms, and can significantly modify marine ecosystems.     

Isolation of new brominated sesquiterpene feeding deterrents from tropical green algaNeomeris annulata (Dasycladaceae: Chlorophyta)

We investigated the natural products chemistry and feeding deterrent effects of brominated sesquiterpenes produced by Pacific collections of the calcareous tropical green algaNeomeris annulata. Assays conducted with whole algae showed thatN. annulata was not susceptible to grazing by natural populations of herbivorous fishes on Guam. Crude extracts and column chromatography fractions containing the brominated sesquiterpenes deterred feeding by herbivorous fishes at natural concentrations in field assays on Guam. Two majorN. annulata sesquiterpenes isolated from Guam collections and three related sesquiterpenes previously reported from Bermuda all deterred fish feeding at the high end of their natural concentration ranges, with the exception of one metabolite from Bermuda collections of the alga that differed structurally from the other compounds. The results support our hypothesis that the compounds produced byNeomeris function as chemical defenses against herbivores. The alga produces both structural defenses (CaCO₃ in the form of aragonite) and secondary metabolites that defend against herbivory by reef fishes.     

Dynamics of Algal Secondary Metabolites in Two Species of Sea Hare

The function of acquired algal secondary metabolites in sea hares is the subject of debate, in part because the dynamics/processing of metabolites by sea hares is poorly understood. This study investigates the dynamics of red algal secondary metabolites in two sea hares, Aplysia parvula and Aplysia dactylomela. Secondary metabolite levels were quantified for the dietary red algae Laurencia obtusa and Delisea pulchra and for sea hares collected from these seaweeds in the field. The patterns and dynamics of algal secondary metabolites were further investigated in the laboratory by quantitative analysis of secondary metabolites in sea hares grown on diets of L. obtusa, D. pulchra, or the green alga Ulva sp. Sea hares accumulated the most abundant metabolites from each red alga, the terpene palisadin A from L. obtusa, and the halogenated furanone 3 from D. pulchra, and stored a greater proportion of these metabolites than other algal metabolites. A. parvula accumulated D. pulchra metabolites at much higher levels than L. obtusa metabolites. A. dactylomela accumulated similar concentrations of L. obtusa metabolites to A. parvula. The loss of L. obtusa metabolites by A. dactylomela matched that expected for dilution of metabolites via growth of the sea hares. However, the loss of L. obtusa metabolites by A. parvula was faster than predicted for growth alone, suggesting that metabolites were actively metabolized or excreted. Data for the loss of D. pulchra metabolites by A. parvula was equivocal. The secretions of A. parvula fed D. pulchra or L. obtusa were analyzed for the presence of algal secondary metabolites to investigate one possible path of excretion. L. obtusa secondary metabolites were detected in the mucous and opaline secretions of A. parvula, but D. pulchra metabolites were not detected in any secretions. The deployment of L. obtusa secondary metabolites in secretions by A. parvula may explain the more rapid rate of loss of these compounds and is consistent with a possible defensive role for acquired metabolites.     

Chemical Deterrence of a Cyanobacterial Metabolite against Generalized and Specialized Grazers

Pitipeptolide A is the major secondary metabolite in a persistent population of the cyanobacterium Lyngbya majuscula from Guam. Because this cyanobacterium is of low preference to many sympatric consumers, we tested the hypothesis that this compound acted as a chemical defense. Pitipeptolide A was deterrent to urchins, two species of amphipods, and small herbivorous crabs, whereas it did not deter feeding by the sea hare Stylocheilus striatus, which readily consumes cyanobacteria. Although our comparison included various small invertebrates, which are often tolerant to algal chemical defenses, this cyanobacterial compound proved deterrent at natural concentrations to all but one of the consumer species tested. Thus, the production of defensive secondary metabolites is likely related to the common occurrence of L. majuscula on this coral reef despite the abundance of diverse grazers.     

Detection of secondary metabolites in marine macroalgae using the marsh periwinkle,Littorina irrorata say,as an indicator organism

Twenty-four chemically profiled organic macroalgal extracts and seven purified natural products were bioassayed using the mesogastropodLittorina irrorata Say to determine if the presence of secondary metabolites in crude algal extracts could be correlated with the activity of the snail. Avoidance behavior by snails in the assay was highly correlated with the presence of secondary metabolites in the macrophyte extracts.     

The Sesterterpene Variabilin as a Fish-Predation Deterrent in the Western Atlantic Sponge Ircinia strobilina

The furanosesterterpene variabilin was identified in an unpalatable crude extract and proved to be a feeding deterrent when offered at 0.23% of artificial diet dry weight to reef fishes in field assays. The icthyodeterrent property of variabilin was expressed when food pellets for the palatability assays were made with calcium alginate, but not carrageenan. Variabilin probably failed to show deterrent activity in the carageenan matrix because of decomposition during the necessary heat treatment.     

Palatability of aposematic queen butterflies (Danaus gilippus) feeding onSarcostemma clausum (Asclepiadaceae) in Florida

Queen butterflies (Danaus gilippus) are generally considered unpalatable to predators because they sequester and store toxic cardenolides from their larval food plants. However, a major queen food plant in Florida, the asclepiadaceous vineSarcostemma clausum, is shown here to be a very poor cardenolide source, and queens reared on this plant contain no detectable cardenolide. A direct evaluation of queen palatability using red-winged blackbirds indicates thatS. clausum-reared butterflies are essentially palatable to these predators (85% of abdomens entirely eaten), indicating little protection from either cardenolides, other sequestered phytochemicals, or de novo defensive compounds. Wild-caught queens that presumably fed as larvae uponS. clausum and also had access to adult-obtained chemicals, such as pyrrolizidine alkaloids (PAs), were relatively palatable as well (77% of abdomens eaten); they did not differ significantly in palatability from the labreared butterflies. Together, these findings suggest that; (1)S. clausumfed queens are poorly defended against some avian predators, and (2) for the particular queen sample examined, PAs do not contribute substantially to unpalatability. The discovery thatS. clausum-feeding queens are essentially palatable is of additional significance because it compels a reassessment of the classic mimicry relationship between queen and viceroy butterflies. Viceroys have been shown recently to be moderately unpalatable; therefore, the established roles of model and mimic may be reversed in some cases.     

Variation in Secondary Metabolite Concentrations in Yellow and Grey Morphs of the Red Sea Soft Coral Parerythropodium fulvum fulvum: Possible Ecological Implications

Secondary metabolites in yellow and grey morphs of the soft coral Parerythropodium fulvum fulvum were compared between colonies collected from different depths and reef sites along the Red Sea. The concentrations of fulfulvene, the major metabolite in the yellow morph, varied considerably among samples, with significant differences between shallow and deep colonies. The concentrations of 5-hydroxy-8-methoxy-calamenene and 5-hydroxy-8-methoxy-calamenene-6-al, the major metabolites in the grey morph, also exhibited significant differences between shallow and deep colonies. The ecological implications of these variations in secondary metabolites are discussed.     

Chemical Defense of the Caribbean Reef Sponge Axinella corrugata Against Predatory Fishes

Field and laboratory experiments were performed to investigate the palatability to predatory fishes of organic extracts and purified compounds from the Caribbean reef sponge Axinella corrugata (=Teichaxinella morchella). When incorporated into artificial foods at the same volumetric concentration as found in sponge tissue, crude extracts of the sponge, as well as a butanol-soluble partition of the crude extract, deterred feeding of the Caribbean reef fish Thalassoma bifasciatum in laboratory aquarium assays and deterred feeding of a natural assemblage of fishes in assays performed on reefs where A. corrugata is found. Bioassay-directed fractionation of the butanol-soluble partition led to the isolation of a single compound responsible for feeding deterrency, stevensine, a previously described dibrominated alkaloid. The mean concentration of stevensine in A. corrugata, as determined by quantitative NMR analysis, was 19.0 mg/ml (N = 8, SD = 7.2 mg/ml). Stevensine deterred feeding in laboratory aquarium assays at concentrations >2.25 mg/ml, and deterred feeding in field assays at ~12 mg/ml. Stevensine represents another in the oroidin class of brominated pyrrole derivatives that function as chemical defenses of sponges in the families Axinellidae and Agelasidae.     

The Role of Vanadium in the Chemical Defense of the Solitary Tunicate, <Emphasis Type="Italic">Phallusia nigra</Emphasis>

Ascidians (sea squirts) may defend themselves from predators, biofouling competitors, and bacterial infection by producing secondary metabolites or sequestering acid, but many species also accumulate heavy metals, most notably vanadium. The defensive functions of heavy metals in ascidians remain unclear, and to this end, the solitary Caribbean tunicate, Phallusia nigra, was studied to localize vanadium in its tissues and to assess the defensive properties of vanadium-containing compounds. As determined by flame atomic absorption spectroscopy, the internal tissues and blood contained the highest vanadium concentrations (mean values of 2,280 and 1,886 ppm dry mass, respectively), followed by the tunic surface (871 ppm dry mass). Results of laboratory feeding assays with the bluehead wrasse, Thalassoma bifasciatum, confirmed outcomes of past studies that demonstrated that vanadyl sulfate (VOSO₄·6H₂O) and sodium vanadate (Na₃VO₄) were unpalatable to fish, although these salts do not accurately reflect the chelation environment or oxidation state of vanadium in living tunicates. Fresh preparations of whole tunic, internal tissues, and blood were unpalatable to fish, but freezing and thawing of internal tissues and blood rendered them palatable. Crude organic extracts of whole tunic and internal tissues contained vanadium metabolites (225 and 750 ppm dry mass, respectively) and were palatable to T. bifasciatum; crude extracts also exhibited no antimicrobial effects against a panel of four marine bacteria known to be pathogens of marine invertebrates (Vibrio parahaemolyticus, Vibrio harveyi, Leucothrix mucor, and Deleya marina). Nonacidic vanadium (+3) complexes neither deterred predation nor inhibited microbial growth, whereas acidic aqua vanadium (+3 and +4) complexes were unpalatable to T. bifasciatum and exhibited antimicrobial activity. Difficulties in decoupling low pH from oxidation state and chelation environment of vanadium prevent definitive conclusions about the importance of some vanadium metabolites, but low pH appears to be the principal agent of chemical defense for P. nigra.     

Quantitative assessment of natural toxicity in sponges: toxicity bioassay versus compound quantification

Microtox^® assay was used to assess the natural toxicity of two sponges, Dysidea avara and Ircinia variabilis. The activity of crude extracts and major metabolites were compared. Methanol extract of D. avara was more toxic than that of acetone and was as toxic as pure avarol, thus suggesting that the toxicity of the sponge was mainly due to this metabolite. We also quantified palinurin, the major metabolite of I. variabilis, in specimens from several habitats. With the same methanol extracts used for palinurin quantification, we ran the Microtox^® assay and found a positive significant regression between toxicity and concentration of this metabolite. Pure palinurin was tested at the same concentration present in the extract, and the toxicity recorded was higher than that of the methanol extract. As with avarol from D. avara, palinurin is the main secondary metabolite that confers toxicity to I. variabilis. The results confirm that the standardized Microtox^® assay is an accurate and reproducible tool for assessing the toxicity of crude extracts and pure metabolites of marine organisms. These results also suggest that methanol is more suitable than acetone for the detection of species toxicity by Microtox^® The method is faster and easier to perform than chemical quantification even when the sponge chemistry is known, and is appropriate for studies on variation in natural toxicity over a range of environmental conditions.     

Chemistry and chemical ecology of the Bahamian spongeAplysilla glacialis

Chemical investigation of the secondary metabolites of the marine spongeAplysilla glacialis collected at French Wells, Crooked Island, Bahamas, resulted in the isolation and characterization of four diterpenes, two sterol endoperoxides, and two methylated primary metabolites. Some of these compounds, along with crude extracts of the sponge, were investigated for their ability to deter fish predation, affect the fouling of surfaces, and inhibit the growth of marine microorganisms. The diterpene manoöl (3), cholesterol endoperoxide (4), and the crude nonpolar extract ofA. glacialis were shown to deter feeding by a natural assemblage of fish predators in an in situ assay conducted at French Wells. Pure secondary metabolites and crude extracts ofA. glacialis also were tested in a laboratory fish feeding assay employing the wrasseThalassoma lunare. A mixture of sterol endoperoxides was isolated from the mucus that coats the surface ofA. glacialis and is exuded in large quantities when the sponge is disturbed. These compounds are thereby distributed in a manner in which they can best serve a defensive role for the sponge. An in situ assay was designed to determine the effect that pure secondary metabolites and crude extracts have on the fouling of surfaces. Manoöl (3) and cholesterol endoperoxide (4) were determined to increase the rate of fouling when compared to control surfaces. 1-Methyladenine (5) was identified as an antimicrobial constituent ofA. glacialis that inhibited the growth of four marine bacteria isolated from seawater samples collected at French Wells.This work was presented at the Gordon Research Conferences (Marine Natural Products Chemistry), February 1990, Oxnard, California.     

Effects of Sequestered Iridoid Glycosides on Prey Choice of the Prairie Wolf Spider, Lycosa carolinensis

Specialist insect herbivores that sequester allelochemicals from their host plants may be unpalatable to potential predators. However, the host-plant species used may determine the degree of palatability. Spiders, including members of the family Lycosidae, are important predators of invertebrate prey. We fed buckeye caterpillars, Junonia coenia (Nymphalidae), reared on Plantago lanceolata (containing high levels of iridoid glycosides) or P. major (containing low levels of iridoid glycosides) to prairie wolf spiders, Lycosa carolinensis (Lycosidae), to determine whether the spiders found insects that sequester iridoid glycosides unpalatable. In a field experiment, spiders ate caterpillars reared on P. major significantly more often than caterpillars reared on P. lanceolata, although they attacked equal numbers of both types of prey. Spiders that bit caterpillars behind their heads or along the middle of their backs prevented caterpillars from implementing deterrent defensive strategies such as regurgitating or defecating. In a laboratory experiment, we presented spiders with P. lanceolata-reared and P. major-reared caterpillars simultaneously for eight consecutive trials. Spiders consumed P. major-reared buckeyes significantly more often than P. lanceolata-reared caterpillars. We found no evidence that the spiders learned to avoid the unpalatable prey.     

Organic Sulfur Compounds from Dictyopteris spp. Deter Feeding by an Herbivorous Amphipod (Ampithoe longimana) but Not by an Herbivorous Sea Urchin (Arbacia punctulata)

Brown seaweeds in the genus Dictyopteris produce several C₁₁ sulfur metabolites that appear biosynthetically related to the C₁₁ compounds known to attract sperm to female gametes of many brown algae. All four of the C₁₁ sulfur compounds that we tested strongly deterred feeding by the amphipod Ampithoe longimana but had no effect on feeding by the sea urchin Arbacia punctulata, even when tested at concentrations that were two to eight times greater than those that deterred amphipods. In numerous previous investigations, a variety of seaweed compounds have been shown to deter feeding by large mobile herbivores such as fishes and urchins but to be relatively ineffective against mesograzers, such as the amphipod of our study. Our results for the C₁₁ sulfur compounds from Dictyopteris thus contrast sharply with patterns from previous studies and suggest that these metabolites may be defenses specifically targeted against small mesograzers such as amphipods. The occurrence of C₁₁ metabolites in brown algal eggs could allow these defenses to be especially important in defending gametes, zygotes, or young sporelings from herbivorous mesograzers.     

Influence Of Diet-Related Chemical Cues from Predators on the Hatching of Egg-Carrying Spiders

Previous studies have shown that animals may make adaptive adjustments in response to chemical cues from predators, but hatching responses to diet-related chemical cues from predators have not been previously demonstrated. In the system studied here, the predator is an araneophagic jumping spider (Salticidae), Portia labiata, and the prey organism is a subsocial spitting spider, Scytodes pallida (Scytodidae). The spitting spider carries its eggs in its chelicerae, and carrying eggs is known to make it more vulnerable to predators. It is also known from an earlier study that the prior diet of the predator alters how dangerous the individual predator is to the spitting spider. In the experiments reported here, incubation time was shorter when volatile cues from the predator were present and longer in control tests when no chemical cues from the predator were present. The previous predators diet also influenced incubation time: when in the presence of volatile cues from individuals of P. labiata that had previously fed on individuals of S. pallida, incubation time was shorter than when in the presence of volatile cues from individuals of P. labiatathat had been feeding instead on house flies.     

Comparative Metabolism of Dietary Terpene, p-Cymene, in Generalist and Specialist Folivorous Marsupials

The urinary metabolites of the monoterpene, p-cymene, were studied in three marsupial species: a generalist herbivore, the brushtail possum (Trichosurus vulpecula), and two specialist folivores, the greater glider (Petauroides volans) and the ringtail possum (Pseudocheirus peregrinus), as well as in the laboratory rat (Rattus norvegicus). Each animal was dosed, intragastrically, with single doses of p-cymene (0.37 mmol/kg and/or 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods. Quantitative analysis of urinary metabolites by extraction, gas chromatography, and mass spectrometry gave a mean recovery of 64% (range 52–74%) of the administered dose in 48 hr for the four species. No fecal metabolites were found. A species-specific pattern of metabolite excretion was evident and reflected the natural occurrence of p-cymene (and terpenes in general) in the diet. If the metabolites excreted are grouped according to the total number of oxygen atoms added (one to four), then the rat excreted metabolites encompassing all degrees of oxidation, but predominantly a monooxygenated metabolite. The brushtail possum excreted metabolites having two, three, and four oxygen atoms added. The ringtail possum and greater glider only excreted metabolites with three or four oxygen atoms. Conjugation played a significant role in the excretion of oxidized metabolites in only the brushtail possums and the rat. These findings indicate that species encountering terpenes, such as p-cymene, in their normal diet have developed efficient oxidation pathways to eliminate them. This oxidative efficiency could also reduce the necessity for subsequent conjugation of metabolites which minimizes further demands on a nutritionally limited diet.     

No Evidence for the Induction of Brown Algal Chemical Defense by the Phytohormones Jasmonic Acid and Methyl Jasmonate

Induced chemical defense reactions are widespread in marine brown algae. Despite the evidence that the biosynthesis of defense metabolites can be up-regulated upon herbivory, we do not know how this regulation of biosynthetic pathways to secondary metabolites is achieved in brown algae. In higher plants, the phytohormone jasmonic acid (JA) is crucial for the mediation of induced chemical defenses, and several findings of this metabolite from marine sources have been reported. We tested the hypothesis that JA or related metabolites play a role in induced brown algal defense. Quantification of oxylipins with a detection limit around 20 ng g⁻¹ algal tissue did not reveal the presence of JA in the seven examined brown algal species Dictyota dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia elongata, Saccharina latissima (formerly Laminaria saccharina), and Sargassum muticum. Moreover, treatment with ecologically relevant concentrations of JA and methyl jasmonate did not lead to a significant change in the profile of medium- and non-polar metabolites of the tested algae. Only when high concentrations of ≥500 μg ml⁻¹ medium of the phytohormones were applied that a metabolic response which could be attributed to unspecific stress was observed. Bioassays with D. dichotoma that focused on medium- and non-polar compounds confirmed the lack of a biological role of JA and methyl jasmonate in the induction of algal induced chemical defenses. The phytohormone-treated samples did not exhibit any increased defense potential towards the amphipod Ampithoe longimana and the isopod Paracerceis caudata. JA and related phytohormones, known to be active in higher plants, thus appear to play no role in brown algae for induction of the defense chemicals studied here.     

Reduction in diet breadth results in sequestration of plant chemicals and increases efficacy of chemical defense in a generalist grasshopper

The lubber grasshopper,Romalea guttata, is a generalist feeding on a broad diet of many herbaceous plant species and has a metathoracic defensive secretion normally containing phenolics and quinones synthesized by the insect. When insects were reared on a restricted diet of wild onion, they sequestered sulfur volatiles from the plant into their defensive secretions. These compounds were not detected by gas chromatography-mass spectroscopy in secretions of insects on an artificial diet or a natural, generalist diet of 26 plants that included wild onion as a component, nor were they present in secretions from field-collected insects. Defensive secretions of insects reared on wild onion were significantly more deterrent, by as much as an order of magnitude, to two species of ant predators than secretions from insects on either of the other two diets, despite a reduction in the concentration of autogenous defensive chemicals in secretions of insects on the onion diet. Sequestration of plant chemicals that increased defensive efficacy occurred when diet breadth was reduced. We suggest that this occurs because under conditions of specialization, plant secondary metabolites are more likely to be ingested and bioaccumulated in sufficient concentrations to have biological activity against predators. What we define as casual bioaccumulation of bioactive plant chemicals following dietary specialization may lead to evolution of sequestered defense syndromes in insects, and this process may not necessarily require specific adaptation to or coevolution with a toxic host plant.     

Production and Bioactivity of Common Lichen Metabolites as Exemplified by <Emphasis Type="Italic">Heterodea muelleri</Emphasis> (Hampe) Nyl.

Based on results of a former study in 2005, this investigation aimed at quantifying UV- and cold temperature stress-induced changes within the secondary metabolite production of the cultured mycobiont of the lichen Heterodea muelleri (Hampe) Nyl. The chemical profiles of the mycobiont cultures and the lichen thallus were determined by high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) analyses. The voucher specimen of H. muelleri produced diffractaic acid as a major polyketide and barbatic acid as a satellite compound, whereas the untreated mycobiont did not contain any detectable secondary metabolites. While UV-C stress caused a general increase in substance formation, cold temperature stress resulted in a strong activation of barbatic acid biosynthesis. A further series of experiments focused on the effect of diffractaic and barbatic acids on the growth of the symbiotic photobiont Trebouxia jamesii; usnic acid was similarly tested. Pure substances were obtained from mycobiont cultures by performing preparative TLC. A determined quantity of algae was incubated on BBM plates that contained three different concentrations of the pure lichen metabolites. The growth of the photobionts was monitored over a period of 1 mo to evaluate the impact of each substance on the cultured algae. While diffractaic and usnic acid had no noticeable effect, barbatic acid strongly inhibited algal growth and resulted in cell death.     

The Fate of <Emphasis Type="Italic">Lyngbya majuscula</Emphasis> Toxins in Three Potential Consumers

Blooms of Lyngbya majuscula have been reported with increasing frequency and severity in the last decade in Moreton Bay, Australia. A number of grazers have been observed feeding upon this toxic cyanobacterium. Differences in sequestration of toxic compounds from L. majuscula were investigated in two anaspideans, Stylocheilus striatus, Bursatella leachii, and the cephalaspidean Diniatys dentifer. Species fed a monospecific diet of L. majuscula had different toxin distribution in their tissues and excretions. A high concentration of lyngbyatoxin-a was observed in the body of S. striatus (3.94 mg/kg⁻¹) compared to bodily secretions (ink 0.12 mg/kg⁻¹; fecal matter 0.56 mg/kg⁻¹; eggs 0.05 mg/kg⁻¹). In contrast, B. leachii secreted greaterconcentrations of lyngbyatoxin-a (ink 5.41 mg/kg⁻¹; fecal matter 6.71 mg/kg⁻¹) than that stored in the body (2.24 mg/kg⁻¹). The major internal repository of lyngbyatoxin-a and debromoaplysiatoxin was the digestive gland for both S. striatus (6.31 ± 0.31 mg/kg⁻¹) and B. leachii (156.39 ± 46.92 mg/kg⁻¹). D. dentifer showed high variability in the distribution of sequestered compounds. Lyngbyatoxin-a was detected in the digestive gland (3.56 ± 3.56 mg/kg⁻¹) but not in the head and foot, while debromoaplysiatoxin was detected in the head and foot (133.73 ± 129.82 mg/kg⁻¹) but not in the digestive gland. The concentrations of sequestered secondary metabolites in these animals did not correspond to the concentrations found in L. majuscula used as food for these experiments, suggesting it may have been from previous dietary exposure. Trophic transfer of debromoaplysiatoxin from L.majuscula into S. striatus is well established; however, a lack of knowledge exists for other grazers. The high levels of secondary metabolites observed in both the anaspidean and the cephalapsidean species suggest that these toxins may bioaccumulate through marine food chains.