A diterpene from Laurencia obtusa

Laurencia obtusa collected at Kimmeridge Bay, Dorset contained 15-bromo-2,16-diacetoxy-7-hydroxy-9(11)-paraguarene as a major metabolise. Closely related compounds have recently been isolated from the sea hare Aplysia dactylomela collected at La Parguera, Puerto Rico.     

Structural diversity and geographical distribution of halogenated secondary metabolites in red algae,Laurencia nangii Masuda (Rhodomelaceae,Ceramiales), in the coastal waters of North Borneo Island

The red algae genus Laurencia (Rhodomelaceae, Ceramiales) is known as a prolific producer of halogenated secondary metabolites with a high level of species diversity and geographical distribution. In North Borneo Island, Malaysia, there are four main Laurencia species: Laurencia snackeyi, Laurencia majuscula, Laurencia similis and L. nangii. Although the chemistry of Laurencia is well studied, the diversity of compounds in L. nangii has not been thoroughly investigated. Therefore, we studied the chemical constituents of seven populations of L. nangii from Tunku Abdul Rahman Marine Park (two populations), Dinawan Island (one population), Tun Mustapha Marine Park (two populations) and Tun Sakaran Marine Park (two populations). Halogenated compounds were isolated and the structures determined via spectroscopic methods. A total of 20 metabolites belonging to the classes of sesquiterpenes, acetylenes, bromoallenes, diterpenes and triterpenes were identified. Populations from Tunku Abdul Rahman Marine Park and Dinawan Island contained non-chamigrane-type sesquiterpenes, acetylenes and diterpenes. Populations from Tun Mustapha Marine Park contained chamigrane-type sesquiterpenes, acetylenes and diterpenes. However, the chemical compositions of populations from Tun Sakaran Marine Park were found to differ significantly, containing chamigrane-type and non-chamigrane-type sesquiterpenes, bromoallenes and triterpenes. This investigation has revealed the presence of interesting chemotaxonomical markers in populations of L. nangii and the existence of chemical races in this species.     

Additional analysis of chemical diversity of the red algal genus Laurencia (Rhodomelaceae) from Japan

The halogenated secondary metabolite constitution of four species of the red algal genus Laurencia (Rhodomelaceae) from southern Japan is reported. Laurencia composita Yamada from Tanegashima Island (Kagoshima Prefecture) bears five sesquiterpenoids (2,10‐dibromo‐3‐chloro‐α‐chamigrene and 2,10‐dibromo‐3‐chloro‐9‐hydroxy‐α‐chamigrene, in addition to pre‐pacifenol epoxide, johnstonol and pacifenol, which are known in other populations of this species). Laurencia intricata Lamouroux from Chinzei (Saga Prefecture) and Oomura Bay (Nagasaki Prefecture) bear a C₁₅ aceto‐genin, okamurallene. Laurencia majuscula (Harvey) Lucas from Tanegashima Island produces three sesquiterpenoids, (Z)‐10,15‐dibromo‐9‐hydroxy‐chamigra‐1, 3(15),7(14)‐triene, 10‐bromo‐7‐hydroxylaurene and 10,11‐dibromo‐7‐hydroxylaurene, corresponding to those of one of its chemical races. Laurencia venusta Yamada from Tanegashima Island produces two sesquiterpenoids, cupalaurenol and cyclolaurenol, which were known only from a sea hare, Aplysia dactylomela Rang. This strongly suggests that Aplysia consumes L. venusta and concentrates these halogenated compounds.     

Studies on the nutrition and feeding preferences of Aplysia: Development of an artificial diet

An artificial diet made up principally of chemicals set in agar with a small amount of water extract of the green alga Ulva fasciata Delile has given good growth and spawn production in the sea hare Aplysia dactylomela Rang. The potential use of such a diet in the study of nutrition of sea hares and of other marine invertebrate herbivores is discussed and its possible rôle in identifying the characteristics of seaweeds which govern natural food choices in these animals is considered.     

An experimental approach to study the biosynthesis of brominated metabolites by the red algal genus Laurencia

The production of labeled brominated metabolites with radioactive ⁸²Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales). Radiobromide [⁸²Br], thin-layer chromatography (TLC), and TLC–autoradioluminography (ARLG) were used. When cultured in artificial seawater medium (ASP₁₂NTA including Na⁸²Br) under 16:8 h light:dark (LD) illumination cycles for 24 h, each of the strains of Laurencia, Laurencia japonensis Abe et Masuda, Laurencia nipponica Yamada (laurencin-producing race and laureatin-producing race), and Laurencia okamurae Yamada, produced species- (or race-) specific ⁸²Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [⁸²Br] in combination with the TLC–ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.     

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.     

Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI = 5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0-12.0), after heating to 55 °C for 30 min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H₂O₂ and other reactive components since it was not counteracted by H₂O₂ scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.     

Acidity enhances the effectiveness of active chemical defensive secretions of sea hares, <Emphasis Type="Italic">Aplysia californica</Emphasis>, against spiny lobsters, <Emphasis Type="Italic">Panulirus interruptus</Emphasis>

Sea hares such as Aplysia californica, gastropod molluscs lacking a protective shell, can release a purple cloud of chemicals when vigorously attacked by predators. This active chemical defense is composed of two glandular secretions, ink and opaline, both of which contain an array of compounds. This secretion defends sea hares against predators such as California spiny lobsters Panulirus interruptus via multiple mechanisms, one of which is phagomimicry, in which secretions containing feeding chemicals attract and distract predators toward the secretion and away from the sea hare. We show here that ink and opaline are highly acidic, both having a pH of ∼5. We examined if the acidity of ink and opaline affects their phagomimetic properties. We tested behavioral and electrophysiological responses of chemoreceptor neurons in the olfactory and gustatory organs of P. interruptus, to ink and opaline of A. californica within their natural range of pH values, from ∼5 to 8. Both behavioral and electrophysiological responses to ink and opaline were enhanced at low pH, and low pH alone accounted for most of this effect. Our data suggest that acidity enhances the phagomimetic chemical defense of sea hares.     

Vulnerability of sea hares to fish predators: importance of diet and fish species

Many sea hares (Opisthobranchia, Anaspidea) sequester secondary metabolites from their algal diets. Tests of the hypothesis that sequestered metabolites deter predators have yielded ambiguous results. We manipulated secondary metabolites in vivo by collecting the sea hare Stylocheilus striatus from different host algae, and by raising it in the laboratory on artificial diets containing or lacking secondary metabolites (malyngamides A and B). Sea hares were then offered to fish. Fish identity affected vulnerability of sea hares to predation far more than did diet history. Wrasses and bream ate most sea hares tasted; damselfishes, goatfishes, and other fishes rejected most. Diet history affected vulnerability of sea hares to wrasses in one of four experiments, and if data from all experiments were pooled, but the effect was modest. Diet history did not affect vulnerability of sea hares to other fish taxa. Despite several studies, evidence for diet-derived defenses against predators in sea hares remains equivocal.     

Influence of proximal stimuli on swimming in the sea hare Aplysia brasiliana

Although the neurobiology and physiology of sea hares are extensively studied, comparatively little is known about their behaviour or ecology. Several species of sea hares swim, but the function of swimming is unclear. In this paper, we tested the hypotheses that swimming in Aplysia brasiliana serves to find food and mates, and to escape predators. Our data strongly support the hypothesis that swimming in A. brasiliana is related to feeding. Sea hares deprived of food overnight swam 12 times longer than ones that had been fed. When sea hares contacted food while swimming they invariably stopped, while those contacting a plastic algal mimic mostly continued to swim. Our experiments provided no evidence to support the hypothesis that swimming in sea hares is related to social behaviour. Sea hares deprived of copulatory mates for 3 days did not swim longer than ones held in copulating groups. Moreover, swimming sea hares never stopped swimming upon encountering a conspecific. Our experiments also supported the hypothesis that swimming in sea hares is related to predation. Sea hares stimulated with a standardised tail pinch and exposed to ink of conspecifics swam four times longer than control individuals, and tail-pinched sea hares that released ink swam five times longer than ones that did not release ink. However, because predators of adult sea hares are mostly lacking and because sea hares often swim spontaneously without predators being present, we conclude that swimming behaviour in A. brasiliana is primarily related to food-finding.     

Multiple components in ink of the sea hare Aplysia californica are aversive to the sea anemone Anthopleura sola

Sea hares of the genus Aplysia rely on an array of behavioral and chemical defenses, including the release of ink and opaline, to protect themselves from predation. While many studies have demonstrated that ink and opaline are repellent to predators, very little is known about which components of these secretions are active against predators. Ink was previously shown to facilitate the escape of Aplysia from predatory anemones (Anthopleura) by eliciting tentacle retraction and/or shriveling, and gastrovascular eversion, but the metabolites mediating this interaction were not identified. We investigated the metabolites in Aplysia californica secretions that were aversive to the anemone Anthopleura sola, as demonstrated by tentacle shriveling and/or retraction. We found that ink elicited tentacle shriveling and/or retraction, while opaline elicited a feeding response. The active components in ink do not appear to be diet-dependent, as ink was aversive regardless of diet (natural seaweed diet vs. Gracilaria ferox). Furthermore, metabolites extracted from G. ferox were not aversive, suggesting that the aversive components are produced by the sea hares. We then examined escapin, a protein in ink with antimicrobial properties. Escapin quickly forms reaction products when mixed with the amino acids l-lysine and l-arginine, which would occur when ink and opaline are released into the sea hare mantle cavity. Neither escapin alone nor escapin mixed with its amino acid substrate l-lysine elicited aversive behaviors either immediately before or 2 min before applying to the tentacles. In addition, escapin mixed with opaline and applied to tentacles after 2 min did not elicit a significant aversive response. Using bioassay-guided fractionation, we attempted to isolate the components in A. californica ink that are aversive to A. sola. We determined that multiple components in ink, including both lipophilic and hydrophilic constituents, elicited aversive responses. We hypothesize that these components may facilitate A. californica's escape from A. sola by eliciting tentacle shriveling and/or retraction, which lead to anemones dropping ensnared sea hares.     

Chemical composition of Laurencia spp. collected from the Seto Inland Sea of Japan

The chemical composition of three Laurencia spp., Laurencia sp., L. okamurae and L. saitoi, which were collected from the Seto Inland Sea of Japan, has been examined. Laurencia sp. collected from the coast of Matoba Park, Takehara, Hiroshima Prefecture, contained a brominated chamigrane-type sesquiterpene (1), named matobol, as the main metabolite. The structure of matobol was determined as (+)-(2R,3R,6R,10S)-2,10-dibromochamigr-7(14)-en-3-ol (1). This is the first time that the optically active 1 has been isolated from Laurencia. On the other hand, L. okamurae from the coast of Ikunoshima Island, Hiroshima Prefecture, produced laurinterol (2) that is a known cyclolaurane-type sesquiterpene characteristic to this species in Japan. L. saitoi from the coast of Matoba Park contained a known bromoallenic C₁₅-acetogenin, neolaurallene (3).     

Brominated metabolites from an Okinawan Laurencia intricata

Two halogenated C₁₅ acetogenins, itomanallenes A and B, with a terminal bromoallene moiety along with a halogenated sesquiterpene, itomanol, have been isolated from the red alga Laurencia intricata collected in Okinawan waters. Their structures were deduced from 1D and 2D NMR experiments including ¹H–¹H COSY, HSQC, HMBC, and NOESY methods. The alcohol corresponding to itomanallene B seems to be a plausible precursor of itomanallene A, which has an unusual 2,10-dioxabicyclo[7.3.0]dodecene skeleton. Itomanol was found to be a selinane-type bromosesquiterpenoid, and is the first example of a selinane to be isolated from Japanese Laurencia species.     

Sesquiterpenoids of Laurencia majuscula (Ceramiales, Rhodophyta) from the Ryukyu Islands, Japan

Two populations of the red alga Laurencia majuscula (Harvey) Lucas (Rhodomelaceae, Ceramiales) from Taketomi Island and Hateruma Island, the Ryukyu Islands, Japan, have been characterized on the basis of both morphological features and halogenated secondary metabolite content. These populations have smaller and more slender thalli than those of other regions. Furthermore, the populations contain two chamigrane-type Sesquiterpenoids, (2R, 3R, 5S)-5-acetoxy-2-bromo-3-chlorochamigra-7(14),9-dien-8-one and (2R, 3R)-2-bromo-3-chlorochamigra-7(14), 9-dien-8-one, and a laurane-type sesquiterpenoid, debromoisolaurinterol, as secondary metabolites which are different from those previously reported from other populations. These results are consistent with the concept of ‘chemical races’ within a single species of Laurencia.     

CHEMOTAXONOMY IN MARINE ALGAE: CHEMICAL SEPARATION OF SOME LAURENCIA SPECIES (RHODOPHYTA) FROM THE GULF OF CALIFORNIA1

Species separation in the genus Laurencia (Rhodomelaceae, Rhodophyta) is complicated by the high degree of morphological variation within the species. Chemical investigations on a worldwide basis of over 15 species indicate that 1 or more of the halogenated natural products synthesized by Laurencia are unique to each species. Our chemical investigations of Laurencia pacifica, as presently understood from the Gulf of California, indicate that more than 1 species had been included under this name. Thin layer chromatographic (TLC) comparisons of the halogenated components of 3 recognizable forms of “L. pacifica” were completed. The results revealed 3 distinct forms, with halogenated products unique to each form. In each form the observed chemical characters had been previously isolated and identified and could now be positively assigned to their algal source. Comparisons were also conducted with L. pacifica Kylin (1941) from the type locality of the species, La Jolla, California, and revealed that it contained another halogenated product different from those isolated from the Gulf species. We conclude that 3 species of Laurencia have been elucidated in the Gulf of California and these are separate from L. pacifica Kylin. Each species can be distinguished by its characteristic array of halogenated compounds. Comparative thin layer chromatography of the lipid components of morphologically similar Laurencia species should prove to be a useful new taxonomic aid.     

The relative importance of nutrient enrichment and herbivory on macroalgal communities near Norman's Pond Cay, Exumas Cays, Bahamas: a “natural” enrichment experiment

The simultaneous effects of grazing and nutrient enrichment on macroalgal communities were experimentally investigated using plastic mesh enclosure/exclosure cages along a natural nutrient (DIN, SRP) gradient from the discharge of a tidal mangrove creek on the west side of Norman's Pond Cay, Exumas Cays, Bahamas. Nutrient enrichment was the only factor affecting total biomass of macroalgae whereas selective herbivory moderated species composition. Biomass ranged from >2 kg dry weight m⁻² at the DIN-enriched Waterfall to <600 g dry weight m⁻² in the DIN-limited Algal Halo. Grazing by the queen conch Strombus gigas and the sea hare Aplysia dactylomela significantly reduced biomass of the epiphyte Laurencia intricata, but not its host Digenea simplex at the DIN-enriched Waterfall. These two rhodophytes dominated the macroalgal community at the DIN-enriched Waterfall and inner Algal Halo, whereas the chlorophytes Microdictyon marinum and Cladophora catenata dominated the relatively DIN-depleted outer Algal Halo. Higher grazing rates by S. gigas, A. dactylomela, and juvenile parrotfish and surgeonfish at the DIN-enriched Waterfall compared to the more oligotrophic Algal Halo suggested selective grazing on higher quality (nitrogen) diets by these herbivores. A pronounced increase in the carbon/nitrogen (C:N) ratios of D. simplex and L. intricata from the Waterfall to the Algal Halo indicated increased DIN-limitation of growth in the more offshore, lower DIN macroalgal communities. In contrast, only modest increases in C:N ratios of M. marinum and C. catenata between the Waterfall and Algal Halo suggested that these species are better adapted to growth in reef environments with lower levels of DIN enrichment. Low coral cover (<2%) co-occurred with high macroalgal cover (>35%) at DIN-enriched reef sites around Norman's Pond Cay (Waterfall, Patch Reef, North Perry Reef) compared to relatively high coral cover (10-20%) and low macroalgal cover (<20%) at the lower DIN reef sites (Rainbow Reef, Lang's Reef). These results support ecological theory that bottom-up control via nutrient enrichment is a primary factor regulating overall biomass and taxonomic assemblages of macroalgae on coral reefs, whereas grazing is more important in controlling relative species composition via dietary preferences.     

Interspecific variation in chemical defenses in the sea hares (Opisthobranchia: Anaspidea)

Most species of sea hares (Opisthobranchia: Anaspidea) sequester secondary metabolites from their algal diets in their digestive glands. Aplysia Juliana Quoy and Gaimard, 1832, a sea hare that feeds on Ulva spp. and Enteromorpha spp., algae with few or no secondary metabolites, were compared with the sympatric sea hares A. oculifera Adams and Reeve, 1850, A. kurodai (Baba, 1937), and Dolabella auricularia (Solander, 1786), all of which eat chemically rich algae, to see if sequestered secondary metabolites afford protection to sea hares from potential predators. Organic extracts of whole A. Juliana stimulated, and organic extracts of whole D. auricularia deterred feeding by crabs. However, tests with organic extracts of individual body parts indicated that this pattern was due almost exclusively to unpalatability of extracts of D. auricularia digestive glands. Tests with pieces of tissue from the exterior of the animals, or with extracts of such tissues, showed no consistent patterns indicating that A. Juliana were more palatable than other sea hares. Sea hare egg masses do not appear to contain diet-derived secondary metabolites. However, pieces of egg masses of A. Juliana and D. auricularia were universally rejected by crabs and reef fish, and extracts of A. Juliana egg masses deterred feeding by some reef fish. Finally, both opaline secretion of A. Juliana and ink of A. kurodai, but not ink of D. auricularia, none of which appear to contain diet-derived secondary metabolites, deterred feeding by crabs. Opaline secretion from A. Juliana, but not ink from A. kurodai, induced avoidance behavior in crabs. Although sequestered secondary metabolites clearly can affect the palatability of the digestive gland, there is little evidence that they affect the palatibility of the ink, opaline secretion, eggs, or skin, suggesting that sequestered secondary metabolites may not play a key role in anti-predator defense of sea hares.     

Nonhalogenated organic molecules from Laurencia algae

The marine red algae of the genus Laurencia have produced more 700 secondary metabolites and exhibited high molecular diversity and intriguing bioactivity. Since the halogenated structures have been comprehensively reviewed previously, this review, covering up to the end of 2012, mainly focuses on the source, structure elucidation, and bioactivity of nonhalogenated organic molecules from Laurencia spp. as well as the relationship between nonhalogenated and halogenated products. Overall, 173 new or new naturally occurring compounds with 58 skeletons, mainly including sesquiterpenes, diterpenes, triterpenes, and C₁₅-acetogenins, are described.     

Antifouling Brominated Diterpenoids from Japanese Marine Red Alga Laurencia venusta Yamada

The marine red algal genus Laurencia has abundant halogenated secondary metabolites, which exhibit novel structural types and possess various unique biological potentials, including antifouling activity. In this study, we report the isolation, structure elucidation, and antifouling activities of two novel brominated diterpenoids, aplysin-20 aldehyde ( 1 ), 13-dehydroxyisoaplysin-20 ( 2 ), and its congeners. We screened marine red alga Laurencia venusta Yamada for their antifouling activity against the mussel Mytilus galloprovincialis. Ethyl acetate extracts of L. venusta from Hiroshima and Chiba, Japan, were isolated and purified, and the compound structures were identified using 1D and 2D NMR, HR-APCI-MS, IR, and chemical synthesis. Seven secondary metabolites were identified, and their antifouling activities were evaluated. Compounds 1 , 2 , and aplysin-20 ( 3 ) exhibited strong activities against M. galloprovincialis. Therefore, these compounds can be explored as natural antifouling drugs.