Raman Spectroscopic Insights into the Chemical Gradients within the Wound Plug of the Green Alga Caulerpa taxifolia

The invasive unicellular green macroalga Caulerpa taxifolia has spread dramatically in the Mediterranean Sea over the last decades. Its success is based on rapid plug formation after wounding, to prevent the loss of cell content. This quick and efficient process involves the rapid transformation of the secondary metabolite caulerpenyne to the reactive 1,4‐dialdehyde oxytoxin 2, which acts as a protein crosslinker. The main metabolites of the wound plug were identified as proteins, caulerpenyne derivatives, and sulfated polysaccharides. Because of a methodological deficit, however, the detailed distribution of the compounds within the wound plug of C. taxifolia was unknown. This study demonstrates the suitability of FT‐Raman spectroscopy for the noninvasive in vivo determination of caulerpenyne and its derivatives, as well as β‐carotene, from signals with special spectral features within the wound plug and the adjacent intact alga tissue, with a resolution of 100 μm. FT‐Raman spectra allowed four different zones with distinct chemical compositions around the region of wounds to be characterized. Gradients of the investigated metabolites within the wound plug and the alga could be determined. Moreover, various caulerpenyne derivatives could be identified spectroscopically, and this has led to a mechanistic proposal for the internal and the external wound plug formation.     

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.     

Evidence for chemical defense in tropical green algaCaulerpa ashmeadii (Caulerpaceae: Chlorophyta): Isolation of new bioactive sesquiterpenoids

Results of field feeding preference studies with 12 species of tropical green algae of the genusCaulerpa showed thatC. ashmeadii was preferred least by herbivorous fishes. Chemical investigations ofC. ashmeadii demonstrated the presence of high concentrations of sesquiterpenoid metabolites. The chemical isolation and structural elucidation of five majorC. ashmeadii metabolites, as well as the results of field feeding preference, antimicrobial, and ichthyotoxicity assays demonstrating the biological activities of these metabolites are reported here.     

Screening of lipase inhibitors from marine algae

The possible presence of an inhibitor of pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was screened in 54 marine algae. An active inhibitor, caulerpenyne, was purified from an extract of Caulerpa taxifolia, using ethyl acetate extraction, followed by successive chromatographies on ODS and silica gel columns. The purified inhibitor was identified by thin-layer chromatography, infrared and nuclear magnetic resonance spectroscopy. Caulerpenyne competitively inhibited lipase activities using emulsified triolein and dispersed 4-methylumbelliferyl oleate (4-MU oleate) as substrates. The concentrations producing 50% inhibition against triolein and 4-MU oleate hydrolysis were 2 mM and 13 μM, respectively. In vivo, oral administration of corn oil with or without caulerpenyne to rats demonstrated a reduced and delayed peak plasma triacylglycerol concentration with caulerpenyne.     

Variations in Caulerpenyne Contents in Caulerpa taxifolia and Caulerpa racemosa

Caulerpenyne (CYN) contents was measured in two Chlorophyceae algae, Caulerpa taxifolia and Caulerpa racemosa, between July 1999 and July 2000. Sampling was performed at three stations exhibiting increasing levels of competition with the seagrass Posidonia oceanica. Significant differences were observed as a function of the Caulerpa species, the season, and the level of competition. CYN concentrations were always greater in C. taxifolia, regardless of either season or level of competition (35–80 times greater, according to the season). For a given species, maximum concentrations were recorded in autumn (September/November) and minimum values occurred in spring (April/May). CYN contents decreased with increasing level of competition, whereas frond length increased over this same gradient. It would appear that when the algae are in competition with P. oceanica, Caulerpa is more inclined to accelerate vegetative growth (competition for light) than to produce secondary metabolites.     

Effects of secondary metabolites from marine algae on feeding by the sea urchin,Lytechinus variegatus

A bioassay was developed to test the hypothesis that secondary metabolites from marine algae affect feeding by sea urchins. During experiments measuring chemoreception and gustation, feeding by the regular sea urchin,Lytechinus variegatus (Lamarck), was inhibited by extracts from the green marine alga,Caulerpa prolifera (Forsskal) Lamouroux andCymopolia barbata (Linneaus) Lamouroux. Cymopol, a monoterpene-bromohydroquinone component ofC. barbata, was partially responsible for the inhibited feeding observed in tests of theCymopolia crude extract. Caulerpenyne, an oxygenated sesquiterpene fromC. prolifera, was responsible for all of the urchin feeding inhibition observed in tests of theC. prolifera crude extract. Feeding was not affected by: (1) extracts from several otherCaulerpa species includingC. mexicana (Sonder) J. Agardh,C. ashmaedii Harvey,C. racemosa v.macrophysa (Kutzing) Taylor, andC. racemosa v.laetevirens (Montagne) Weber-van Bosse; (2) caulerpin, an indole-containing pigment isolated from all of theCaulerpa species exceptC. mexicana; and (3) an extract from the red marine alga,Gracilaria foliifera v.angustissima (Harvey) Taylor, which has no known secondary metabolites. Feeding inhibition was independent of the test diameter which correlated with the reproductive state of the urchins. Feeding inhibition was also independent of the starvation periods between experiments, and the temperature and salinity in ranges tolerated byL. variegatus obtained from the Florida Gulf Coast. The data strongly suggest that at least one alga,Caulerpa prolifera, is chemically defended against a dominant omnivore in its community.     

Activated Chemical Defense in Marine Sponges—a Case Study on <Emphasis Type="Italic">Aplysinella rhax</Emphasis>

Activated chemical defense, i.e., the rapid conversion of precursor molecules to defensive compounds following tissue damage, has been well documented for terrestrial and marine plants; but evidence for its presence in sessile marine invertebrates remains scarce. We observed a wound-activated conversion of psammaplin A sulfate to psammaplin A in tissue of the tropical sponge Aplysinella rhax. The conversion is rapid (requiring only seconds), the turnover rate increases with increasing wounding activity (e.g., ~20% after tissue stabbing vs. ~85% after tissue grinding), and is likely enzyme-catalyzed (no reaction in the absence of water and inhibition of the conversion by heat). Fish feeding assays with the pufferfish Canthigaster solandri, an omnivorous sponge predator, revealed an increased anti-feeding activity by the conversion product psammaplin A compared to the precursor psammaplin A sulfate. We propose that the wound-activated formation of psammaplin A in A. rhax is an activated defense targeted against predator species that are not efficiently repelled by the sponge's constitutive chemical defense. Recent observations of conversion reactions also in other sponge species indicate that more activated defenses may exist in this phylum. Based on the findings of this study, we address the question whether activated defenses may be more common in sponges-and perhaps also in other sessile marine invertebrates-than hitherto believed.     

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.     

Geographic Variation and Tissue Distribution of Endogenous Terpenoids in the Northeastern Pacific Ocean Dorid Nudibranch Cadlina luteomarginata: Implications for the Regulation of De Novo Biosynthesis

Gas chromatography (GC) analyses of whole animal skin extracts and individual tissue extracts obtained from specimens of Cadlina luteomarginata collected in British Columbia and southern California were used to determine if concentrations of the nudibranch's biosynthetic products—albicanyl acetate (1), cadlinaldehyde (2), and luteone (3)—vary significantly between two populations, among individuals of a population, and among body tissues of individual specimens. The major biosynthetic product, albicanyl acetate (1), has the same concentration in both British Columbia and California populations, while the British Columbia population contains greater total amounts of 2 and 3 than the California population. Within individuals from one population, the largest proportion of endogenous metabolites is in the dorsum, specifically in the mantle dermal formations and margins. The GC analyses show that across geographically separated populations and within geographically localized populations the concentration of endogenous metabolites is inversely correlated with availability of structurally similar compounds from dietary sources. This suggests that the de novo biosynthesis of defensive compounds might be regulated according to need.     

Hydrothermal fractional pretreatment of sea algae and its enhanced enzymatic hydrolysis

BACKGROUND: Sea algae cellulose has been little utilized because the cellulose content in sea algae is low. For the effective utilization of sea algae cellulose, cellulose must be converted without drying into valuable material with a high rate and yield. From this viewpoint, effects of hydrothermal pretreatments of sea algae to enhance the glucose production by enzymatic hydrolysis of sea algae cellulose were investigated. RESULTS: Using hydrothermal pretreatment performed at 423 K for Monostroma nitidum Wittrock (green alga) and at 473 K for Solieria pacifica (red alga) for 30 min, yields of extracted water‐soluble components containing monosaccharides were 0.51 g g^?1 for the green alga and 0.62 g g^?1 for the red alga. The apparent rate of glucose production from hydrothermally pretreated green alga by enzymatic hydrolysis was > 10 times faster than that of the non‐pretreated sample. Yields of glucose from cellulose by enzymatic hydrolysis were 79.9% for the green alga and 87.8% for the red alga. CONCLUSION: Cellulose of sea algae was successfully fractionated by hydrothermal pretreatments, which resulted in high susceptibility of sea algae cellulose to enzyme attack. This process leads to the effective utilization of sea algae cellulose. Copyright © 2008 Society of Chemical Industry     

Reinvestigation of Female Sex Pheromone of Processionary Moth (Thaumetopoea pityocampa): No Evidence for Minor Components

The female sex pheromone of the processionary moth Thaumetopoea pityocampa has been reinvestigated to look for possible minor components. Examination by GC-MS and GC-EAD of the contents of virgin female glands, after stimulation with PBAN (pheromone-biosynthesis-activating neuropeptide), showed that the major component, (Z)-13-hexadecen-11-ynyl acetate (1), appears to be the only pheromone compound present in the gland. Comparison of female attractivity with that of the natural extract and synthetic (Z)-13-hexadecen-11-ynyl acetate showed that this chemical is able to elicit a similar activity to that displayed by virgin females in a wind tunnel. In single cell recording experiments, two specialist receptor cell types were found in the trichoid sensilla. One cell type was tuned to enyne acetate 1. The other one was tuned to (Z,Z)-11,13- hexadecadienal and (Z)-13-hexadecen-11-ynal, the major components of the pheromone blend of other Thaumetopoea spp., and constitutes a further example of interspecific inhibitor receptor cells. Our results show that the processionary moth may not need minor components for successful mate recognition.     

Simultaneous Removal of Algae and Their Secondary Algal Metabolites from Water by Hybrid System of DAF and PAC Adsorption

Abstract

The feasibility of a hybrid system consisting of powdered activated carbon (PAC) adsorption and dissolved air flotation (DAF) processes was examined for the simultaneous removal of algae (anabaena and mycrocystis) and their secondary algal metabolites (2‐methylisoboneol and geosmin). Before studying the hybrid system, adsorption equilibrium and kinetics of organics (2‐methylisoboneol and geosmin) produced from algae on three powdered activated carbons (wood‐based, coal‐based, coconut‐based) were studied. The flotation efficiency of algae and PAC in DAF process was evaluated with zeta potential measurements. Interestingly, we found that the agglomerate of bubble and PAC particle can be successfully floated by DAF. In addition, the simultaneous removal of algae and organics (i.e., secondary algal metabolites) dissolved in water can be achieved by using the hybrid system of adsorption/DAF processes.     

Evidence for Metabolic Turnover of Polyphenolics in Tropical Brown Algae

Polyphenolic chemical defenses of plants have traditionally been classified as immobile or quantitative and as such are believed to have low to negligible rates of turnover. This assumption is an important element in many ecological theories of chemical defense that invoke cost versus benefit relationships, because (1) turnover increases the metabolic cost of maintaining an effective level of defense, and (2) changes in the rate of turnover could affect the conclusions of studies that rely upon static concentration (standing crop) measurements, since changes in compound synthesis may not emerge as corresponding changes in compound concentration. By using a stable-isotope labeling technique, we measured rates of synthesis and turnover for the polyphenolic compounds of marine brown algae in laboratory and field experiments. During the laboratory experiment, we observed the relatively rapid turnover of phlorotannins in a population of the tropical brown alga Lobophora variegata. In order to determine if such metabolic turnover in brown algae occurred under natural conditions, we then measured in situ rates of synthesis, polymerization, and turnover for extractable phlorotannins in two species of tropical marine brown algae, Sargassum hystrix var. buxifolium (Fucales) and Dictyopteris justii (Dictyotales), over a 17-day period in the field. We found that phlorotannins in L. variegata and S. hystrix var. buxifolium demonstrated rapid rates of turnover in laboratory culture and in situ field experiments, respectively. The trends for D. justii also support the presence of turnover. Results indicate that (1) the assumption that algal polyphenolics can be grouped with the tannins of vascular plants as "immobile" defenses needs to be reevaluated, (2) estimates of the metabolic cost of algal polyphenolics that presume negligible rates of turnover may significantly underestimate the total cost of defense, and (3) studies designed to test the predictions of ecological theories for the phlorotannin concentrations of tropical brown algae may be affected by changes in the rates of metabolic turnover.     

Novel detection of formylated phloroglucinol compounds (FPCs) in the wound wood of Eucalyptus globulus and E. nitens

This study characterized the chemical responses of Eucalyptus globulus and Eucalyptus nitens to artificial inoculation with a basidiomycete decay fungus. Nine-year-old trees responded to mechanical wounding or inoculation with the decay fungus by producing new wound wood characterized by the presence of dark extractives 17 months after wounding. Analysis of crude wound wood extracts by HPLC coupled to negative ion electrospray mass spectrometry revealed the presence of a complex mixture of many unidentified formylated phloroglucinol compounds (FPCs), in addition to a diverse range of other polyphenolic compounds (hydrolyzable tannins, proanthocyanidins, flavanone glycoside, stilbene glycosides). Prior to this study, FPCs have only been reported from leaves and buds of Eucalyptus spp. Unequivocal evidence for the presence of macrocarpal A and B, and sideroxylonal A and B in the crude extracts was obtained, as well as evidence for a wide range of as yet unreported FPCs. Subsequent preliminary in vitro fungal and bacterial bioassays did not support an antimicrobial role for FPCs in host-pathogen interactions in eucalypts.     

Phytogrowth Properties of Limonoids Isolated from Cedrela ciliolata

An epimeric mixture of the limonoid photogedunin, isolated from the heartwood of C. ciliolata, as well as its R- and S-acetate derivative were evaluated for phytotoxicity in mono- and dicotyledoneous plants. A mixture of epimeric photogedunin 1 and 2, a mixture of epimeric photogedunin acetate, (R)-photogedunin acetate 3, and (S)-photogedunin acetate 4 inhibited seed germination, seedling growth, and root and hypocotyl/coleoptyle growth in all species assayed. The concentration of phytochemicals required for 50% inhibition ranged from 4.5 to 300 M. Inhibitory plant responses appeared to require that the OH-group at C-23 be acetylated in photogedunin, since the nonacetylated compound showed less phytotoxic activity.     

(Z,Z)-4,7-tridecadien-(S)-2-yl acetate: sex pheromone of Douglas-fir cone gall midge,Contarinia oregonensis

Our objectives were to identify and field test the sex pheromone of female Douglas-fir cone gall midge, Contarinia oregonensis (Diptera: Cecidomyiidae). Coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses of pheromone extract revealed a single compound (A) that elicited responses from male antennae. Hydrogenation of pheromone extract, followed by renewed GC-EAD analysis, revealed a new EAD-active compound with chromatographic characteristics identical to those of tridecan-2-yl acetate on five fused silica columns (DB-5, DB-210, DB-23, SP-1000, and Cyclodex-B). Syntheses, chromatography, and retention index calculations of all possible tridecen-2-yl acetates suggested that the candidate pheromone A was a tridecadien-2-yl acetate with nonconjugated double bonds. Synthetic candidate pheromone component (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) cochromatographed with A on all analytical columns and elicited comparable antennal activity. In GC-EAD analyses that separated the enantiomers (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7) and (Z,Z)-4,7-tridecadien-(R)-2-yl acetate (2R-Z4Z7) with baseline resolution, only 2S-Z4Z7 as a component in a racemic standard or in pheromone extract elicited antennal responses. In Douglas-fir seed orchards, sticky traps baited with 2S-Z4Z7 captured male C. oregonensis, whereas 2R-Z4Z7 was behaviorally benign. Comparable catches of males in traps baited with racemic Z4Z7 (50 g) or virgin female C. oregonensis suggested that synthetic pheromone baits could be developed for monitoring C. oregonensis populations in commercial Douglas-fir seed orchards.     

Behavioral responses ofSpodoptera littoralis males to sex pheromone components and virgin females in wind tunnel

The major component of the sex pheromone of femaleSpodoptera littoralis, (Z,E)-9,11-tetradecadienyl acetate (1), elicited all steps of the male behavioral sequence, i.e., wing fanning and taking flight, oriented upwind flight and arrival to the middle of the tunnel, close approach and contact with the source. The activity was equivalent to that elicited by virgin females. In the range of doses tested, the dosage of1 had no significant effect on the number of source contacts. Male response was significantly affected by light intensity, being optimum at 3 lux. Activity of the minor components (Z)-9-tetradecenyl acetate (2), (E)-11-tetradecenyl acetate (3), tetradecyl acetate (4), (Z)-11-tetradecenyl acetate (5), and (Z,E)-9,12-tetradecadienyl acetate (6) was significantly lower than that of the major component when assayed individually. In multicomponent blends compound4 appeared to strongly decrease the number of males arrested at the source, the effect being particularly important when compound5 was present in the blend. Results of single sensillum experiments confirmed the existence of two main physiologically distinct sensillar types. The most common type of sensilla contained a neuron that responded specifically to compound1. A second type of sensilla, located laterally on the ventral sensory surface, contained two receptor neurons responding to compound6 and to (Z)-9-tetradecenol. Among short sensilla, one hair responded to compound4 and could represent a minor sensillar type. No sensory neuron was found to detect the other minor pheromone compounds2, 3, and5.Dedicated to the memory of the late Prof. Félix Serratosa (1925–1995).     

Insights into Removal of Phenol from Aqueous Solutions by Low Cost Adsorbents: Clays Versus Algae

Abstract

Exchanged clays and cross-linked algae were compared based on their properties for the removal of phenol from aqueous solutions. Algae Lessonia nigrescens Bory (A1) and Macrocystis integrifolia Bory (A2) were cross-linked with CaCl₂ to enhance their physical and mechanical properties. The natural clays were chemically-exchanged with salts of tetramethyl ammonium (B1), hexadecyltrimethyl ammonium (B2), and bencyltriethyl ammonium (B3) ions to increase their affinity towards organic substrates. The effects of pH and adsorbent dose were evaluated. pH exhibited a strong effect mainly on the phenol aqueous chemistry. Sorption isotherm results were modelled on the Langmuir and Freundlich equations and complemented with EDX analysis, indicating that adsorption of phenol from water was mostly driven by hydrophobic forces, with the exchanged bentonites being the adsorbents that reported the maximum adsorption capacities. Conversely, a polar surface adsorption is suggested for algae mostly by means of hydrogen bonding formation. These results provide further insight into the adsorption mechanism of phenol and analogues and their use as powerful and cheap adsorbent for the treatment of phenol-containing real wastewater.     

Chemical Defense in Harvestmen (Arachnida,Opiliones): Do Benzoquinone Secretions Deter Invertebrate and Vertebrate Predators?

Two alkylated 1,4-benzoquinones were identified from the defensive secretion produced by the neotropical harvestman Goniosoma longipes (Gonyleptidae). They were characterized as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. We tested the effectiveness of these benzoquinone secretions against several predator types, including invertebrates and vertebrates. Different predators were exposed to the harvestmen's gland secretion or to distilled water in laboratory bioassays. Our results indicate that secretions containing the 1,4-benzoquinones released by G. longipes can be an effective defense against predation, and that the effectiveness of the secretion is dependent on the predator type. The scent gland secretion repelled seven ant species, two species of large wandering spiders, and one frog species, but was not an effective defense against an opossum. Our study also demonstrates that the scent gland secretion of G. longipes can work as a chemical shield preventing the approach of three large predatory ants for at least 10 min. The chemical shield may protect the harvestman against successive attacks of the same ant worker and also allow the harvestman to flee before massive ant recruitment. Our data support the suggestion that chemical defenses may increase survival with some but not all potential predators. This variation in defense effectiveness may result from many interacting factors, including the attack strategy, size, learning ability, and physiology of the predators, as well as the chemical nature of the defensive compounds, type of emission, and amount of effluent released by the prey.     

Defensive mechanisms of loblolly and shortleaf pine against attack by southern pine beetle,Dendroctonus frontalis Zimmermann,and its fungal associate,Ceratocystis minor (Hedgecock) Hunt

Loblolly and shortleaf pine growing on a single site in the North Carolina piedmont were examined to determine similarities and differences in their defensive mechanisms against the southern pine beetle,Dendroctonus frontalis Zimmermann, and its fungal associate,Ceratocystis minor (Hedgecock) Hunt. Both species responded to wounding and fungal inoculation by forming a hypersensitive lesion around the wound site. There were significantly less soluble sugars and more monoterpenes in the lesion tissue than in unwounded inner bark. The two species were similar in resin flow rate and inner bark soluble sugar content, but the loblolly pines had thicker bark, longer hypersensitive lesions, and a higher concentration of inner bark monoterpenes. Inner bark monoterpene composition was also significantly different between the two pine species. It is hypothesized that two different defensive strategies against southern pine beetle attack may be utilized.