Algal phagostimulants for marine herbivorous gastropods

Methanol extracts of the green algaUlva pertusa contain four kinds of glycerolipids that are active as feeding-stimulants for marine herbivorous gastropods. These compounds are digalactosyldiacylglycerol (DGDG), 1,2-diacylglycerly-4-O-(N,N,N-trimethyl)-homoserine (DGTH), 1-monoacylglyceryl-4'-O-(N,N,N-trimethyl)-homoserine (MGTH), and 6-sulfoquinovosyldiacylglycerol (SQDG). The various gastropods exhibit marked specificity, however, as young abaloneHaliotis discus respond to DGDG and DGTH at minute dosages of 20–30 g/sample zone, but do not respond to 300 g of SQDG, which is a phagostimulant for two other kinds of gastropods,Turbo comutus andOmphalius pfeifferi.Chemical Studies on Phagostimulants for Marine Gastropods. Part VI. For Part V, see Sakata et al. (1986b).     

Biocidal activity of algal toxins against immature mosquitoes

Extracts of two species of green algae, filamentousRhizoclonium hieroglyphicum Kütz and a phytoplankton,Chlorella ellipsoidea Gerneck, obtained with solvents in the laboratory were assayed againstAedes aegypti L.,Culex quinquefasciatus Say, andCuliseta incidens (Thomson). On extraction with petroleum ether, groundR. hieroglyphicum yielded an active crude extract which was chromatographed on a neutral alumina column and eluted consecutively with petroleum ether, benzene, and methanol. All three eluted fractions were found to induce significant mortality in test mosquito species. The benzene-eluted fraction was the least toxic. The methanol-eluted fraction was the most toxic to all species and exhibited juvenile hormone-like activity; it also caused morphogenetic changes in emerging adults. All three fractions delayed the rate of development of mosquito larvae by 2–5 days. Three supernatants ofC. ellipsoidea obtained on different occasions were tested against first instars ofC. quinquefasciatus. After the confirmation of their activity, all supernatants were extracted with diethyl ether, combined, and assayed against first- and fourth-stage larvae of the three mosquito species. The first-stage larvae ofC. quinquefasciatus andC. incidens were approximately three times more suspectible than those ofA. aegypti. However, fourth-stage larvae of the former two species were about twice as susceptible to the extract as those of the latter species. Dead first-stage larvae of all the species had a shrunken appearance. In general,C. ellipsoidea extract was quicker acting than that ofR. hieroglyphicum.     

Phytochemical basis of learning inRhagoletis pomonella and other herbivorous insects

Examples of phytochemically-based learning of host preference in herbivorous insects are reviewed in the context of traditionally important issues: the number and kinds of chemicals involved; which sensory modalities are affected; whether peripheral or central nervous processing is altered; and whether learning is associative or not. A fifth issue addressed here— whether experience enhances a feeding or ovipositing insect's propensity to accept familiar chemical stimuli or to reject novel chemical stimuli-has been ignored in previous studies. Following the review, evidence is presented indicating that female apple maggot flies (Ragoletis pomonella) learn to reject both novel physical and novel chemical stimuli.     

Polyphenols in brown algaeFucus vesiculosus andAscophyllum nodosum: Chemical defenses against the marine herbivorous snail,Littorina littorea

Polyphenols from two brown algae,Fucus vesiculosus (L.) andAscophyllum nodosum (L.) Le Jolis, inhibited feeding by the herbivorous snail,Littorina littorea. The active compounds were characterized as phloroglucinol polymers with a wide molecular weight range (mol wt <30,000 to >300,000) by spectroscopic, Ultrafiltration, thin-layer chromatographic, and chemical degradation data. As little as 1% (dry wt) polyphenol in food reduced feeding by more than 50%, and polyphenolic extracts inhibited feeding entirely when present in concentrations of 2–5% (dry wt). Commercially available phloroglucinol dihydrate and gallotannin, which are known herbivore feeding deterrents in terrestrial plants, inhibitedL. littorea feeding when added to food media in concentrations similar to those above. We conclude that polyphenols inF. vesiculosus andA. nodosum are functionally similar to terrestrial plant polyphenols (tannins) in providing chemical defenses against herbivores. This research is the first demonstration that chemical compounds defend these two dominant, perennial marine algae from the major herbivore found in their community.     

Comparison of three methods for quantifying brown algal polyphenolic compounds

Three methods of quantifying total phenolic compounds in marine brown algae were compared for their ability to measure accurately known concentrations of phloroglucinol in the presence of a protein, bovine serum albumin (BSA). These methods were: (1) a Folin-Ciocalteu assay for compounds dissolved in 80% methanol, (2) a Folin-Ciocalteu assay for compounds dissolved in 75% methanol-25% trichloroacetic acid, and (3) an assay in which polyvinylpolypyrrolidone (PVPP) was used to remove phenolic compounds in order to quantify nonphenolic, Folin-Ciocalteu-reactive materials. The Folin-Ciocalteu assay used with compounds dissolved in 80% methanol was the method that produced the most consistent results. It was also the assay in which the presence of proteins interfered least with the measurements of phloroglucinol and the assay that was easiest to conduct. The presence of TCA decreased the pH of the extracting solution and increased the amounts of proteins in solution. These proteins later interfered with the Folin-Ciocalteu reaction. TCA effectively bound proteins when protein concentrations were low but not when protein concentrations were high. The amount of phloroglucinol removed by PVPP varied widely and was dependent on phloroglucinol concentration, making this an unreliable method for quantifying phenolic concentrations. The Folin-Ciocalteu assay will measure some nonphenolic substances when brown algae are extracted in 80% methanol; however, inFucus gardneri, nonphenolic substances were at most 5% of the material quantified by the assay.     

Photocatalytic inactivation of algal growth in eutrophic water with hollow glass beads

Photocatalytic inactivation of algae,Anabaena, Microcystis, andMelosira, was performed with TiO₂-coated pyrex glass beads under the illumination of UV light (370 nm wavelength). After being irradiated with UV light in the presence of the TiO₂-coated pyrex glass beads,Anabaena andMicrocystis, known as typical cyanobacteria, lost their photosynthetic activity, and the string ofAnabaena cells and the colonies ofMicrocystis cells were completely separated into individual spherical ones. In the case ofMelosira, which is a typical diatom, however, somewhat lower photocatalytic inactivation efficiency was obtained, which was believed to be due to the presence of the inorganic siliceous wall surrounding the cells ofMelosira.     

Light-mediated allelochemical effects of naturally occurring polyacetylenes and thiophenes from asteraceae on herbivorous insects

Polyacetylenes and their thiophene derivatives, characteristic secondary metabolites of the Asteraceae, were examined for their effects on herbivorous insects. Three thiophenes (a monothiophene, a bithiophene, and -terthienyl) and four polyacetylenes (phenylheptatriyne, phenylheptadiynene, phenylheptadiyene acetate, and matricaria lactone) were studied for their phototoxicity and light-independent toxicity to (1) a polyphagous lepidopteran,Ostrinia nubilalis, whose host range includes a number of phototoxic Asteraceae, (2) a polyphagous lepidoteran,Euxoa messoria, whose host range includes very few species of Asteraceae, and (3) an oligophagous lepidopteran,Manduca sexta, which is a specialist on Solanaceae. Several compounds were phototoxic toM. sexta andE. messoria even at very low irradiance levels, but behavioral adaptations, including spinning silk and boring into diet, allowedO. nubilalis to avoid photosensitization. Light-independent activity of the compounds to all three species involved feeding deterrence increasing in the orderO. nubilalis, E. messoria, andM. sexta, and longterm metabolic toxicity in the form of impaired nutrient utilization. The biosynthetically derived thiophenes were more toxic than their acetylenic precursors, and toxicity increased with increasing number of thiophene rings. The results are discussed in terms of plant-insect coevolution.     

Alarm response of the marine mud snail,Nassarius obsoletus: Specificity and behavioral priority

Nassarius obsoletus, N. vibex, andN. trivittatus were exposed to juices of conspecifics and taxonomically and ecologically related gastropod species. Conspecific juices caused the strongest alarm responses, while those from taxonomically related species (congeners) induced less pronounced responses. Juices from nonrelated species induced no alarm response with one notable exception, the strong alarm response ofN. trivittatus toUrosalpinx cinerea juices. Nonrelated gastropod species induced stronger feeding responses than more closely related species. The strongest feeding responses, however, were induced by juices from nongastropod mollusks. The apparent balance between feeding attraction to less related species and alarm response to more related species was further examined by manipulating the snails' diet. Starvation resulted in a great reduction of conspecific alarm responsiveness. Following the resumption of feeding, the original responsiveness was again observed. Although the alarm response is likely an antipredator device, conclusive evidence for its effectiveness in reducing predation is lacking. One of the predators ofNassarius obsoletus which may be responsible for maintenance of an alarm response is the green crab,Carcinus maenas.This paper is based in part upon a thesis submitted by D.S. to Boston University for the degree of Master of Arts.     

Genetic Characterization of Five Hatchery Populations of the Pacific Abalone (Haliotis discus hannai) Using Microsatellite Markers

The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient F(IS) ranged from 0.054 to 0.184 (mean F(IS) = 0.121 ± 0.056). The genetic differentiation across all populations was low but significant (overall F(ST) = 0.009, P < 0.01). Pairwise multilocus F(ST) tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry.     

Invert emulsion as a medium for fungal lipase activity

An extracellular lipase from the fungusPythium ultimum was active in an invert [water-in-oil] emulsion consisting of 4% water emulsified into edible oils with taurocholic acid as the surfactant. The pH range for optimum lipolytic activity was 7.5–8.5, and the optimum temperature for activity was 45°C. Specific activity of the purified lipase was 919.5 μmol/min/mg protein in the invert emulsion. Water content of the invert emulsion influenced activity of the lipase differently, depending on the substrate. The rate of olive oil hydrolysis with thePythium lipase decreased with time, possibly due to inactivation of the enzyme and inhibition by free fatty acid products of the reaction. Total hydrolysis of olive oil by thePythium lipase was compared with that by lipases fromCandida rugosa andRhizopus arrhizus in the invert emulsion. Hydrolysis essentially ceased within 24 h or less for the lipases from each source. However, the addition of aqueous solution at 8 h from the beginning of incubation stimulated hydrolysis byC. rugosa andR. arrhizus lipases by 1.8-fold and 2.5-fold, respectively, but not by theP. ultimum lipase, over corresponding controls after 48 h.     

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.     

Antifungal activity of volatile fractions of essential oils from four aromatic wild plants in israel

Essential oils were extracted fromMajorana syriaca, Satureja thymbra, Micromeria fruticosa, andSalvia triloba, and their volatile fractions were tested for their antifungal activity against the soil-borne pathogensFusarium oxysporum andMacrophomina phaseolina and the foliar pathogensBotrytis cinerea andExserohilum turcicum. Results showed a fungistatic effect of 1, 2.5, and 5l of the various essential oils on fungal mycelium growth. The most significant effect was exerted by essential oils extracted fromM. syriaca, which inhibited the growth ofB. cinerea by 44% and of all the other fungi tested by 100%.Contribution from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel. No. 3555-E, 1992 series.     

Enzymatic adaptations of herbivorous insects and mites to phytochemicals

A variety of oxidases, reductases, esterases, epoxide hydrolases, and group transferases in herbivorous insects and mites detoxify and facilitate the excretion of toxic phytochemicals (allelochemicals). Current theory indicates that the cytochrome P-450-dependent mixed-function oxidases (MFOs) are by far the most important enzymes because they have many attributes that are essential for an effective detoxification system. Data presented here on the midgut microsomal MFO activity of larvae of the gypsy moth,Lymantria dispar, are discussed in the light of previous work and support the theory. In the gypsy moth, the MFO levels exhibit a parallel trend with changes in specific feeding rates, and changes in the specific activity of the enzyme appear to be regulated ontogenetically and by inductive effect of chemicals in the diet. The specific activity of the MFOs rises more sharply on leaves of a highly preferred type-1 plant, the pin oak, than on an artificial wheat germ diet; the increase from mid-second instar to mid-fifth is 4.5- and 1.8-fold, respectively. The relationship of food consumption rate to increase in body mass (W) was slightly in excess of a 11 ratio for both pin oak and the artificial diet, indicating that the feeding rate surpasses the increase in W (a rare phenomenon in insects). Moreover, the surface-to-volume ratios are fairly constant for combined data of gut lumen and epithelium in second to fifth instars, because the volume occupied by the epithelial cells is much larger than in older ones. Thus, it is concluded that greater specific activity of the MFO is necessary with larval advancement to higher instars in order that they may process dietary allelochemicals with an efficiency comparable to younger larvae. Additional data suggest that MFO level increases reflect further adaptation to: (1) normal, seasonal changes in plants' allelochemical composition and concentration; (2) increase in allelochemical concentration in response to leaf damage; and (3) the risk faced by dispersing larvae of encountering a greater amount and variety of allelochemicals on suboptimal/ less suitable plants. Evidence also has emerged recently for MFO-catalyzed metabolism/deactivation of numerous plant allelochemicals, including compounds that induce the enzyme. MFOs are further adapted for participation in the biogenesis of substances physiologically important to insects. Moreover, the catalytic center of the MFO system, cytochrome P-450, occurs in multiple forms; the significance of this important feature is discussed.Paper presented at the Symposium Bioorganic Chemistry of Communication Systems, at the NERM-15 ACS meeting, SUNY College at New Paltz, New York, June 1985.     

Synthesis and pheromonal activity of 6,10,13-trimethyl-1-tetradecanol for predatory stink bug,Stiretrus anchorago (Heteroptera: Pentatomidae)

Racemic 6,10,13-trimethyl-1-tetradecanol has been synthesized. Successive elaboration of the molecule using 5-methyl-2-hexanone as the carbon-terminal fragment gave the desired alcohol in a 4% overall yield over 13 steps. Neglecting stereochemistry, the product was shown to be identical to the alcohol previously identified from the sternal glands of maleStiretrus anchorago. It attracts both adult sexes and larvae of this insect in the field, confirming its postulated identity as an aggregation pheromone. The corresponding aldehyde is more than 10 times more concentrated in airbornetrapped samples (5–7% by gas chromatography) than in extracts of glandular setae (0.4–0.5%). This alcohol or its isovalerate ester has been found in other predaceous pentatomids, providing further evidence for pheromones in these species.     

Concentration and preliminary characterization of a chemical attractant of the oyster drill,Urosalpinx cinerea

Predatory muricid gastropods,Urosalpinx cinerea, respond to specific chemical stimuli by creeping upcurrent. Attractant substances originate from living barnacles. Newly hatched snails have no prior predatory experience but respond strongly to attractants. We report here methods for rapidly extracting and desalting attractants from seawater. Attractants from living barnacles are relatively large, at least partially proteinaceous, heat-stable molecules (> 1000 but < 10000 dallons) that adsorb onto Amberlite XAD-7, a polyacrylate water purification resin, at neutral pH. Attractants remain adsorbed to the resin during a wash with deionized water and can be eluted in a small volume with 100% methanol. Attractant substances are effective in the bioassay in g/liter concentrations (octa- to nanomolar range). Potency is destroyed by nonspecific proteases (carboxy-peptidase and pronase) but not by trypsin. Attractant is not sequestered within barnacles.     

Advanced techniques for nanocharacterization of polymeric coating surfaces

Surface properties of a polymeric coating system have a strong influence on its performance and service life. However, the surface of a polymer coating may have different chemical, physical, and mechanical properties from the bulk. In order to monitor the coating property changes with environmental exposures from the early stages of degradation, nondestructive techniques with the ability to characterize surface properties with micro- to nanoscale spatial resolution are required. In this article, atomic force microscopy has been applied to study surface microstructure and morphological changes during degradation in polymer coatings. Additionally, the use of AFM with a controlled tip-sample environment to study nanochemical heterogeneity and the application of nanoindentation to characterize mechanical properties of coatings surfaces are demonstrated. The results obtained from these nanometer characterization techniques will provide a better understanding of the degradation mechanisms and a fundamental basis for predicting the service life of polymer coatings. Presented at the 81st Annual Meeting of the Federation of Societies for Coatings Technology on November 12–14, 2003, in Philadelphia, PA.     

Alarm substance of the marine mud snail,Nassarius obsoletus: Biological characterization and possible evolution

The gastropod snailNassarius obsoletus shows a dramatic self-burial response to the presence of crushed conspecifics. After it was shown that this burial alarm response could be reliably replicated in laboratory tests, a further characterization of the alarm substance was undertaken. Dilution experiments showed a very high response threshold resulting in a short effective radius of the substance in agreement with earlier field reports. Longevity experiments showed that the substance had lost some activity after 16 hr standing over marsh mud in sea water at room temperature; it became inactive after 24 hr. Superthreshold concentration in sea water was not necessary to keep the snails buried: Mud apparently provides an adsorption surface which can remain a stimulus source for previously unalarmed snails, and snails tend to remain buried after a short exposure to alarm substance, even when given a fresh environment. The substance is present in the snail's blood and tissues and is passively released. A potential natural predator capable of such release isCarcinus maenas, the green crab. Predator odor alone did not cause burial alarm responses. Preliminary chemical analysis indicates that the substance is water soluble, heat stable, and of high apparent molecular weight (over 100,000). A comparison with fish alarm substance and response is made in a discussion of the possible evolution of chemically triggered alarm responses. It is argued thatN. obsoletus may have developed an alarm response to an existing nonspecific substance rather than a true alarm pheromone.This paper is based in part upon a thesis submitted by D.S. to Boston University for the degree of Master of Arts.     

Insecticidal activity of hypericin towardsManduca sexta larvae

The toxic effect of hypericin (HYP), a photodynamic quinone that occurs in certain species of the genusHypericum, towardsManduca sexta third-instar larvae was investigated. The LD₅₀ of purified hypericin was 16 g/g larval initial fresh weight in constant light (22 W/m²). Reduced irradiance resulted in decreased mortality. Sublethal applications retarded larval growth (body fresh weight) in a dose-dependent manner. Toxicity had an absolute light dependence at the dose levels used, the active wavelengths being >500 nm. The phototoxic effect was rapidly lost when larvae were maintained in darkness (>8 hr) prior to irradiation. However, the potential for light-dependent mortality was retained if larvae were starved prior to irradiation.     

Homarine as a feeding deterrent in common shallow-water antarctic lamellarian gastropodMarseniopsis mollis: A rare example of chemical defense in a marine prosobranch

The common bright yellow antarctic lamellarian gastropodMarseniopsis mollis was examined for the presence of defensive chemistry. Proton nuclear magnetic resonance (NMR) spectroscopy indicated that a major component of ethanolic extracts purified by reversed-phase column chromatography was homarine. Further high-performance liquid chromatography (HPLC) analysis of the mantle, foot, and viscera verified the presence of homarine in all body tissues at concentrations ranging from 6 to 24 mg/g dry tissue. A conspicuous macroinvertebrate predator of the shallow antarctic benthos, the sea starOdontaster validus, always rejected live individuals ofM. mollis, while readily feeding on pieces of fish tail muscle. Filter paper disks treated with shrimp elicited a broad range of feeding behaviors in the sea starO. validus (movement of disc to mouth, extrusion of cardiac stomach, humped feeding posture). Shrimp disks treated with homarine (0.4 and 4 mg/disk) were rejected byO. validus significantly more frequently than control disks treated with solvent carrier and shrimp or shrimp alone. The highest concentration of homarine tested not only caused feeding deterrence, but in several sea stars a flight response was noted. Homarine was not detected in the tunic of the antarctic ascidianCnemidocarpa verrucosa, a presumed primary prey ofM. mollis. Nonetheless, crude extracts of the epizooites that foul the tunic (primarily the bryozoans and hydroids) contain homarine, suggestingM. mollis may ingest and derive its chemistry from these organisms. This appears to be only the third example of chemical defense in a member of the Order Mesogastropoda. As the vestigial internalized shell ofM. mollis is considered a primitive condition, the findings of this study lend support to the hypothesis that chemical defense evolved prior to shell loss in shell-less gastropods.     

Biological activity of the shrubBoscia senegalensis (PERS.) LAM. ex Poir. (Capparaceae) on stored grain insects

Biological activity of leaves, fruits and extract of the African shrubBoscia senegalensis (PERS.) LAM. ex Poir. was evaluated against five stored-grain insects. When added to cowpeas at 2–4% (w/w), fresh ground fruits and leaves caused 80–100% mortality inCallosobruchus maculants (F.) adults and significantly reduced both emergence and damage of the F₁ progeny. Acetone fruit extract exhibited a potent fumigant effect onProstephanus truncatus Horn, C.maculatus, andSitotroga cerealella OLIV.; with LT₅₀ values of 3.8, 2.3, and below 1.5 hr, respectively. LC₅₀ determination forB. senegalensis fruits and leaves as well as pure methylisothiocyanate (MITC) onTribolium castaneum HERBST,Sitophilus zeamais MOTSCH. andC. maculatus showed a differential response of the insects to plant parts or MITC. Quantitative dosage ofBoscia active components and LC₅₀ values obtained for the plant tissues, compared to those of pure molecules, indicate that the biological activity ofB. senegalensis is due to the liberation of MITC from a glucosinolate precursor glucocapparin contained inBoscia fruits and leaves.