Chemical Defenses Against Fish Predation in Three Brazilian Octocorals: 11β,12β-Epoxypukalide as a Feeding Deterrent in Phyllogorgia dilatata

The feeding-deterrence properties of crude extracts of three Brazilian octocoral species, Neospongodes atlantica Kükenthal (Alcyonacea, Nephtheidae), Plexaurella regia Castro (Gorgonacea, Plexauridae), and Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae), were investigated. All the extracts were incorporated into food strips at the concentrations occurring in the living organisms. Crude extract and its ethyl acetate fraction obtained from P. dilatata collected in Armação dos Búzios (Rio de Janeiro State), when incorporated into artificial diets and tested in the habitat of origin, reduced consumption of food strips by fishes, relative to controls. Crude extracts from two octocoral species collected at the National Marine Park of Abrolhos (Bahia State), N. atlantica and P. regia, had no apparent feeding-deterrence properties; in fact, they seemed to stimulate feeding. Bioassay-guided fractionation of the bioactive P. dilatata crude extract revealed that the deterrent property was restricted to a medium polarity fraction. Field palatability experiments with two pure compounds isolated from this fraction revealed that the furanocembranolide 11,12-epoxypukalide is a potent feeding deterrent produced by P. dilatata against fish. Apparently, furanocembranolides are a particular class of compounds with feeding deterrent properties, protecting some octocorals from potential fish predator species in both tropical and temperate environments.     

Does the odor from sponges of the genus Ircinia protect them from fish predators?

Caribbean sponges of the genus Ircinia contain high concentrations of linear furanosesterterpene tetronic acids (FTAs) and produce and exude low-molecular-weight volatile compounds (e.g., dimethyl sulfide, methyl isocyanide, methyl isothiocyanate) that give these sponges their characteristic unpleasant garlic odor. It has recently been suggested that FTAs are unlikely to function as antipredatory chemical defenses, and this function may instead be attributed to bioactive volatiles. We tested crude organic extracts and purified fractions isolated from Ircinia campana, I. felix, and I. strobilina at naturally occurring concentrations in laboratory and field feeding assays to determine their palatability to generalist fish predators. We also used a qualitative technique to test the crude volatile fraction from I. felix and I. strobilina and dimethylsulfide in laboratory feeding assays. Crude organic extracts of all three species deterred feeding of fishes in both aquarium and field experiments. Bioassay-directed fractionation resulted in the isolation of the FTA fraction as the sole active fraction of the nonvolatile crude extract for each species, and further assays of subfractions suggested that feeding deterrent activity is shared by the FTAs. FTAs deterred fish feeding in aquarium assays at concentrations as low as 0.5 mg/ml (fraction B, variabilin), while the natural concentrations of combined FTA fractions were >5.0 mg/ml for all three species. In contrast, natural mixtures of volatiles transferred from sponge tissue to food pellets and pure dimethylsulfide incorporated into food pellets were readily eaten by fish in aquarium assays. Although FTAs may play other ecological roles in Ircinia spp., these compounds are effective as defenses against potential predatory fishes. Volatile compounds may serve other defensive functions (e.g., antimicrobial, antifouling) but do not appear to provide a defense against fish predators.     

Shield Defense of a Larval Tortoise Beetle

Larvae of the folivorous tortoise beetle, Plagiometriona clavata, carry shields formed from feces and exuviae above their bodies. We used an ecologically relevant predatory ant, Formica subsericea, in a bioassay to determine if shields functioned as simple barriers, as previous studies indicated, or whether they were chemical defenses. Shields were necessary for larval survival; shield removal rendered larvae vulnerable. Shields produced by larvae reared on a substitute diet failed to provide protection. Solvent-leached shields also failed to deter ants, indicating the shield had a host-derived chemical component likely located in the feces, not in the exuviae. Solanum dulcamara, the larval host plant, contained free phytol, steroidal glycoalkaloids, and saponins. Shields contained partially deglycosylated metabolites of host steroidal glycoalkaloids and saponins, a suite of fatty acids, and derivatives of phytol, which together formed a deterrent barrier against ant attack. We compared the mobile shield of P. clavata to the stationary shield of another S. dulcamara-feeding leaf beetle, Lema trilinea. Both larval shield defenses were formed from a very similar array of host-derived compounds with deterrent properties. We concluded that convergent patterns of limited chemical transformation and selective incorporation of particular deterrent metabolites in shield defenses of two unrelated taxa represented responses to selection from invertebrate predators.     

Leaf surface extracts ofSolanum berthaultii hawkes deter colorado potato beetle feeding

Leaf rinses ofS. berthaultii PI 473334 with methylene chloride were deterrent to feeding by the Colorado potato beetle when applied toS. tuberosum tuber and leaf disks. When the leaf rinse was separated into its nonvolatile and volatile fractions and applied to tuber disks, the nonvolatile fraction was highly deterrent, while the volatile fraction reduced consumption, but not significantly compared to the controls. A hexane leaf rinse was not deterrent to feeding, while an acetone rinse was approximately twofold more deterrent than the methylene chloride rinse when applied to leaf disks. Three cycles of bioassay-guided, reversed-phase open-column fractionation of an acetone leaf rinse yielded a relatively polar fraction with low deterrent activity, and two nonpolar fractions exhibiting higher specific activity. Reversed-phase preparative HPLC of these fractions yielded seven active fractions among the 10 assayed. Subsequent analytical HPLC indicated that two fractions each contained a single UV-absorbing compound, while another represented a mixture of at least four compounds. The remaining fractions were composed of complex mixtures of possibly ionic or polymeric compounds that were poorly resolved by HPLC.     

Phytotoxic and Antifungal Compounds from Two <Emphasis Type="Italic">Apiaceae</Emphasis> Species, <Emphasis Type="Italic">Lomatium californicum</Emphasis> and <Emphasis Type="Italic">Ligusticum hultenii</Emphasis>, Rich Sources of Z-ligustilide and Apiol,Respectively

The seeds of two Apiaceae species, Ligusticum hultenii and Lomatium californicum, were investigated. Preliminary bioassays indicated that methylene chloride extracts of seeds of both species contained selective phytotoxic activity against monocots and antifungal activity against Colletotrichum fragariae. Active constituents were isolated by bioassay-guided fractionation, and the structures were elucidated by NMR and GC-MS as apiol and Z-ligustilide, isolated from L. hultenii and L. californicum, respectively. Apiol and Z-ligustilide had I₅₀ values of about 80 and 600 μM, respectively, for inhibition of the growth of Lemna paucicostata. The methylene chloride (CH₂Cl₂) extracts of the seeds and the isolated and purified compounds were tested against the 2-methylisoborneol-producing cyanobacterium (blue-green alga) Oscillatoria perornata, and the green alga Selenastrum capricornutum. The CH₂Cl₂ extracts of both Apiaceae species and apiol were weakly toxic to both species of phytoplankton, while Z-ligustilide was toxic to both with a lowest complete inhibitory concentration (LCIC) of 53 μM. Seeds of L. californicum and L. hultenii were found to be rich sources of Z-ligustilide (97 mg/g of dry seed) and apiol (40 mg/g of dry seed), respectively.     

Defensive role ofAllium sulfur compounds for leek mothAcrolepiopsis assectella Z. (Lepidoptera) against generalist predators

It has been shown previously that sulfur volatiles produced byAllium plants affect the behavior of their specialist phytophages and of their specialist entomophages. The action of these compounds in protecting the leek mothAcrolepiopsis assectella against generalist entomophages was studied in comparison to the proposed original defensive role of these compounds against generalist herbivorous insects. Two ants species,Formica selysi andF. fusca, were used as generalist predators. Six behavioral criteria of the predatory behavior of the ants were studied in presence of the last-instar caterpillars (C₅). C₅ reared on artificial diets with or without leek components were tested, as well as C₅ soaked in frass of leek-reared caterpillars or disulfide solutions. In addition, the response of the ants to pure chemicals found in leek was studied using honey solutions with or without sulfur compounds. The sulfur allelochemicals ofAllium plants have a negative action on predatory ants. Interestingly, the nonvolatile precursors of sulfur volatiles ofAllium plants seem to have a protective role for their phytophagous insects against generalist entomophages.     

Responses of maxillary sensilla styloconica inBombyx mori to glucosides fromOsmunda japonica,a nonhost plant

We isolated glucosides from the royal fern,Osmunda japonica, which elicit a deterrent response in larvae ofBombyx mori. These compounds were absent in taro (Colocasia antiquorum) and castor-oil plant (Ricinus communis) leaves and did not evoke responses of sensory cells in the lateral and medial sensilla styloconica ofSpodoptera litura. This glucoside extract of the royal fern leaves stimulates receptors generally associated with deterrent. It is also possible that this compound may act as a behavioral deterrent, and from actual feeding tests, it is suggested that this compound may prevent feeding in some monophagous insects, such asBombyx mori. The deterrent glucoside possesses a noncyclic aglycon.     

Antifeedant and toxic effects of sesquiterpenes fromSenecio palmensis to colorado potato beetle

A bioassay-guided fractionation of the aerial parts ofSenecio palmensis resulted in the isolation of two sesquiterpenes, 2,10-bisaboladien-1-one and 11-acetoxy-5-angeloyloxy-silphinen-3-one. The bisabolene and the silphinene represented 0.012% and 0.024% of the plant dry weight, respectively. Both compounds showed antifeedant activity againstLeptinotarsa decemlineata larvae and adults in short-term choice and no-choice bioassays. Both compounds were also tested against different species of phytopathogenic fungi. The beetles were more sensitive to these compounds in choice than in no-choice assays, with a gradient of increasing sensitivity from second instars to adults. Bisabolene was 45 times less active as an antifeedant than juglone, which was tested as a positive control. The silphinen was more active than the bisabolene, with a range of activity similar to juglone. Furthermore, exposure of fourth instars to these compounds over a 24-hr period resulted in reduced feeding and growth rates. To distinguish between antifeedant and toxic effects, growth efficiencies were calculated as the slope of the regression of relative growth rate on relative consumption rate. The comparison of these results with those of antifeedant simulation and contact toxicity bioassays indicates that feeding inhibition is the primary mode of action of the bisabolene, while the silphinene shows both antifeedant and toxic effects.     

The Chlorophyll Catabolite,Pheophorbide <Emphasis Type="Italic">a</Emphasis>, Confers Predation Resistance in a Larval Tortoise Beetle Shield Defense

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 μg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 μg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 μg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 μg added Pha were comparable to intact shields, even though the Pha concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.     

Nerolidol: An antifeeding sesquiterpene alcohol for gypsy moth larvae fromMelaleuca leucadendron

A systematic procedure is reported for the isolation of a feeding deterrent, (E,S)-nerolidol (I), fromMelaleuca leucadendron leaves for the gypsy moth larvae. Testing of the related alcohols, farnesol (II) and geraniol (III) showed them to be deterrent, but the simpler isoprene-related compounds, 2-methyl-3-buten-2-ol andt-amyl alcohol were inactive.     

Synergistic Effects of Three Piper Amides on Generalist and Specialist Herbivores

The tropical rainforest shrub Piper cenocladum, which is normally defended against herbivores by a mutualistic ant, contains three amides that have various defensive functions. While the ants are effective primarily against specialist herbivores, we hypothesized that these secondary compounds would be effective against a wider range of insects, thus providing a broad array of defenses against herbivores. We also tested whether a mixture of amides would be more effective against herbivores than individual amides. Diets spiked with amides were offered to five herbivores: a naïve generalist caterpillar (Spodoptera frugiperda), two caterpillar species that are monophagous on P. cenocladum (Eois spp.), leaf-cutting ants (Atta cephalotes), and an omnivorous ant (Paraponera clavata). Amides had negative effects on all insects, whether they were naïve, experienced, generalized, or specialized feeders. For Spodoptera, amide mixtures caused decreased pupal weights and survivorship and increased development times. Eois pupal weights, larval mass gain, and development times were affected by additions of individual amides, but increased parasitism and lower survivorship were caused only by the amide mixture. Amide mixtures also deterred feeding by the two ant species, and crude plant extracts were strongly deterrent to P. clavata. The mixture of all three amides had the most dramatic deterrent and toxic effects across experiments, with the effects usually surpassing expected additive responses, indicating that these compounds can act synergistically against a wide array of herbivores.     

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.     

Structure–Activity Relationship of Inhibition of Fish Feeding by Sponge-derived and Synthetic Pyrrole–Imidazole Alkaloids

We investigated the relationship between the structures of pyrrole-containing alkaloids from marine sponges of the genus Agelas and their capacity to deter feeding by the omnivorous Caribbean reef fish, Thalassoma bifasciatum. Seven natural products were assayed at volumetric concentrations of 1, 5, and 10 mg/ml: dispacamide A, keramadine, oroidin, midpacamide, 4,5-dibromopyrrole-2-carboxylic acid, 4,5-dibromopyrrole-2carboxamide, and racemic longamide A. We also assayed 14 structural analogs obtained mostly by chemical synthesis. Of the seven natural products, only rac-longamide A was not significantly deterrent at any of the assay concentrations. The pyrrole moiety was required for feeding inhibition activity, while the addition of the imidazole group enhanced this activity. Variously functionalized imidazoles lacking the pyrrole moiety were not deterrent. Combinations of the natural products appeared to have an additive effect on feeding inhibition; there was no evidence of synergy. Given their high concentrations in sponge tissue, dispacamide A and oroidin most probably serve as the primary chemical defenses of many Agelas sp., while minor compounds such as keramadine are not present in high enough concentrations to contribute much to chemical defense.     

Vertebrate Herbivory on Eucalyptus—Identification of Specific Feeding Deterrents for Common Ringtail Possums (Pseudocheirus peregrinus) by Bioassay-Guided Fractionation of Eucalyptus ovata Foliage

Factors determining the acceptance of Eucalyptus ovata foliage by common ringtail possums (Pseudocheirus peregrinus) were studied. Bioassay-guided fractionation was used with foliage from both browser-susceptible and browser-resistant trees to identify the chemical components underlying the resistance. In foliage from browser-resistant trees, the deterrent principles were contained in the base-soluble fraction of the chloroform extract. Further fractionation of this material yielded polar and nonpolar fractions that contained acylphloroglucinol derivatives, and from the polar fraction we isolated macrocarpal G. Addition of this compound to an artificial diet at a concentration of 2.1% of dry matter resulted in a 90% reduction of voluntary food intake compared with solvent-treated controls. This is the first time that a specific compound in Eucalyptus has been shown to inhibit feeding of any marsupial folivore.     

Larval Beetles Form a Defense from Recycled Host-Plant Chemicals Discharged as Fecal Wastes

Larvae of the leaf-feeding beetles Neolema sexpunctata and Lema trilinea carry feces on their backs that form shields. We used the generalist predatory ant, Formica subsericea, in a bioassay to determine whether shields were a physical barrier or functioned as a chemical defense. Fecal shields protected both species against ant attack. Larvae of both species reared on lettuce produced fecal shields that failed to deter ants. Commelina communis, N. sexpunctata's host, lacks noxious secondary compounds but is rich in phytol and fatty acids, metabolites of which become incorporated into the fecal defense. In contrast, the host plant of L. trilinea, Solanum dulcamara, contains steroidal glycoalkaloids and saponins, whose partially deglycosylated metabolites, together with fatty acids, appear in Lema feces. Both beetle species make modifications to host-derived precursors before incorporating the metabolites into shields. Synthetic chemicals identified as shield metabolites were deterrent when applied to baits. This study provides experimental evidence that herbivorous beetles form a chemical defense by the elimination of both primary and secondary host-derived compounds. The use of host-derived compounds in waste-based defenses may be a more widely employed strategy than was hitherto recognized, especially in instances where host plants lack elaborate secondary compounds.     

Direct and mediated effects onBactrocera oleae (Gmelin) (Diptera; Tephritidae) of natural polyphenols and some of related synthetic compounds: Structure-activity relationships

Among the main polyphenols occurring in olive oil vegetation waters (VW), catechol showed the most deterrent action on the oviposition ofBactrocera oleae (Gmelin); 4-methylcatechol was less active, whereas hydroxytyrosol and tyrosol were inactive. In contrast, synthetico-quinone was found to be stimulant at 7.5 × 10^–2 M. Two other synthetic derivatives of catechol, diacetylcatechol and guaiacol, were also deterrent, suggesting these compounds undergo a biochemical transformation into catechol by means of the bacterial symbionts ofB. oleae. VW and their phenolic extracts showed deterrence only when highly concentrated, while natural olive juice was strongly deterrent. Experiments carried out to evaluate the effect of olive juice and catechol on the fecundity ofB. oleae showed that they strongly reduce this function. Moreover, the possible utilization of VW and their bioactive polyphenols in protection of olives againstBatrocera oleae is discussed.     

Insect feeding and oviposition deterrents from western red cedar foliage

The feeding deterrent activity of fractions from the foliage of western red cedar,Thujaplicata Donn, was studied in laboratory bioassays using the white pine weevil,Pissodes strobi Peck, as a test insect. The most active fraction was the volatile mixture that comprises the leaf oil of this tree species. Further fractionation of the leaf oil indicated feeding deterrent activity in the monoterpene hydrocarbon, thujone, and terpene alcohol fractions. When tested alone, both (–)-3-isothujone and (+)-3-thujone, which made up 75–88% and 5–10% of the leaf oil, respectively, deterred feeding by the weevils. Western red cedar leaf oil also showed antifeedant activity with the alder flea beetle,Altica ambiens (Le Conte), and served as an oviposition deterrent for the onion root maggot,Hylemya antiqua Meigen. The leaf oil, however, had no inhibitory effect on the feeding of the leaf roller,Epinotia solandriana L., and the red-backed sawfly,Eriocampa ovata L.Research supported by the Natural Science and Engineering Research Council, Canada, Co-op Grant No. A0243 and Operating Grant Nos. A3881 and A3706, and by the British Columbia Ministry of Labour Youth Employment Program.     

Antifeedant activity of extracts from neem,Azadirachta indica,to strawberry aphid,Chaetosiphon fragaefolii

Leaf disk choice test bioassays demonstrated that formulated neem seed oil (NSO) was equally deterrent to first- and third-instar nymphs and adult strawberry aphids,Chaetosiphon fragaefolii (Cockerell). Concentrations of NSO resulting in 50% feeding deterrence were approximately 1.1% for this species. The rapid disruption of aphid feeding (<1 hr) was not related to the presence of the limonoid azadirachtin, and deterrence likely results from the combined activity of several compounds. Activity toC. fragaefolii disappeared within 12–24 hr following application to strawberry in the greenhouse. NSO was deterrent to only half of the six aphid species tested. The antifeedant properties of neem do not appear to contribute significantly to the control of aphids and the viruses they transmit.     

Cardenolides as oviposition deterrents to twoPieris species: Structure-activity relationships

Oviposition responses ofPieris rapae andP. napi oleracea to 18 cardenolides were compared under the same conditions. Effects of different concentrations of selected cardenolides were also tested. Most of the compounds were deterrent to oviposition by both insects, but to significantly different degrees.P. rapae were strongly deterred by K-strophanthoside, K-strophanthin-, cymarin, convallatoxin, oleandrin, erysimoside, erychroside, and gitoxigenin. The most deterrent compounds forP. napi oleracea were erychroside, cymarin, erysimoside, convallatoxin, and K-strophanthoside. Strophanthidin-based glycosides were more deterrent than digitoxigenin-based ones, and the number and type of sugar substitutions can have profound effects on activity. Both similarities and contrasts were found in responses ofP. rapae andP. napi oleracea to these cardenolides. Cymarin was equally deterrent to bothPieris species at all concentrations tested. However, when compared withP. rapae, P. napi oleracea was less sensitive to most of the cardenolides.P. napi oleracea was insensitive to K-strophanthin- and oleandrin at 0.5 × 10^–4 M, which were highly deterrent toP. rapae.     

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.