Prevalence of Chemical Defenses among Freshwater Plants

Although macrophyte–herbivore interactions in freshwater systems were generally disregarded for many years, recent data suggest that herbivory can be intense and important in structuring freshwater communities. This has led to the hypothesis that chemical defenses should be common among freshwater plants, but few studies have reported such chemical defenses, and no previous studies have assessed the frequency of chemical defenses among a substantial number of freshwater plant species. In a study of 21 macrophyte species co-occurring with the omnivorous crayfish Procambarus acutus in a southeastern USA wetland environment, we found that extracts of 11 species (52%) deterred feeding by P. acutus when tested in artificial foods at natural concentrations. Of these 11 chemically defended species, one species, Eupatorium capillifolium, consistently had a more unpalatable extract following mechanical damage to plant tissue, indicative of an activated chemical defense. Because herbivores are commonly nitrogen-limited and select food based on several plant traits, including plant nutritional value, it might be expected that chemical defenses would be especially important for protein-rich plants. However, we found no relationship between soluble protein concentration and deterrence of plant extracts.     

Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.     

Chemical Defenses Promote Persistence of the Aquatic Plant Micranthemum umbrosum

Five of the most common macrophytes from an aquaculture facility with high densities of the herbivorous Asian grass carp (Ctenopharyngodon idella) were commonly unpalatable to three generalist consumers—grass carp and the native North American crayfishes Procambarus spiculifer and P. acutus. The rooted vascular plant Micranthemum umbrosum comprised 89% of the total aboveground plant biomass and was unpalatable to all three consumers as fresh tissues, as homogenized pellets, and as crude extracts. Bioassay-guided fractionation of the crude extract from M. umbrosum led to four previously known compounds that each deterred feeding by at least one consumer: 3,4,5-trimethoxyallylbenzene (1) and three lignoids: β-apopicropodophyllin (2); (−)-(3S,4R,6S)-3-(3′,4′-methylenedioxy-α-hydroxybenzyl)-4-(3″,4″-dimethoxybenzyl)butyrolactone (3); and (−)-hibalactone (4). None of the remaining four macrophytes produced a chemically deterrent extract. A 16-mo manipulative experiment showed that the aboveground biomass of M. umbrosum was unchanged when consumers were absent, but the biomass of Ludwigia repens, a plant that grass carp preferentially consumed over M. umbrosum, increased over 300-fold. Thus, selective feeding by grass carp effectively eliminates most palatable plants from this community and promotes the persistence of the chemically defended M. umbrosum, suggesting that plant defenses play critical yet understudied roles in the structure of freshwater plant communities.     

Chemical Defenses of Cryptic and Aposematic Gastropterid Molluscs Feeding on their Host Sponge Dysidea granulosa

Numerous opisthobranchs are known to sequester chemical defenses from their prey and use them for their own defense. Information on feeding biology is critical for understanding the ecology and evolution of molluscs, yet information on feeding biology is still scarce for many groups. Gastropterid molluscs are often found on sponges, but there is controversy as to whether they are true sponge feeders. On Guam, we found the gastropterids Sagaminopteron nigropunctatum and S. psychedelicum on the sponge Dysidea granulosa. They seem to rely on contrasting defense strategies as S. psychedelicum has vivid colors, consistent with the warning coloration found in many chemically defended opisthobranchs, whereas S. nigropunctatum is highly cryptic on the sponge. S. nigropunctatum is avoided by the pufferfish Canthigaster solandri in aquarium assays. We analyzed the secondary metabolites of the two species and found that both share polybrominated diphenyl ethers (BDEs) with their host sponge D. granulosa. S. psychedelicum and S. nigropunctatum sequester the major BDE in the sponge and accumulate it in the mantle at approximately the same concentration as in the sponge (4.03 and 2.37%, respectively), and concentrate it in their parapodia at over twice the sponge concentration (7.97 and 10.10%, respectively). We also detected trace amounts in the mucus secretion of S. psychedelicum, and quantified significant amounts in the mucus (1.84%) and egg masses (2.22%) of S. nigropunctatum. Despite contrasting color patterns displayed by the two gastropterid species, they seem to share a similar chemical defense strategy, i.e., they feed on D. granulosa and accumulate the major BDE of the sponge in their tissues.     

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.     

Geographic and Seasonal Variation in Alkaloid-Based Chemical Defenses of Dendrobates pumilio from Bocas del Toro, Panama

Poison frogs contain an alkaloid-based chemical defense that is derived from a diet of certain alkaloid-containing arthropods, which include mites, ants, beetles, and millipedes. Variation in population-level alkaloid profiles among species has been documented, and more than 800 different alkaloids have been identified. In the present study, we examine individual alkaloid variation in the dendrobatid poison frog Dendrobates pumilio among seven populations and between two seasons on Isla Bastimentos, located in the Bocas del Toro archipelago of Panama. Alkaloid profiles vary among populations and between seasons, illustrating that chemical defense in this species can vary on a small spatial and temporal scale. Alkaloid variation among populations is marginally correlated with geographic distance, and close populations have profiles more similar to each other than to distant populations. Individuals within populations also vary in alkaloid profiles. Differences are attributed to both spatial and temporal variations in the availability of alkaloid-containing arthropods. Many of the alkaloids present in the skin of D. pumilio appear likely to be of ant origin, supporting the importance of myrmecophagy in chemical defense among poison frogs. However, a variety of frog skin alkaloids was recently detected in mites, suggesting that mites may also play an important role in chemical defense.     

Defoliation Affects Chemical Defenses in All Plant Parts of Rye Seedlings

The effect of defoliation and consecutive defoliation (condefoliation) of rye seedlings on the allocation patterns of biomass and hydroxamic acids (Hx) was evaluated five days after treatment. Growth of condefoliated seedlings was lower than that of defoliated and nondefoliated ones. Concentration of Hx decreased in shoots of condefoliated seedlings compared to nondefoliated ones, while concentration of Hx in root exudates increased. Allocation of Hx to roots and root exudates increased at the expense of allocation to the shoots in condefoliated seedlings. The ratio of Hx-aglucone to Hx-glucoside was higher in shoots of defoliated and condefoliated seedlings. The decrease in quantity of defense in shoots was accompanied by an increase in its quality, given that aglucones are more toxic than glucosides. The increase in concentration of Hx—an allelopathic compound also involved in mineral uptake by roots—in root exudates of condefoliated seedlings was suggested to lead to an advantage in the acquisition of resources for the regeneration of lost biomass.     

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

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

Inter- and Intraspecific Comparisons of Antiherbivore Defenses in Three Species of Rainforest Understory Shrubs

Plants defend themselves against herbivores and pathogens with a suite of morphological, phenological, biochemical, and biotic defenses, each of which is presumably costly. The best studied are allocation costs that involve trade-offs in investment of resources to defense versus other plant functions. Decreases in growth or reproductive effort are the costs most often associated with antiherbivore defenses, but trade-offs among different defenses may also occur within a single plant species. We examined trade-offs among defenses in closely related tropical rain forest shrubs (Piper cenocladum, P. imperiale, and P. melanocladum) that possess different combinations of three types of defense: ant mutualists, secondary compounds, and leaf toughness. We also examined the effectiveness of different defenses and suites of defenses against the most abundant generalist and specialist Piper herbivores. For all species examined, leaf toughness was the most effective defense, with the toughest species, P. melanocladum, receiving the lowest incidence of total herbivory, and the least tough species, P. imperiale, receiving the highest incidence. Although variation in toughness within each species was substantial, there were no intraspecific relationships between toughness and herbivory. In other Piper studies, chemical and biotic defenses had strong intraspecific negative correlations with herbivory. A wide variety of defensive mechanisms was quantified in the three Piper species studied, ranging from low concentrations of chemical defenses in P. imperiale to a complex suite of defenses in P. cenocladum that includes ant mutualists, secondary metabolites, and moderate toughness. Ecological costs were evident for the array of defensive mechanisms within these Piper species, and the differences in defensive strategies among species may represent evolutionary trade-offs between costly defenses.     

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.     

Expression of Constitutive and Inducible Chemical Defenses in Native and Invasive Populations of <Emphasis Type="Italic">Alliaria petiolata</Emphasis>

The Evolution of Increased Competitive Ability (EICA) hypothesis posits that invasive plants in introduced habitats with reduced herbivore pressure will evolve reduced levels of costly resistance traits. In light of this hypothesis, we examined the constitutive and inducible expression of five chemical defense traits in Alliaria petiolata from four invasive North American and seven native European populations. When grown under common conditions, significant variation among populations within continents was found for trypsin inhibitors and peroxidase activity, and glucosinolates and trypsin inhibitors were significantly jasmonate-inducible across populations. Across populations, constitutive levels of glucosinolates and trypsin inhibitors were negatively correlated with their degree of induction, with three North American populations tending to have lower constitutive levels and higher inducibility of glucosinolates than the seven European populations. Alliarinoside and isovitexin 6″-O-β-d-glucopyranoside levels were both higher in North American plants than in European plants, but levels of these compounds were generally increased by jasmonate in European plants and decreased by the same treatment in North American plants. Aside from the tendency for invasive populations to have reduced constitutive glucosinolate levels coupled with increased inducibility, little support for the predictions of EICA was evident in the chemical defenses that we studied.     

Chemical Composition of Inks of Diverse Marine Molluscs Suggests Convergent Chemical Defenses

Some marine molluscs, notably sea hares, cuttlefish, squid, and octopus, release ink when attacked by predators. The sea hare Aplysia californica releases secretions from the ink gland and opaline gland that protect individuals from injury or death from predatory spiny lobsters through a combination of mechanisms that include chemical deterrence, sensory disruption, and phagomimicry. The latter two mechanisms are facilitated by millimolar concentrations of free amino acids (FAA) in sea hare ink and opaline, which stimulate the chemosensory systems of predators, ultimately leading to escape by sea hares. We hypothesize that other inking molluscs use sensory disruption and/or phagomimicry as a chemical defense. To investigate this, we examined concentrations of 21 FAA and ammonium in the defensive secretions of nine species of inking molluscs: three sea hares (Aplysia californica, Aplysia dactylomela, Aplysia juliana) and six cephalopods (cuttlefish: Sepia officinalis; squid: Loligo pealei, Lolliguncula brevis, Dosidicus gigas; octopus: Octopus vulgaris, Octopus bimaculoides). We found millimolar levels of total FAA and ammonium in these secretions, and the FAA in highest concentration were taurine, aspartic acid, glutamic acid, alanine, and lysine. Crustaceans and fish, which are major predators of these molluscs, have specific receptor systems for these FAA. Our chemical analysis supports the hypothesis that inking molluscs have the potential to use sensory disruption and/or phagomimicry as a chemical defense.     

Chemical Deterrence of a Cyanobacterial Metabolite against Generalized and Specialized Grazers

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

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

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

Chemical Defenses of the Sacoglossan Mollusk Elysia rufescens and Its Host Alga Bryopsis sp.

Sacoglossans are a group of opisthobranch mollusks that have been the source of numerous secondary metabolites; however, there are few examples where a defensive ecological role for these compounds has been demonstrated experimentally. We investigated the deterrent properties of the sacoglossan Elysia rufescens and its food alga Bryopsis sp. against natural fish predators. Bryopsis sp. produces kahalalide F, a major depsipeptide that is accumulated by the sacoglossan and that shows in vitro cytotoxicity against several cancer cell lines. Our data show that both Bryopsis sp. and Elysia rufescens are chemically protected against fish predators, as indicated by the deterrent properties of their extracts at naturally occurring concentrations. Following bioassay-guided fractionation, we observed that the antipredatory compounds of Bryopsis sp. were present in the butanol and chloroform fractions, both containing the depsipeptide kahalalide F. Antipredatory compounds of Elysia rufescens were exclusively present in the dichloromethane fraction. Further bioassay-guided fractionation led to the isolation of kahalalide F as the only compound responsible for the deterrent properties of the sacoglossan. Our data show that kahalalide F protects both Bryopsis sp. and Elysia rufescens from fish predation. This is the first report of a diet-derived depsipeptide used as a chemical defense in a sacoglossan.     

Plant Chemical Defense Induced by a Seed-Eating Pollinator Mutualist

Plant-seed parasite pollination mutualisms involve a specific pollinator whose larvae develop by consuming a fraction of the host plant seeds. These mutualisms are stable only if the plant can control seed destruction by the larvae. Here, we studied the chemical response of the European globeflower Trollius europaeus to infestation by an increasing number of Chiastocheta fly larvae. We used liquid chromatographic analysis to compare the content of phenolic compounds in unparasitized and parasitized fruits collected in two natural populations of the French Alps, and mass spectrometry and nuclear magnetic resonance to elucidate the structure of adonivernith, a C-glycosyl-flavone. This compound is present in many of the organs of T. europaeus, but not found in other Trollius species. Furthermore, it is overproduced in the carpel walls of parasitized fruits, and this induced response to infestation by fly larvae is density-dependent (increases with larval number), and site-dependent (more pronounced in the high-altitude site). Mechanical damage did not induce adonivernith production. This tissue-specific and density-dependent response of T. europaeus to infestation by Chiastocheta larvae might be an efficient regulation mechanism of seed-predator mutualist population growth if it decreases survival or growth of the larvae.     

Chemical vapor deposition of aluminum for ulsi applications

Aluminum has been used widely as a conducting material in the fabrication of integrated circuits, and chemical vapor deposition process for Al has been actively investigated for the application in ultra large scale integration. In this review, various precursors, mainly alkyl aluminum and alane compounds, and reaction mechanisms of these precursors in Al CVI) are described. Epitaxial growth and selectivity of the deposition are also discussed. In addition to thermal reactions, plasma and photochemical reactions are also briefly described.     

Geographic and Temporal Variation of Cardenolide-Based Chemical Defenses of Queen Butterfly (Danaus gilippus) in Northern Florida

The cardenolide-based chemical defenses of danaine butterflies vary macrogeographically. This study demonstrates that these defenses also vary both microgeographically and temporally. We sampled 280 queen butterflies (Danaus gilippus) at 11 sites in northern Florida during the summer of 1993 and determined their cardenolide concentrations and thin-layer chromatography profiles. Queens captured in coastal salt marshes and xeric inland sites were low in cardenolide concentration, while those from hydric inland sites had much higher concentrations. Concentrations also varied temporally, especially at the coastal sites. Thin-layer chromatography analyses of wild-captured queens and those reared on local milkweeds indicated that microgeographic and temporal variation in the cardenolide chemistry of the butterflies is mediated by their host plants. The large differences in cardenolide concentrations among queen populations must result in strong differences in palatability spectra to vertebrate predators. This finding has major implications both for interspecific mimicry and for automimicry.     

Chemical Ecology of the Defense of Two Nymphalid Butterfly Larvae Against Ants

We analyzed the behavioral responses of the ants Camponotus rufipes and Solenopsis geminata towards all instars of Dione junio and Abananote hylonome. We also analyzed ant behavior towards hexane extracts of larvae and extracts of the spines and neck glands of the fifth instars of both species and identified the chemical compounds present. Larvae of both species were repellent to ants from the first instar onward. Later instars survived ant attacks better than earlier instars. The spines and neck glands of the larvae influenced the behavior of C. rufipes. The chemical compounds contained in the hexane extracts of whole first and fifth instars and in the spines and neck glands of fifth instars were principally carboxylic acids and terpenes. Further bioassays confirmed the repellent effect of some of these acids toward ants.     

Comparison of the wound-activated transformation of caulerpenyne by invasive and noninvasive Caulerpa species of the Mediterranean

The invasive green alga, Caulerpa taxifolia, that has spread rapidly after its introduction into the Mediterranean and the North American Pacific, reacts to wounding by transforming its major metabolite caulerpenyne (1). This wound-activated reaction involves the transformation of the bis-enol acetate moiety of 1, releasing reactive 1,4-dialdehydes. The ability to perform this transformation is found also in both the noninvasive Mediterranean C. prolifera and the invasive C. racemosa. Trapping experiments, as well as transformation of the model substrate geranyl acetate, suggest that all three investigated Caulerpa spp. rely on esterases that act upon wounding of the algae by subsequently removing the three acetate residues of caulerpenyne. The resulting reactive 1,4-dialdehyde oxytoxin 2 (9) can be identified by liquid chromatography–mass spectrometry and is unstable in the wounded tissue. Caulerpenyne transformation occurs rapidly, and severe tissue damage caused degradation of more than 50% of the stored caulerpenyne within 1 min in all three algae. Prevention of the enzymatic reaction before extraction, by shock freezing the tissue with liquid nitrogen, was used for the determination of the caulerpenyne content in intact algae. It gives about twofold higher values compared to an established methanol extraction protocol. The speed and mechanism of the wound-activated transformation, as well as the caulerpenyne content in intact tissue of invasive and noninvasive Caulerpa spp., are comparable. Thus, this enzymatic , transformation, despite being fast and efficient, is likely not the key for the success of the investigated invasive species.