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.     

Leaf-tying by tortricid larvae as an adaptation for feeding on phototoxicHypericum perforatum

Hypericin, an anthrone dimer inHypericum perforatum (Guttiferae), is shown for the first time to be phototoxic to generalist lepidopteran larvae; survivorship was reduced and development prolonged forHeliothis zea (Noctuidae) andPlatynota flavedana (Tortricidae) that consumed hypericin diets in the presence of light. Survivorship for both species is enhanced when photoactivating wavelengths are excluded by filters. In natureP. flavedana successfully survives onH. perforatum by tying together leaves and feeding inside the ties. Shielded from light, the larvae are protected by their mode of feeding from phototoxic compounds.     

Attraction of sphingid moths (Lepidoptera: Sphingidae) to 10,12-hexadecadienyl aldehydes and acetates: Evidence of pheromone components

A field survey of 10,12-hexadecadienyl alcohols, acetates, and aldehydes showed attraction to a wide range of sphingid moths. Data are presented showing the attraction ofSmerinthus jamaicensis, Smerinthus cerisyi, Pachysphinx modesta, Hemaris diffinis, andProserpinus flavofasciata to these compounds. Mass spectral, EAG, and EAD data show evidence for the presence of these dienes in female extracts ofS. cerisyi, Hyles gallii, andSphinx drupiferarum. NRCC No. 25857.In this paper the chemical nomenclature is abbreviated as follows: Z and E indicate thecis andtrans configurations of the double bond, the number followingZ orE indicates the position of that double bond, the last number indicates the carbon chain length, the terminal functional groups are: Ac=acetate; :OH=alcohol; :Aid=aldehyde.     

Ecological adaptation of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous,to aristolochic acids

Seven analogs of aristolochic acids (I, II, III, B, C, D, and E) were isolated from the leaves ofAristolochia debilis and characterized as the larval feeding stimulants of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous. Aristolochic acids showed synergistic activity in combination with the water-soluble components in the leaf extract. Aristolochic acids were detected in the body tissues and specialized organs throughout all life stages ofA. alcinous. Larval osmeterial fluid contained aristolochic acids as high as 2% of the secretions, the compositions of which were similar to that found in the leaf extracts. In contrast, the eggs selectively contained aristolochic acids I and II. These two acids were present both in the egg yolk and egg coating material as well as in the collateral glands (glandulae sebaceae) of the adult females. The cannibalistic activity of the larvae against the eggs and pupae seemed to be triggered by aristolochic acids present on the egg surface and pupal cuticle, suggesting a possible adaptive mechanism in this species. Aristolocic acid I deterred feeding of tree sparrows, which suggested a defensive role against vertebrate predators.     

Evidence of an oviposition stimulant for peachtree borer

Peachtree borer (PTB) cocoons, peach tree bark, and a mixture of frass and gum obtained arount PTB wounds were extracted with organic solvents of increasing polarity. Female PTB moths,Synanthedon exitiosa (Say), laid the greatest number of eggs on filter paper substrates treated with acetone extracts of PTB cocoons, ethyl acetate extracts of peach tree bark and acetone extracts of frass and gum mixtures. The extracts of cocoons or frass and gum mixture were separated by liquid chromatography and thin layer chromatography.Synanthedon exitiosa (Say).Mention of a commercial or proprietory product does not constitute an endorsement of this product by the USDA.     

Evidence for diglycerides as attractants in an ant-seed interaction

The chemical basis of an ant-seed interaction was investigated for the antAphaenogaster rudis and the ant-dispersed violetViola odorata. A laboratory behavioral bioassay was developed to chemically identify the attractant responsible for the interaction. The ant attractant, localized in the elaiosome, was classified as a lipid by both field and laboratory bioassays. Assays of partially purified lipid extracts revealed that the principal attractant may be a diglyceride. Gas-liquid chromatography analysis of the hydrolyzed diglyceride fraction revealed oleic acid as the major fatty acid present, suggesting that either 1,2- or 1,3-diolein may be the attractant. Structure-activity correlations for lipid standards demonstrated a clear preference for the diglyceride 1,2-diolein. The data also suggest that ricinoleic acid is not the lipid eliciting the ant response toViola odorata, as had been previously suggested.     

Evidence for the controlled release of a crustacean sex pheromone

Evidence for the controlled release of a crustacean sex pheromone: mature malePortunus sanguinolentus (Herbst) did not give the characteristic display described by Ryan (1966) to water from a container with a carried or artificially restrained premolt female. However, the sex pheromone was still present in the urine of the restrained female, suggesting that she inhibits the emission of the sex pheromone by regulating her urine flow.Based on part of a dissertation presented by the author in partial fulfillment of the requirements for the doctoral degree in zoology at the University of Hawaii.     

Direct Evidence for Membrane Transport of Host-Plant-Derived Pyrrolizidine Alkaloid N-Oxides in Two Leaf Beetle Genera

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).     

Evidence for Four-Component Close-Range Sex Pheromone in the Parasitic Wasp Glyptapanteles flavicoxis

Females of the parasitic wasp Glyptapanteles flavicoxis (Hymenoptera: Braconidae) deposit a close-range sex pheromone from their abdominal tip that attracts conspecific males and elicits wing-fanning behavior. In this study, we isolated the pheromone components and determined their role in the males' behavior. In coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses of the females' body extract, four components (below GC detection) elicited strong responses from male antennae. Monitored by GC-EAD, the components were separated by flash silica gel and high-performance liquid chromatography. Y-tube olfactometer experiments with one or more components revealed that all are necessary to elicit short-range attraction and wing-fanning responses by males. These components remained below detection threshold of the mass spectrometer (∼10 pg) even when 4500 female equivalents were analyzed in a single injection, which attests to the potency of the pheromone and the insects' sensitivity to it.     

Evidence for presence and nature of female sex pheromone ofBrithys crini fabricius (Lepidoptera: Noctuidae)

The existence of a female sex pheromone of the noctuid mothBrithys crini Fabricius was confirmed in both laboratory bioassay and field tests. Crude extracts and airborne volatiles from females were analyzed by gas chromatography and mass spectrometry and the data compared with authentic compounds. The primary sex pheromonal compound was Z11-16: Ald. Scanning electron microscopy showed that the external surface of the sex pheromone gland was covered with folds that might increase the sex pheromone evaporation area.     

Evidence for allelochemical attraction of the coffee berry borer,Hypothenemus hampei,by coffee berries

Petri dish choice tests conducted on the coffee berry borer (CBB),Hypothenemus hampei, showed that females were able to discriminate between coffee berries at different ripening stages. A Y-shaped glass olfactometer was used to demonstrate that coffee berries emitted volatile chemicals that elicited upwind movement by female CBB. Olfactometer tests with three different solvent extracts of berries showed that at least some of the attractive chemical(s) released by the coffee berries could be extracted with acetone.     

Active and selective copper catalysts supported on alkali-doped silica for the dehydrogenation of cyclohexanol to cyclohexanone

Copper supported on alkali-doped silica was found to be the most effective among various types of copper-containing catalysts tested for the dehydrogenation of cyclohexanol to cyclohexanone. This catalyst could be used with lower copper content than others, and showed the highest conversion and selectivity and good resistance against thermal sintering.     

Toxicity of linear furanocoumarins toSpodoptera exigua: Evidence for antagonistic interactions

The linear furanocoumarins psoralen, bergapten, and xanthotoxin were tested for toxicity to the beet armywormSpodoptera exigua (Hübner) under short ultraviolet (UVB) radiation. Increased dietary concentrations of each furanocoumarin significantly decreased insect larval weight, extended generation time, and induced higher mortality. Xanthotoxin was the most toxic, followed by psoralen and bergapten. Combining psoralen with bergapten, xanthotoxin, or both resulted in significantly antagonistic effects on insect mortality. The combination of bergapten and xanthotoxin, however, produced additive effects. The implications of these observations forS. exigua resistance in the wild plant accession ofApium prostratum and the enigma the findings represent for plant-insect relationships are discussed.     

Anaerobic microflora of the anal sac of the red fox (Vulpes vulpes)

Anal sac fluid from the red fox (Vulpes vulpes) was cultured for anaerobic bacteria by two techniques. The more strictly anaerobic technique yielded a wider range of species. Considerable biochemical differences were noted within named species, but no correlation could be found between the foxes and the organisms of their anal sac fluids.     

Effect of predator odors on heart rate and metabolic rate of wapiti (Cervus elaphus canadensis)

We measured the heart rate (HR) and oxygen consumption ( ) of wapiti (Cervus elaphus canadensis) before, during, and after presentation of biologically irrelevant odors (pentane, thiophene, and a perfume), artificial predator odors (an ether extract of cougar feces, and PDT, a compound found in mustelid anal gland secretion), stale predator odors (dog feces and urine and fox urine, kept at ambient temperature for a few weeks), and fresh predator odors (wolf, coyote, and cougar feces and the odor of a dead coyote, kept frozen between collection and test). Overall, responses to odors were small compared to other stressful stimuli. Individual variability was high among scents and among wapiti, but two of the fresh predator odors (cougar and wolf feces) produced larger HR and responses than the other scents and were more often successful at producing responses. As a group, fresh predator odors produced larger tachycardias and elicited a larger number of significant HR responses than biologically irrelevant novel odors. although the two classes of odors did not differ in their effect on . Although several other studies have shown that ungulates have reduced feeding levels when their food is scented with predator odors, it is not clear if this is due to reduced palatability or antipredator behavior. This study is the first demonstration that a wild ungulate species reacted more strongly to predator odors than to other odors in a nonfeeding situation.     

From Terpenoids to Aliphatic Acids: Further Evidence for Late-Instar Switch in Osmeterial Defense as a Characteristic Trait of Swallowtail Butterflies in the Tribe Papilionini

We compared the chemical compositions of the osmeterial secretions of fourth and fifth (last) instars of eight swallowtail species of the tribe Papilionini. Four species (Papilio demoleus, P. polytes, P. paris, and P. macilentus) are Asian Rutaceae-feeding swallowtails. The other four (Chilasa epicydes, C. agestor, P. troilus, and P. glaucus) represent more distant clades within the Papilionini and species with larval hosts in other plant families. We conducted a quantitative analysis for six species, but only qualitative analysis for P. glaucus and C. agestor. In all eight species, regardless of larval host plant, secretions of the fourth instar principally consisted of mono- and sesquiterpene hydrocarbons, whereas those of the fifth instar comprised aliphatic acids and their esters. Consistent with earlier findings, our results suggest that this “heterogeneous” pattern of osmeterial chemistry, not seen in other tribes, may characterize the Papilionini as a whole. Unlike those of most Papilio species, the fourth and fifth instars of Chilasa species resemble each other in body coloration. Thus, the heterogeneous osmeterial pattern is not necessarily associated with color change in papilionid larvae. The major terpenoids identified in fourth instar larval secretions from the six species were α-pinene, sabinene, β-myrcene, limonene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, p-mentha-1,4(8)-diene, β-elemene, β-caryophyllene, (E)-β-farnesene, (3Z,6E)-α-farnesene, (Z)-α-bisabolene, germacrene-A, (E)-α-bisabolene, and germacrene-B. The profiles for individual species differed both qualitatively and quantitatively from one another, and certain species also secreted methyl 3-hydroxy-n-butyrate and oxygenated sesquiterpenes in relatively large proportions. Secretions from fifth instars were composed of varying proportions of isobutyric, 2-methylbutyric, and acetic acids, and methyl and ethyl (minor) esters of both isobutyric and 2-methylbutyric acids. The heterogeneity of osmeterial chemistry in the tribe Papilionini may represent fine-tuning of chemical defense in response to shifting predation pressures as the larvae age and grow.     

Attraction of male beetles to grubs: Evidence for evolution of a sex pheromone from larval odor

Females of the scarabaeid beetleCyclocephala lurida produce a volatile sex pheromone which attracts conspecific males. Field experiments demonstrated that larvae of both sexes also emit volatile chemicals that stimulate similar responses in adult males, including attempts by the attracted males to mate with the nonreproductive immature stage. Significantly more adult males were caught in traps baited with conspecific male or female larvae or adult females than in blank control traps. Hexane extracts of both male and female grubs were at least as effective as live larvae in trapping male adults, demonstrating that the behavioral responses are mediated by volatile chemicals. Sensory and behavioral responses of males to sex pheromones emitted by adult females are part of the functional communication system. However, their response to grubs is not functional, because grubs are normally temporally and spatially inaccessible to mate-seeking males. In theory, the evolution of a communication system is problematic because it requires the development of a signal in one sex and the sensory and behavioral attributes to respond to that signal in the other sex. The ontogeny of sex pheromone communication inC. lurida suggests a partial solution to this evolutionary problem. We propose that this sex pheromone communication system is probably derived from noncommunicative volatile chemicals that are lost in adult males and retained by adult females.     

Cucurbitacins fromCucurbita texana: Evidence for the role of isocucurbitacins

In addition to cucurbitacins E andI, cucurbitacins D,1, 3-epi-isocucurbitacin D,2, and B,3, were isolated from the fruits ofCucurbita texana and structurally identified by UV, IR,¹H NMR,¹³C NMR, and MS, and 2-O--glucopyranosylcucurbitacin I was identified. These compounds have not been reported previously as constituents of this species. The isolation of 3-epi-isocucurbitacin D2 together with normal cucurbitacins suggests that isocucurbitacins occur naturally. Evidence is also discussed that isocucurbitacins are biosynthesized one step ahead of normal cucurbitacin.     

Characterization of and male adaptation to pheromone of femaleTrichostrongylus colubriformis (Nematoda)

Males of the ruminant nematodeTrichostrongylus colubriformis were significantly attracted to an incubate from their females during in vitro assay. Exposure of males to sera from uninfected and infected animals or selected neurotransmitters had no effect on their subsequent responsiveness to the female's pheromone. In contrast, exposure to 1500 female-hours of pheromone decreased male responsiveness after moderate rinsing prior to bioassay. Rinsing of the males with larger volumes of Tyrode's solution prior to in vitro assay increased their subsequent response to the female's pheromone. High-performance liquid chromatography yielded a presumptive pheromone peak with a fivefold increase in biological activity. This peak was soluble in alcohols and tetrahydrofuran, based on elution from reverse-phase Sep-Pak cartridges. Stability of the peak was increased by EDTA or ascorbic acid (10 mM). Storage for six weeks in ascorbic acid at 4 °C allowed recovery of 47.3% of the original material.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Evidence for a short-range sex pheromone in female Maladera matrida beetle

Laboratory studies with live and frozen Maladera matrida female and male beetles showed that males were attracted to chemical substances emanating from the females at dusk. Beetles exhibited sexual activity (including mating) at dusk towards frozen females but not towards frozen males. Frozen females that had been extracted with methanol together with either hexane or dichloromethane or with a mixture of all three solvents did not elicit male sexual activity. Activity was fully restored when a concentrate of the extract was applied to the previously extracted frozen females. Males also responded with vigorous sexual activity to frozen males to which female extract had been applied. Deterrent chemicals appear to be absent from the male body. Males exposed to females that had been frozen during the morning displayed weak sexual activity, indicating that females lack active semiochemicals. Differences between dusk and morning extracts were found with respect to more than 20 compounds, some of which were present in much higher concentrations at dusk than in the morning, while others were not detected in the morning extract. The active component(s) of the short-range sex pheromone of female M. matrida is (are) presumably to be found among these compounds.