Rapid Analysis of Abietanes in Conifers

Diterpene resin acids are major constituents of conifer oleoresin and play important roles in tree defense against insects and microbial pathogens. The tricyclic C-20 carboxylic acids are generally classified into two groups, the abietanes and the pimaranes. The abietanes have conjugated double bonds and exhibit characteristic UV spectra. Here, we report the analysis of abietanes by reversed-phase high-performance liquid chromatography using multiwavelength detection to optimize quantification of underivatized abietic, neoabietic, palustric, levopimaric, and dehydroabietic acids. The utility of the method is demonstrated with methanol extracts of white spruce (Picea glauca) phloem, and representative concentrations are reported.     

Mechanism of feeding discrimination between matured and juvenile foliage by two species of pine sawflies

Two species of pine sawflies,Neodiprion rugifrons Midd. andN. swainei Midd., feed only on matured foliage of jack pine,Pinus banksiana Lamb., and leave juvenile or current-season foliage intact. This unique form of adaptation was studied from the viewpoint of the chemical ecology of this insect-host plant relationship. It was first determined that the differential larval feeding behavior reflects the presence of feeding deterrents. Two major biologically active substances were isolated and identified as 13-keto-8(14)-podocarpen-18-oic acid and dehydroabietic acid. These account for 63.5% and 24.6% of the total deterrency, respectively. The content of the former substance in current-year foliage decreases, as the foliage begins to mature, to the levels that become acceptable toN. swainei by August (60 days old) and to the second generationN. rugifrons by September (90 days old). The timing of their acceptance of juvenile foliage indicates the high levels of adaptation by these insects to allow oviposition for the on-coming adults and acceptable needles for the next generation of larvae. The level of dehydroabietic acid, on the other hand, does not change appreciably during the same time period: this indicates that the component does not play a significant role in the mechanism of differentiating juvenile from matured foliage by the sawfly larvae.     

Fatty and resin acids of spanishpinus pinaster Ait. Subspecies

The qualitative and quantitative composition of the acid fraction of wood extractives from three SpanishPinus pinaster Ait. subspecies (Atlantic, mountain Mediterranean and plain Mediterranean) were studied. Seven samples of each subspecies were prepared according to Technical Association of Pulp and Paper Standards, extracted with petroleum ether (b.p. 40–60°C) in a Soxhlet apparatus and saponified with ethanolic 0.4N potassium hydroxide. The acid fraction was methylated with diazomethane and studied by combined gas chromatography/mass spectrometry. The major constituents found were palmitic, oleic and linoleic among the fatty acids and pimaric, sandaracopimaric, levopimaric, isopimaric, abietic and dehydroabietic among the resin acids. Neoabietic and palustric acids were not found. Variations in the quantitative composition of the acid fraction enable us to distinguish the three subspecies. Main quantitative differences are found between the Atlantic subspecies and the two Mediterranean subspecies.     

松香树脂酸的单离与应用

分别综述了枞酸、左旋海松酸、脱氢枞酸、新枞酸等松香树脂酸在医药、农药、表面活性剂制备以及在其他精细化学品合成中的应用,指出纯粹的松香树脂酸作为绿色化学合成的原料有广阔的工业应用前景。介绍了松香树脂酸单离的胺盐结晶方法,提出对松香树脂酸单离的研究重点是加快胺化反应速度、强化结晶传递过程、采用反应与分离耦舍的方法,以提高松香树脂酸的提取率。     

Field and electroantennogram responses to sex pheromone optical isomers by monophagous jack pine sawflies (Hymenoptera: Diprionidae)

Several species of monophagous jack pine sawflies (Hymenoptera: Diprionidae) were tested in the field and by electroantennograms (EAG) for activity toward the optical isomers of a pine sawfly sex pheromone, the acetate and propionate esters of 3,7-dimethylpentadecan-2-ol.Neodiprion rugifrons andNeodiprion dubiosus were attracted to a mixture of the propionate esters of the 2S,3R,7R and 2S,3R,7S isomers, whereasNeodiprion swainei was attracted to the 2S,3S,7S propionate isomer. Samples containing the 2S,3R,7S propionate isomer elicited the strongest EAG responses in these three species andNeodiprion nigroscutum. The 2S,3S,7S propionate isomer was equally active (EAG) in the case ofN. swainei.     

Influence of diterpene resin acids on feeding and growth of larch sawfly,Pristiphora erichsonii (Hartig)

Diterpene resin acids significantly affect consumption rates, feeding efficiencies, and growth rates of the larch sawfly,Pristiphora erichsonii (Hartig) when topically applied to their natural food, tamarackLarix laricina (DuRoi) K. Koch. Abietic acid, neoabietic acid, dehydroabietic acid, and isopimaric acid significantly reduced consumption rates, feeding efficiencies, and growth rates. Sandaracopimaric acid reduced growth and efficiency but did not influence consumption rate. Two-way analysis of variance indicates a significant interaction between chemical and concentration for growth rate, feeding efficiency, and consumption rate. This interaction indicates that increasing chemical concentrations do not influence the larch sawfly in a uniform manner, supporting the concept of concentration-dependent biological activity of allelochemics.Research supported by School of Natural Resources College of Agriculture and Life Sciences, University of Wisconsin-Madison and School of Forestry, Northern Arizona Univeristy under grants from the McIntire-Stennis Program.     

Allelochemics and nutritional indices for larch sawfly,Pristiphora erichsonii (Hartig): A specialist feeding onLarix spp.

Nutritional indices for larch sawfly,Pristiphora erichsonii (Hartig), larvae fed single and tufted needles of fourLarix spp. are reported. Larvae offered only single needles ofL. lancina, L. russica, andL. decidua had lower relative growth rates than larvae fed tufted needles of the same species. There was no significant reduction in larval growth for larvae fedL. kaempferi single needles as compared to tufted needles. Abietic acid-treated foliage reduced consumption but did not lower relative growth rate. These findings are discussed with respect to the mechanism of preferential feeding of the larch sawfly and current hypotheses of host plant herbivore interaction.Research supported by the School of Natural Resources, College of Agricultural and Life Sciences, University of Wisconsin—Madison under a grant from the McIntire-Stennis Program, Project 2243.     

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.     

气相色谱用马尾松松香标准样品的定值

系统地介绍了气相色谱用马尾松松香标准样品的定值方法。采用单一方法通过6家实验室联合定值,得到标准样品中7种树脂酸的GC含量(%)的特性值和扩展不确定度:海松酸8.2683±0.3203;山达海松酸:1.7433±0.3199;异海松酸:0.7652±0.3118;长叶松酸:30.8201±1.3668;去氢枞酸:3.2731±0.8693;枞酸:34.9275±1.2664;新枞酸:18.7469±1.8494。     

Diterpene resin acids: Major active principles in tall oil against Variegated cutworm,Peridroma saucia (Lepidoptera: Noctuidae)

Tall oil, a by-product of the kraft process for pulping softwood, has been shown to have insecticidal properties. In the present study, the active principles in tall oil against the variegated cutworm,Peridroma saucia Hübner, were investigated. GC-MS analysis showed that abietic, dehydroabietic, and isopimaric acids were major resin acid components of crude tall oil and depitched tall oil. When crude tall oil samples of differing resin acid composition were incorporated into artificial diet at a concentration of 2.0% fresh weight, they suppressed larval growth by 45–60% compared to controls. This suppression was significantly (P0.05) correlated with the equivalent contents of abietic, dehydroabietic, isopimaric, and total resin acids. These results were also evident from a diet choice test, showing that the second-instar larvae obviously selected diets with low levels of resin acids when different diets were randomly arranged in a Petri dish. Bioassays with pure resin acids (abietic, dehydroabietic, and isopimaric acids) demonstrated that all individual chemicals have similar bioactivity against this insect. Comparison of the bioactivities of depitched tall oil and an equivalent mixture of pure resin acids in thePeridroma chronic growth bioassay indicated that pure resin acids and depitched tall oil share a common mode of action to this insect. This study confirms that resin acids are major active principles in tall oil against the variegated cutworm, but other chemicals likely also contribute to the bioactivity of tall oil.     

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.     

Effects of nitrogen fertilization on secondary chemistry and ectomycorrhizal state of Scots pine seedlings and on growth of grey pine aphid

Effects of nitrogen availability on secondary compounds, mycorrhizal infection, and aphid growth of 1-year-old Scots pine (Pinus sylvestris L.) seedlings were studied during one growing season. Seedlings were fertilized with nutrient solutions containing low, optimum, and two elevated (2 × and 4 × optimum) levels of NH₄NO₃. At the end of growing season foliar nitrogen concentration, needle biomass, needle length, water contents of needles, root collar diameter, and number of buds increased with enhanced nitrogen availability. Addition of nitrogen did not have effect on concentrations of monoterpenes in growing needles, but in mature needles significantly decreased concentrations of some individual and total monoterpenes were detected. In growing needles the concentrations of some individual resin acids decreased, and in mature needles concentrations of some individual and total resin acids increased with increased nitrogen fertilization. Higher numbers of resin ducts were found in mature needles with nitrogen fertilization. Nitrogen fertilization decreased total phenolic concentrations in growing and mature needles of the current year, but in needles of the previous year no significant differences occurred. Mycorrhizal infection was highest at medium (optimum and 2 × optimum) nitrogen fertilization levels. The relative growth rate (RGR) of grey pine aphid [Schizolachnus pineti (F.)] responded positively to the increase in foliar nitrogen content. However, the increase in aphid performance between optimum and the highest fertilization level was slight. This may indicate a deterring effect of resin acids on aphids. The results indicate that carbon/nutrient balance hypothesis fails to predict directly the effects of nitrogen availability on concentrations of carbon-based defensive compounds in mature foliage. Altered nitrogen supply affects allocation to secondary metabolites differently, depending on the developmental state of the plant and the biosynthesis pathway, cost of synthesis, and storage of compounds.     

Feeding behavior of graminivorous grasshoppers in response to host-plant extracts,alkaloids, and tannins

Secondary metabolites exhibit the potential to direct food selection by grass-feeding (graminivorous) grasshoppers. We examined the effects of plant extracts and representative secondary metabolites on the feeding behavior of two such grasshoppers,Ageneotettix deorum (Scudder) andPhoetaliotes nebrascensis (Scudder). Three alkaloids and two tannins were bioassayed for their activity as feeding deterrent allelochemicals, as were extracts from the foliage of the graminoids commonly eaten by these grasshoppers:Agropyron smithii Rydb.,Andropogon hallii Hack.,Andropogon scoparius Michx.,Bouteloua gracilis (H. B. K) Lag. ex Griffiths,Carex heliophila Mack. andStipa comata Trin. & Rupr. Alkaloids strongly deterred feeding but tannins only exhibited a weak effect, even when present at four times the concentration of total phenolics typical for these graminoids. Host-plant extracts also exhibited weak effects, such that we found no evidence for either strong deterrence or phagostimulation. Our results for alkaloids and host-plant extracts are consistent with the view that grass-feeding grasshoppers may be restricted to graminoids because of: (1) the presence of deterrents in nonhosts and (2) the absence of deterrents in hosts. However, our data for tannins show that these are unlikely to be effective barriers to herbivory by these grasshoppers.     

Chemical basis of differential feeding behavior of the larch sawfly,Pristiphora erichsonii (Hartig)

The machanisms by which the larvae of the larch sawfly,Pristiphora erichsonii (Hartig), are prevented from feeding on the single needles of the new shoot of tamarack,Larix laricina (DuRoi) K. Koch, were studied. As a result of extensive purification attempts, five deterrent chemicals were isolated and identified. They are: abietic, dehydroabietic, 12-methoxyabietic, sandaracopimaric, and isopimaric acid. These chemicals, particularly the first two, are abundant in mid-July to August in the single needles and, apparently, provide the basis for the deterrency against the larval feeding.Research supported in part by the School of Natural Resources, College of Agricultural and Life Sciences, University of Wisconsin-Madison, under a grant from the McIntire Stennis Program. Project No. 2243.     

Weak responses of pine sawfly larvae to high needle flavonoid concentrations in scots pine

Responses of sawfly larvae (Hymenoptera, Diprionidae) to the flavonoid taxifolin glucoside in their host plant were studied in a laboratory experiment. Larvae ofNeodiprion sertifer andDiprion pini were raised from egg hatch to cocoon spinning on two Scots pine (Pinus sylvestris) chemotypes, one without needle taxifolin glucoside (–) and the other containing 2–4% taxifolin glucoside (+). The (+) chemotype had somewhat lower concentrations of needle terpenoids (resin acids) than the (–) chemotype. Current-year needles had higher taxifolin glucoside concentrations than mature needles. There were no differences in survival or body size betweenN. sertifer larvae that fed on the (+) chemotype and those that fed on the (–) chemotype. FemaleD. pini larvae raised on (+) needles developed 6% more slowly than larvae fed (–) needles. The results from this study are contrary to earlier findings showing that flavonoid glucosides have strong negative effects on insect performance. Possible explanations for the different outcomes are discussed.     

Susceptibility to vole attacks due to bark phenols and terpenes inPinus contorta provenances introduced into Sweden

Seedlings of North AmericanPinus contorta introduced to Sweden and Finland are severely gnawed by voles, e.g.,Microtus agrestis. The level of damage varies between provenances. Chemical analyses of various phenolic compounds, monoterpenes, and resin acids of different provenances and of damaged and undamaged stems showed that some phenolic substances in the bark increased after damage without deterring the animals, that monoterpene differences between provenances were not related to vole damage, and that certain resin acids occurred in larger amounts in lightly than in severely damaged provenances. Levopimaric and neoabietic acid, and possibly abietic acid, may be important for a partial resistance to vole browsing.     

Insecticidal Effects of Extracts from Two Rice Varieties to Brown Planthopper, Nilaparvata lugens

Ether extracts from a resistant rice variety, Jingxian 89, and a susceptible variety, Qidaizhan, were significantly toxic to Nilaparvata lugens adult females, causing 98% and 73.5% mortalities, respectively. The area covered honeydew droplets excreted by N. lugens adult females while feeding on Qidaizhan was 28.5-fold more than the area of those feeding on the resistant variety, Jingxian 89. The honeydew covered areas were reduced 29.7- and 8.8-fold, respectively, after feeding on plants treated with the ether extracts from both varieties. Among the fractions from each variety, fractions 9 (three sterols) from both varieties and fraction 10 (3-nitraphthalic acid) from Jingxian 89 showed significant repellent effects on the planthoppers. Further bioassays revealed that fraction 9 from extracts of both varieties at 0.02 mg/ml or higher deterred the feeding activity of the planthoppers, whereas the deterrent effects of fraction 10 from the extract of Jingxian 89 were 100- to 500-fold stronger than those of fraction 9. Fraction 10, identified as 3-nitraphthalic acid, unique in Jingxian 89, was extremely toxic to N. lugens adult females, and the LC₅₀ and LC₉₀ were 0.00045 and 0.00525 mg/ml, respectively. After extensive bioassays and analysis, we concluded that 3-nitraphthalic acid plays a key role in the resistance to N. lugens adult females.     

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.     

Variation in Growth, Chemical Defense, and Herbivore Resistance in Scots Pine Provenances

Variation in the growth, chemical defense, and susceptibility to monophagous and polyphagous sucking herbivores was assessed for four Scots pine (Pinus sylvestris) provenances in Petri dish and pot experiments. Seed provenances, Muonio (MU), Suomussalmi (SU), Korpilahti (KO), and Saaremaa (SA) represented a 1200-km N–S gradient. Scots pines from SU were least preferred as a host plant by pine-feeding aphids Schizolachnus pineti Fabr. and Eulachnus agilis, and in pot experiments, pines from SA were most preferred. S. pineti also produced the fewest nymphs on pines from SU. Polyphagous Lygus rugulipennis laid significantly fewer eggs on pines from MU provenance, which were smaller than seedlings from other provenances. The total and some individual terpene concentrations increased, while the total resin acid concentration decreased, towards the north. The absolute concentrations of total or individual terpenes and resin acids in the needles of unfavored SU plants did not differ significantly from the other provenances. Only the proportional quantity of dehydroabietic acid was higher in SU plants than in KO plants, and it correlated, together with sandaracopimaric acid, negatively with the number of S. pineti nymphs in the reproduction experiment. Conversely, absolute concentrations of some terpenes correlated negatively and some resin acids positively with the number of S. pineti nymphs. The length and weight of shoots decreased towards the north, adequately explaining the number of Lygus eggs and the final numbers of both aphids. The results suggest that seedling size is a better estimator of susceptibility of pine seedlings to both specialist and generalist sucking insects than relative or absolute concentrations of monoterpenes and resin acids.     

A neolignoid feeding deterrent againstLuehdorfia puziloi larvae (lepidoptera: Papilionidae) fromHeterotropa aspera,a host plant of sibling species,L. japonica

A feeding deterrent against larvae of a papilionid butterfly,Luehdorfia puziloi (Parnassiinae), a specialist onAsiasarum plants (Aristolochiaceae), was isolated from another aristolochiaceous plant,Heterotropa aspera. Antifeedant activity was exhibited by then-hexane-soluble fraction that proved to contain at least two active components. One of the deterrents was identified as a neolignan compound, asatone. The concentration of asatone inH. aspera was estimated at approximately 225 ppm, and larval feeding ofL. puziloi was significantly deterred at concentrations over 90 ppm. By contrast, asatone was not detectable (< 1 ppm, if any) in its host plant,Asiasarum sieboldii.