Journal of Chemical Ecology, Vol. 18, No. 3, 1992




P. O H M A R T , 1'3 and R O L F G R E F 4

IDepartment of Plant and Forest Protection Swedish University of Agricultural Sciences P. O. Box 7044, S- 750 07 Uppsala, Sweden 2Department of Chemistry Swedish University of Agricultural Sciences P.O. Box 7015, S-750 07 Uppsala, Sweden 4Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences S-90! 53 Ume~, Sweden (Received July 9, 1991; accepted October 28, 1991)

Abstract--Responses of sawfly larvae (Hymenoptera, Diprionidae) to the flavonoid taxifolin glucoside in their host plant were studied in a laboratory experiment. Larvae of Neodiprion sertifer and Diprion 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 between N. sertifer larvae that fed on the (+) chemotype and those that fed on the ( - ) chemotype. Female D. 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.

*To whom correspondence should be addressed. 3present address: Scientific Methods, Inc., P.O. Box 599, Durham, California 95938.

271 00984~331/92/03004)271506.50/0 9 1992PlenumPublishingCorporation



Key Words--Plant-insect interaction, Pinus sylvestris, Neodiprion sertifer, Diprion pini, Hymenoptera, Diprionidae, insect performance, flavonoids, taxifolin glucoside, resin acids.


Flavonoids are polyphenolic compounds found in many species of plants, where they usually occur as glycosides (Harborne, 1979). Little is known about the function of flavonoid compounds. Some (e.g., quercitrin, rutin, and maysin) are known to have deleterious effects on agricultural insects (Shaver and Lukefahr, 1969; Waiss et al., 1979; Elliger et al., 1980, 1981; Isman and Duffey, 1982; Hedin et al., 1983; Duffey et al., 1986). However, mtin has also been shown to enhance insect growth (McFarlane and Distler, 1982), and other flavonoid compounds (e.g., morin and kaempherol) act as feeding stimulants for certain insects (Hamamura et al., 1962; Nielsen et al., 1979). Coniferous trees have a great variety of flavonoid compounds in their needles (Nieman, 1988), yet their function in the plant is poorly known. There are some indications that they confer resistance against microorganisms (Nomura and Kishida, 1978; Szweykowski and Urbaniak, 1982). Their effects on the feeding of folivorous insects have not been studied. Two Scots pine (Pinus sylvestris L.) chemotypes exist with respect to one particular flavonoid, the dihydroflavonol glucoside taxifolin 3'-O-B-D-glucopyranoside (taxifolin glucoside) (Lundgren and Theander, 1988; Laracine-Pittet and Lebreton, 1988). One of the chemotypes lacks taxifolin glucoside, while the other can have a needle taxifolin glucoside concentration of 3-4 % dry weight (Lundgren, unpublished data). The two chemotypes cooccur over large parts of Europe, and the relative frequencies in a given tree population seem to vary with climatic conditions (Laracine-Pittet and Lebreton, 1988). These chemotypes offer an interesting opportunity to study flavonoid-insect interactions in natural plant tissue. In some cases, variation in plant phenolics may have an important influence on insect population dynamics. For example, it has been suggested that stress-induced changes in needle chemistry may trigger outbreaks of needle-eating insects (Larsson and Tenow, 1984). Environmental stress can alter phenolic glycoside concentrations in leaves of some woody plants (Larsson et al., 1986b; Bryant et al., 1987). It is not known, however, whether this also applies to flavonoids in conifers. If it can be shown that insect performance is affected by flavonoids, then it would be rewarding to investigate how needle flavonoid concentrations are influenced by environmental factors. In this paper we report the results from an experiment in which two species of pine sawflies [Diprion pini L. and Neodiprion sertifer (Fourcroy)] were fed the two Scots pine chemotypes. Insects were raised on the two types of needles,



and responses were evaluated in terms of larval survival, developmental time, and cocoon weight. METHODS AND MATERIALS

Study Organisms. Plant material was obtained from a 15-year-old Scots pine plantation at Hfigerdal, 30 km north of Uppsala, central Sweden. Trees in this plantation had been investigated earlier with respect to variation in needle flavonoid chemistry (Lundgren unpublished data). Based on these results, trees with [(+) chemotype] and without [ ( - ) chemotype] taxifolin glucoside and chemically related compounds (Figure 1) were selected for use. The experiment was performed in 1986 and 1987, using a different insect species each year (see below). From each study tree (N = 12 trees in 1986, N = 5 trees in 1987) one branch (three branches in 1987) was sampled from the lower half of the crown, which was immediately placed in a bucket of water and taken to the laboratory. Larvae of Neodiprion sertifer, collected from an outbreak area south of Sandviken, central Sweden, were used in the 1986 experiment, and larvae of Diprion pini from a small outbreak at N/irsholmen, Gotland, southeast Sweden, were used in the 1987 experiment. In both years, insects for the experiments were obtained by collecting pine shoots containing egg batches just prior to hatching. The shoots were taken to the laboratory and checked daily for hatched larvae. The transfer of larvae to test shoots began as soon as the first emerging larvae were observed. Larval Performance Experiments. Sawfly larvae were raised from egg hatch to cocoon spinning on experimental shoots in an environmental chamber (18:6 light-dark photoperiod, 18~ 70% relative humidity). Larvae were presented their preferred needle type, i.e., 1-year-old needles for N. sertifer and both R~


O-glc OH O

R=R'=H R=OH; R'=H R=R'=OH

Eriodictyol glucoside Taxifolinglucoside Ampelopsinglucoside

FIG. 1. Flavonoid glucosides present in the needles of the (+) chemotype (cf. Lundgren and Theander, 1988).



i-year-old needles (early instars) and current-year needles (late instars) for D.

pini. Each shoot was placed in a plastic container filled with tap water. To prevent mixing of the larvae, each shoot was enclosed in a transparent plastic Cylinder with a muslin cover. Larvae were given fresh shoots at weekly intervals. At the start of the experiment newly emerged larvae or eggs just about to hatch were transferred to shoots representing each of the two types of trees. About 30 larvae were transferred to each shoot. In 1986, 12 replicates were studied (one shoot from each of 12 trees). In 1987, three shoots from each of five trees were studied (always one terminal and two lateral shoots). Larvae that had started to feed on a terminal shoot were always given terminal shoots, and larvae that had started feeding on lateral shoots were always given lateral shoots. In order to avoid pseudoreplication, means were calculated for male and female larvae fed needles from the same tree. Thus, there were 12 independent observations in 1986 and five in 1987. Statistical analyses were performed on these mean values. Chemical Analyses. Needles intended for chemical analyses were sampled from branches offered to the larvae in the performance experiments. In 1986, 1-year-old needles were sampled fresh from trees on June 17. In 1987, 1-yearold needles were sampled fresh from trees on July 16. After one week in the laboratory, samples were again taken from the same shoots to check whether the laboratory treatment had any effects on needle phenolic chemistry. Currentyear foliage was sampled fresh from trees on July 27, 1987, and again from shoots in the laboratory on July 30. Since no effects of the laboratory treatment on phenolic chemistry were found, data were pooled in all subsequent analyses. Phenolic compounds were analyzed by reverse phase high-performance chromatography (HPLC). In the 1986 experiment, needle extracts were treated with pectinase and the resulting aglycone mixtures were extracted with ethyl acetate and examined by HPLC. The taxifolin content was determined, and the concentration of taxifolin glucoside in the needles calculated, assuming that taxifolin glucoside is quantitatively hydrolyzed and extracted to the organic layer. Acetovanillon was used as internal standard. In 1987, the intact extractives were analyzed, and picein was used as internal standard. The internal standard (0.5 mg) was added to the needle sample (1000 mg fresh weight), and the sample was homogenized with an UltraTurrax with 2 • 5 ml of 95 % ethanol (5 and 0.5 rain, respectively) and with 2 • 5 ml of 80% ethanol in the same way. The combined aqueous ethanol extract was evaporated to dryness, dissolved in water (15 ml) and extracted with light petroleum (bp 40-60~ (2 • 20 ml). In 1987 the aqueous layer was evaporated to dryness and the residue dissolved in methanol (1 ml). In 1986 the aqueous layer was treated with pectinase (0.5 ml, No. P-5146, Sigma) overnight at room temperature, acidified with three drops of 25 % phosphoric acid, and extracted with ethyl acetate (3 • 10 ml). The combined ethyl acetate extract was dried with sodium sulfate and



evaporated to dryness, after which the residue was dissolved in methanol (1 ml). The HPLC separation was performed with a Spectra Physics liquid chromatogram system with a Nova-Pak Rad-Pak C-18 column (Waters Associates). The separated components were detected with a UV detector at 280 nm. For the 1986 experiment, the mobile phase (mixture by volume of 0.01 M, pH 2.8, aqueous sodium phosphate buffer and methanol) consisted of a linear gradient from 20% to 30% methanol for 15 min, then maintained isocratically for 10 min, followed by a linear gradient to 55% methanol for 25 min and to 80% methanol for 10 min. The flow rate was 1.5 ml/min. For the 1987 experiment, the mobile phase (mixture by volume of 0.01 M, pH 2.8, aqueous sodium phosphate buffer and acetonitrile) consisted for the initial 7 min of 5 % acetonitrile. The concentration of acetonitrile was increased linearly to 25 % in 43 min and subsequently to 70% during 10 min. The flow rate was 1.5 ml/min. Diterpenoids were extracted and treated according to the methods reported by Gref and Ericsson (1985). Samples were analyzed on a Varian 3700 gas chromatograph equipped with a fused silica capillary column (15 m • 0.25 mm ID) with 0.25-/~m film of DB-1 (J & W Scientific). The chromatograph was equipped with a split injector and a flame ionization detector. Hydrogen was used as a carder gas at a flow rate of 1.60 ml/min. The chromatograph was operated isothermally at 210~ or temperature programmed to increase from 160~ to 240~ at 4~ Peak areas, relative to the internal standard, and retention times were measured with an electronic integrator. Total nitrogen (on a dry weight basis) was measured with a CHN-elemental analyzer (Perkin-Elmer model 2400 CHN).


Needle Chemistry. As expected, the two types of trees differed in their needle concentrations of taxifolin glucoside. No taxifolin glucoside was found in the ( - ) chemotype, whereas taxifolin glucoside made up 1.7-3.8% of the needle dry weight in the (+) chemotype (Figure 2; Table 1). In 1987, taxifolin glucoside concentrations were twice as high in current-year needles as in 1-yearold needles. Other hydrophilic UV-absorbing compounds (presumably phenolics) were not specifically quantified, but their total chromatogram peak areas were determined. Concentrations of these compounds in 1-year-old needles were higher in the (+) chemotype than in the ( - ) chemotype in both 1986 and 1987, i.e., 26% and 40%, respectively (Table 1). No differences were found with respect to these compounds in current-year needles. In 1986, concentrations of total resin acids were, on average, 69% higher in the ( - ) chemotype compared with the (+) chemotype (Table 1). Pinifolic




I 1 r






























Time (rain)

FIG. 2. HPLC separation of phenolic extractives from needles of (a) a pine tree containing taxifolin glucoside [ = (+) chemotype] and (b) a pine tree lacking taxifolin glucoside [ = ( - ) chemotype]. Peak numbers refer to (1) picein (internal standard), (2) ampelopsin glucoside, (3) taxifolin glucoside, and (4) eriodictyol glucoside (cf. Figure 1).

acid contributed most to the total resin acid content (66.5 %, averaged over the two types of trees). In 1987, a subset of five trees of each type was compared. These trees were selected in a way that minimized variation in resin acid concentrations. As a result, there was much less variation in resin acid concentrations in the 1987 experiment. No differences were found in total resin acid or pinifolic acid concentrations, but there were significantly higher concentrations of 4-epiimbricatolic acid and neoabietic acid in the ( + ) chemotype. There were no differences in concentrations of total nitrogen between the two types of trees (Table 1). Larval Performance. No differences were found in survival or cocoon weight between N. sertifer larvae raised on the ( + ) chemotype and those raised on the ( - ) chemotype (Table 2). No detailed measurements of larval development were made. However, according to a survey made on June 27 (late cocoon spinning stage), the proportion of larvae that had spun cocoons was 79 % on the ( + ) chemotype and 71% on the ( - ) chemotype. D. pini larvae developed more slowly on the ( + ) chemotype (Table 2). The difference was small (6%) and significant for female larvae (P < 0.05), but not significant for male larvae. No effects on body size were found.

0.00(0) 1.10(0.30) 0.186(0.054) 0.095(0.061) 0.105(0.046) 1.060(0.320) 1.540(0.356) 0.176(0.332) no data


3.33b(0.81) 1.39(0.29) 0.104(0.053) 0.109(0.075) 0.113(0.053) 0.586(0.113) 0.913(0.200) 0.040(0.141) no data

(+) 0.001 0.05 0.001 ns ns 0.001 0.001 ns --

p< 0.00(0) 2.55(0.20) 0.068(0.086) nd 0.090(0.024) 0.734(0.154) 1.125(0.476) 0.574(0.533) 1.31(0.13)

(-) 1.67(0.174) 3.57(0.43) 0.191(0.086) nd 0.205(0.079) 0.712(0.234) 0.940(0.301) 0.00(0) 1.35(0.15)


Old needles

0.001 0.01 0.05 -0.05 ns ns ns ns


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...
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