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Host plant influence on the composition of the defensive secretion of Chrysomela vigintipunctata larvae (Coleoptera: Chrysomelidae)
Biochemical Systematics and Ecology 26 (1998) 703 — 712
Host plant influence on the composition of the defensive secretion of Chrysomela vigintipunctata larvae (Coleoptera: Chrysomelidae) Philippe Soetens!,*, Jacques M. Pasteels!, De´sire´ Daloze", Michel Kaisin" aLaboratoire de Biologie Animale et Cellulaire, Faculte´ des Sciences, Universite´ Libre de Bruxelles, Av. F. D. Roosevelt, 50, 1050 Bruxelles, Belgium bLaboratoire de Chimie Bio-Organique, Faculte´ des Sciences, Universite´ Libre de Bruxelles, Av. F. D. Roosevelt, 50, 1050 Bruxelles, Belgium Received 2 December 1997; accepted 8 April 1998
Abstract Phenolic glucosides from willow leaves are used by Chrysomela vigintipunctata larvae as precursors of salicylaldehyde produced in the defensive secretion. When these larvae were fed on different Salix species with high to moderate phenolic glucoside content (S. purpurea, S. myrsinifolia, S. fragilis), their defensive secretion mainly contained salicylaldehyde and only traces of other constituents (benzaldehyde and phenylethanol). The volume of secretions was reduced in larvae fed on host plant with low to very low phenolic glucoside content (S. caprea). Salicylaldehyde remained the predominant constituent in most secretions, but in some, (Z)-3hexen-1-ol, benzaldehyde and phenylethanol, appeared as major constituents besides salicylaldehyde. Quantitative assessments demonstrate that these compounds did not compensate for the reduction in salicylaldehyde as compared with the amount produced when fed on Salix with high phenolic glucoside content. The results are discussed in terms of sequestration ability, defence, and host range observed in nature. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Chrysomela vigintipunctata; Chrysomelidae; Salicaceae; Phenolic glucosides; Salicylaldehyde; Defensive secretion; Host plant influence
1. Introduction Many Chrysomela species and Phratora vitellinae derive their larval defence, salicylaldehyde, from host-plant phenolic glucosides, i.e. salicin (Pasteels et al., 1983) * Corresponding author. 0305-1978/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 8 )00 0 39 -8
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and salicortin (at least in Ph. vitellinae, Soetens, 1993). In Ph. vitellinae, besides salicylaldehyde, no other volatile compound has been reported so far in larval secretions. When fed on leaves of Salix caprea, which do not contain phenolic glucosides or very little amounts (Shao, 1991), no detectable secretion was produced by the larvae (Rowell-Rahier and Pasteels, 1982). On the contrary, in some Chrysomela species, small amounts of benzaldehyde were reported in larval secretions (Matsuda and Sugawara, 1980; Pasteels et al., 1988) and, when fed on S. caprea, the larvae produce an obvious secretion indicating that host-derived salicylaldehyde is not the sole chemical defence. In this paper, we investigate these additional/alternative chemical defences in the larvae of Chrysomela vigintipunctata. This species was selected because it is reported to feed in nature on different willow species containing high to low amounts of phenolic glucosides (Topp and Beracz, 1989; Ko¨pf et al., in press). The larvae were fed with leaves of either Salix myrsinifolia, S. purpurea, S. fragilis or S. caprea; all natural food plants. Concentration of salicin and salicortin in the leaves of these Salix spp. were reported to be high in S. myrsinifolia and S. purpurea, low in S. fragilis and very low or absent in S. caprea (Julkunen—Tiitto, 1986; Julkunen—Tiitto and Tahvanainen, 1989; Shao, 1991). The volume of secretion produced by full-grown third instar larvae was measured. The chemical composition of the secretions, as well as the total amount and concentration of the major constituents in the secretions were determined. Since the amount of phenolic glucosides in leaves varies between individuals of the same species, the phenolic glucoside content in leaves of the willows used in this study was determined when possible. The results are discussed in terms of flexibility in defence and host-affiliation exemplified in the genus Chrysomela.
2. Material and methods 2.1. Biological material Beetles were collected in early spring on S. myrsinifolia (Petite Camargue, Saint Louis, France) and S. fragilis (Rochefort, Belgium). They were reared on their natural host plant in a growth room at 20°C on a 16 : 8 L : D cycle. Egg clusters were placed on fresh leaves of S. myrsinifolia, S. purpurea, S. fragilis and S. caprea and larvae were allowed to develop on these plants. Fresh leaves were provided every 2—3 days. Leaves of S. myrsinifolia (absent in Belgium) were sent from the Petite Camargue and were not available for chemical analysis. The leaves of the other willows were collected around Brussels. Two different shrubs of S. caprea were used in the experiments: one growing on the campus grounds of the Free University of Brussels, the other in Buizingen (20 km South of Brussels). They will be referred to as S. caprea A and B, respectively. Leaves from a single plant were used for all other willow species. From 30 to 60 larvae were reared in each treatment, i.e. on each plant and from beetles of both origins. Secretions of third instar larvae fed on S. myrsinifolia, S. fragilis
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or S. caprea A were collected individually in calibrated glass capillaries, sealed and stored at !30°C until analysis (see quantification of the major secretion components). Pooled secretions of five to ten larvae fed either on S. purpurea or S. caprea B were collected and stored in the same way. Secretions of larvae from each locality were collected separately. 2.2. Chemical analyses 2.2.1. Quantification of phenolic glucosides in host plants The leaves were prepared for analysis according to the method of Lindroth and Hwang (1996): freshly collected leaves were plunged into liquid nitrogen for a few minutes, freeze-dried for 24 h and then stored at !17°C until analyzed. The extraction and quantification of salicin, salicortin, tremulacin and tremuloidin in leaves of S. fragilis, S. purpurea and S. caprea were determined using the method of Meier et al. (1988), slightly modified as follows. A 200—500 mg amount of dried material was extracted in a clipping homogenisator once with 25 ml and then with 40 ml of methanol. The residue was washed with 20 ml of methanol and the solvent was evaporated using a vacuum evaporator, the water-bath being kept in ice/water. The sample was redissolved in 9 ml of methanol—water (7 : 2) and 1 ml of resorcinol solution (internal standard) was added. 0.5 ml of this extract was purified on a Bond Elut C (100 mg) solid-phase extraction column. The cartridge was washed with 18 0.5 ml of methanol—water (7 : 2). HPLC analyses were performed on a Waters LC Module 1 apparatus equipped with a 100]4.6 mm ID Alltech cartridge filled with Spherisorb ODS II (3 lm) as the stationary phase. The injection volume was 10 or 20 ll. The elution gradient used was the same as that of Meier et al. (1988). The quantities of salicin, salicortin, tremulacin and tremuloidin were calculated using reference phenolic glucosides and resorcinol as an internal standard (1 mg ml~1). Salicin and resorcinol are commercially available (SIGMA', respectively, 99.4 and 99.0% purity) and three other compounds (salicortin, tremulacin and tremuloidin, purity '99.0%) were obtained from Dr. Chenault. The percentage of the four phenolic glucosides on a dry weight basis (% D.W.) are reported in Table 1. 2.2.2. GC and GC—MS analyses Secretions collected in capillaries either pooled or from individuals were dissolved into 2 ll hexane. 0.5 or 1 ll of secretion solution were injected in the gas chromatograph. Capillary gas—liquid chromatographic analyses (GC) were performed on a Varian 3700 gas chromatograph with on-column injection, equipped with a 25 m]0.32 mm Carbowax 20M fused-silica column (Rescom) programmed from 50 to 200°C at 7°C min~1, using nitrogen as a carrier gas. The gas chromatography—mass spectrometry (GC—MS) analyses were determined using (a) a Finnigan ion-trap detector (ITD 800), coupled to a Tracor 540 gas chromatograph under the same conditions as the GC analyses, or (b) a Fisons VG AutoSpec coupled to a Fisons GC 8065 gas chromatograph with split injection (injector temperature 220°C), equipped with a 25 m]0.25 mm Carbowax 25M fused-silica column (Rescom) programmed as follows: 50—100°C at 4°C min~1, 100—180°C at 7°C min~1, isothermal
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condition at 180°C during 10 min~1, 180—200°C at 7°C min~1. For the GC—MS analyses, helium was used as the carrier gas. Ion intensities were recorded using either electron impact (EI, 70 eV) or chemical ionization (CI, 70 eV) with isobutane or NH as the reactant gas. Results of the 3 GC—MS analyses of the secretions from larvae reared on S. myrsinifolia, S. fragilis, S. purpurea and S. caprea are reported in Table 2. Identification of individual components based on MS data were confirmed by co-injection in GC with authentic samples obtained from Sigma-Aldrich'. 8-Hydroxylinalool was synthesized following the method described in Behr et al., 1978. 2.2.3. Quantification of the major secretion components The concentrations and the quantities of the major compounds in individual secretions, namely salicylaldehyde, benzaldehyde, 2-phenylethanol and (Z)-3-hexen1-ol were determined by GC and are reported in Table 3. Phenetole was used as an internal standard and calibration factors were determined for each compound using reference material of at least 98% purity from Sigma-Aldrich'. The concentrations given in Table 3 are mean values calculated from analyses of three different secretions of larvae reared on S. myrsinifolia and S. fragilis and of four different secretions of larvae reared on S. caprea. Each sample was analyzed in triplicate.
3. Results 3.1. Phenolic glucosides in leaves of host plants The concentrations of the four major phenolic glucosides in the leaves of the different willows are given in Table 1. Salicin and salicortin, but not tremulacin, are used by the larvae of Ph. vitellinae to derive salicylaldehyde (Soetens, 1993). The amount of salicortin was thus transformed in salicin-equivalent by taking into account their respective mass—weight, and added to the amount of salicin to obtain the total salicin-equivalent. This offers a better basis of comparison between plants than the concentrations of each compound, assuming that C. vigintipunctata derives salicylaldehyde as does Ph. vitellinae.
Table 1 Quantities of phenolic glucosides determined by HPLC in different Salix species (% D.W.)
Salicin Salicortin Tremuloidin Tremulacin Salicin-equivalent
S. myrsinifolia (from Shao, 1991)
S. purpurea
S. fragilis
S. caprea A
S. caprea B
0.46 3.58 0.00 0.00 2.88
0.06 4.97 0.62 3.29 3.41
0.03 0.59 0.03 1.75 0.42
0.004 0.016 0.025 0.061 0.015
0.015 0.064 0.019 0.079 0.058
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As reported in the literature (Shao, 1991) S. purpurea had high contents of phenolic glucosides in its leaves with 3.4 total salicin-equivalent (% D.W.). S. fragilis had a much lower content in phenolic glucosides (0.4 total salicin-equivalent) and S. caprea still less. The two shrubs of S. caprea varied in their content of phenolic glucosides. S. caprea A had the lowest total salicin-equivalent content (0.015, whereas this content reaches 0.058 in S. caprea B). Since S. myrsinifolia was not available in this study, values given in Table 1 are from Shao (1991). 3.2. Volume of the secretions The volume of larval secretion was identical in larvae fed on S. myrsinifolia (0.103 ll $ 0.029; mean $ s.d. n"10) or S. fragilis (0.103 ll $ 0.019, n"10) despite the fact that the total salicin-equivalent content is much higher in the leaves of the first willow than in the leaves of the second. The volume of secretion of larvae fed on S. purpurea was not measured but appeared to be very similar. However, the volume of secretion (0.047 ll $ 0.019, n"20) was reduced in larvae fed on Salix caprea A, with very low salicin-equivalent in its leaves. The volume of secretion also looked reduced in larvae fed on S. caprea B, but was not quantified. 3.3. Qualitative composition of the secretions In Table 2, the compounds identified in the different secretions are listed as well as their relative abundance expressed as percentage of salicylaldehyde, estimated by their relative peak areas in the chromatograms. No obvious differences were observed between larvae of beetles collected in France or Belgium. In all secretions, salicylaldehyde was the major constituent, except for larvae reared on S. caprea. However, the relative abundance of the other constituents varied according to the host plants. In the secretions of larvae reared on S. myrsinifolia, S. fragilis and S. purpurea, salicylaldehyde was the only major constituent, and the others are at best minor compounds (e.g. benzaldehyde in secretions of larvae fed on S. purpurea) or trace constituents (less than 1% of salicylaldehyde). The secretions of larvae fed on S. caprea were more complex and other compounds were either minor constituents (between 1 and 10% of salicylaldehyde) or even major constituents (above 10% of salicylaldehyde). The secretions of larvae fed on the two different S. caprea were quite distinct in the proportion of their constituents. When fed on S. caprea A, but not on S. caprea B, (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol were major constituents besides salicylaldehyde. 3.4. Quantity of salicylaldehyde and other major constituents secreted The higher proportion of some constituents in the secretion of larvae fed on S. caprea A could be due to a decrease in salicylaldehyde production or to an increase in the production of the other constituents, or both. To check these possibilities salicylaldehyde and these constituents were quantified in secretions of individual larvae (Table 3). As expected, the amount and concentration of salicylaldehyde were
Hexanal (E)-2-Hexenal 1-Hexanol (Z)-3-Hexen-1-ol (Z)-2-Hexen-1-ol (E)-3-Hexen-1-ol Benzaldehyde Phenylacetaldehyde Salicylaldehyde Furfuryl alcohol Geraniol Benzylalcohol Phenylethanol Phenol Eugenol 2-Phenoxyethanol Methyl palmitate 8-Hydroxylinalool Methyl stearate Benzoic acid 5(Hydroxymethyl) furfural Hexadecanoic acid # #
0.0061 0.0014 0.0168
#
#
0.0004
#
##
#
0.0704 0.0005
100
0.0606
0.0871 0.0003
0.2355
0.0745 0.1077 0.0003
100
1.6417
0.0024
S. purpurea F (1) Be (2)
#
##
#
S. myrsinifolia F (1) F (2)
tr #
##
#
0.2744
0.0023 0.0288
0.0055
0.2692 0.0166 0.0083
100
0.3809
0.0139
S. fragilis F (1) Be (2)
A and B refer to 2 different bushes of S. caprea. F (France) and Be (Belgium) refer to the origin of the beetles. (1) and (2) refer to the analytical method. (1): ITD 800, tr: trace ((1%), #: minor ((10%), ##: major; (2): VG AutoSpec, proportion calculated as percentage of salicylaldehyde, based on pic area.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Compounds
Table 2 Components of the defensive secretions of C. vigintipunctata larvae fed on different Salix spp.
#
# #
# ## #
##
tr ## tr tr ##
A F (1)
22.48 1.11 100 0.33 0.12 2.33 29.90 7.46 9.25 11.23 1.07 13.92 0.28 1.28 12.60 2.67
2.50 7.17 0.83 38.58
A Be (2)
S. caprea
1.50
0.36 0.02 0.12 6.50
0.01 0.04 2.34 0.06 0.25
1.89 0.02 100
0.36 0.15
B Be (2)
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Table 3 Mean concentrations (C, lg ll~1) ($SE) and quantities (Q, lg/larva) ($SE) of main components determined by GC in the secretions of C. vigintipunctata larvae reared on different Salix species. Host plant
S. myrsinifolia
S. fragilis
S. caprea A
Salicylaldehyde
C Q C Q C Q C Q
403$69 172$7 2.57$0.34 1.10$0.02 1.37$0.36 0.60$0.19 ND ND
0.83$0.35 0.17$0.11 0.81$0.26 0.18$0.14 1.79$0.92 0.37$0.30 1.06$0.23 0.22$0.08
Benzaldehyde 2-Phenylethanol (Z)-3-hexen-1-ol
526$190 188$73 0.04 or NQ 0.02 or NQ NQ NQ ND ND
Note: NQ: too low to be quantified. ND: not detected.
much reduced (by nearly three orders of magnitude) in the secretions of larvae fed on S. caprea A. The production of salicylaldehyde was somewhat lower in larvae fed on S. fragilis which contained small amounts of total salicin-equivalent in its leaves than when fed on S. myrsinifolia containing a higher amount of total salicin-equivalent. However, the difference is not statistically significant (Anova, F"1.11, P"0.351, DF : 4,1). A larger sample should be studied to check for a possible influence of the host plant on the production of salicylaldehyde when the leaves vary in total salicin-equivalent content. In any case, the amount produced was not proportional to the amount of total salicin-equivalent observed in the plants (see Table 1). The amount of (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol in the secretion vary with the food plant. However, these compounds always remain minor constituents compared with salicylaldehyde produced by larvae fed on S. myrsinifolia and S. fragilis.
4. Discussion Chemical defence was much reduced in larvae fed on Salix caprea. Not only was the volume of secretion that was produced about half, but the total amount of secreted volatiles were even more reduced. As expected, it is salicylaldehyde that is considerably reduced in the secretion of these larvae (by three orders of magnitude). However, quite unexpectedly, salicylaldehyde still remained the major constituent in most secretions of larvae fed on S. caprea. This is in sharp contrast with previous results for Phratora vitellinae which did not produce any detectable amounts of secretion when fed on S. caprea (Rowell-Rahier and Pasteels, 1982). Some salicin could be transferred from the females (fed on S. myrsinifolia or S. fragilis) to their eggs and later used by the larvae to produce salicylaldehyde (up to 2 lg/egg when fed on S. purpurea, Pasteels et al., 1986), but this maternal transfer cannot explain the amount of salicylaldehyde
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found in the secretions of 3rd instar larvae since the secretion appeared much reduced after each moult and most of it is probably discarded with the exuvia. We cannot totally exclude the possibility, but it seems doubtful that salicylaldehyde is actually synthesized by the larvae. It seems far more likely that C. vigintipunctata (as well as other Chrysomela species, unpublished results) has a much greater ability than Ph. vitellinae to sequester and transform traces of salicin and salicortin present in S. caprea leaves. When larvae were fed on leaves of S. caprea A, which contain very low levels of salicin-equivalent, salicylaldehyde was only a major constituent among others. Salicylaldehyde was more abundant than (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol in some secretions (Table 2), but not in all (Table 3). However, when fed on S. caprea B containing four times more salicin-equivalent, salicylaldehyde is by far the dominant compound in the secretion. This high sequestering ability could explain why the larvae produced nearly as much salicylaldehyde when fed on S. fragilis as when fed on S. myrsinifolia, although the first plant has much lower amounts of salicin and salicortin in its leaves than the second. Except for benzaldehyde, the other constituents were never reported in Chrysomela species which specialize on Salicaceae. The origin of these compounds remains to be elucidated, but many are probably sequestered from host-plant or derived from host-plant precursors. Indeed some of the identified compounds are frequently encountered in plant leaves: hexanal, (E)-2-hexenal, hexanol, (Z)-3-hexen-1-ol, geraniol, benzylalcohol, phenylethanol, eugenol and 8-hydroxylinalool were reported in extracts of macerated leaves or leaf extracts after glucosidase treatment of S. fragilis (Schulz et al., 1997). Benzaldehyde could be produced by the oxidation of benzylalcohol by an oxidase present in the secretion, as reported for other Chrysomela species (Pasteels et al., 1990). We cannot exclude, however, a more active participation of the insects in the biosynthesis of some compounds. For example, some phenylethanol is derived from phenylalanine in C. lapponica (Schultz et al., 1997) and in C. vigintipunctata (Soetens et al., in prep.). In any case, when fed on S. caprea, the larvae of C. vigintipunctata do not seem to be able to compensate for the loss of salicylaldehyde by an increased neosynthesis. Interestingly, no isobutyric- or 2-methylbutyric acids or esters of these were found in the secretion of C. vigintipunctata. These acids are synthesized by the larvae of C. lapponica, respectively, from valine and isoleucine (Schultz et al., 1997). So far, these isobutyrates and 2-methylbutyrates were observed in the larval secretions of C. interrupta (Blum et al., 1972), C. lapponica (Hilker and Schulz, 1994, and unpublished results) and in C. walshi, C. mainensis and C. knabi (unpublished results). This neosynthesis from amino acids appears to be restricted to members of the Chrysomela interrupta group of species (sensu Brown, 1956). If chemical defence is reduced when the larvae feed on S. caprea, it would be premature to conclude that the risk of predation is increased in all circumstances. Defence against generalist predators is certainly reduced. Salicylaldehyde was shown to be highly effective in deterring ants (Pasteels et al., 1986) or spiders (Palokangas et al., 1992). However, in the field, the level of predation was not correlated with the amount of salicylaldehyde produced by larvae of Ph. vitellinae feeding on different willows (Rank et al., in press). Actually, some predators, e.g. syrphid larvae, which
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specialize in feeding on Chrysomelinae larvae use the larval defensive secretion as a cue to find their prey, (Rank et al., 1996; Ko¨pf et al., 1997). In Chrysomela aenicollis, feeding on willows with low content of phenolic glucoside is correlated with a very low production of salicylaldehyde, possibly as an adaptation to escape intense predation by specialist wasps (Rank, 1994). Flexibility of defence by using a wider range of plant compounds or by the ability to synthesize additional compounds (e.g. isobutyrates and 2-methylbutyrates) could allow leaf beetles to increase their host-range or even to shift on new host-plants, possibly under the selective pressure of specialized natural enemies. Interestingly, several species of the Chrysomela interrupta group in which the ability to synthesize isobutyrates and 2-methylbutyrates evolved, shifted during speciation from Salicaceae to Betulaceae (Brown, 1956).
Acknowledgements We are grateful to the ‘‘Communaute´ franiaise de Belgique’’ (ARC 93-3318) for financial support. We thank Dr J. Chenault (Laboratoire de Chimie Bioorganique et Analytique, Orle´ans) for sending to us reference phenolic glucosides. The authors also thank Dr M. Rowell—Rahier for sending us insects and leaves of S. myrsinifolia from Petite Camargue (Saint Louis, France).
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Meier, B., Julkunen-Tiitto, R., Tahvanainen, J., Sticher, O., 1988 Comparative high-performance liquid and gas—liquid chromatographic determination of phenolic glucosides in Salicaceae species. J. Chromat. 442, 175—186. Palokangas, P., Neuvonen, S., Haapala, S., 1992. Effect of host plants on growth and defence in two Phratora (Coleoptera: Chrysomelidae) species. In: Menkel, S. B. J., Visser, J. H., Harrewijn, P. (Eds.), Proc. 8th Int. Symp. Insect—Plant Relationships Kluwer, Dordrecht, pp. 343—344. Pasteels, J. M., Rowell-Rahier, M., Braekman, J. C., Dupont, A., 1983. Salicin from host plant as precursor of salicylaldehyde in defensive secretion of chrysomeline larvae. Physiol. Entomol. 8, 307—314. Pasteels, J. M., Daloze, D., Rowell-Rahier, M., 1986. Chemical defence in chrysomelid eggs and neonate larvae. Physiol. Entomol. 11, 29—37. Pasteels, J. M., Braekman, J. C., Daloze, D., 1988. Chemical defense in the Chrysomelidae. In: Jolivet, P., Petitpierre, E., Hsiao, T. H. (Eds.), Biology of Chrysomelidae. Kluwer, Dordrecht, pp. 233—252. Pasteels, J. M., Duffey, S., Rowell-Rahier, M., 1990. Toxins in chrysomelid beetles. Possible evolutionary sequence from de novo synthesis to derivation from food—plant chemicals. J. Chem. Ecol. 16, 211—222. Rank, N. E., 1994. Host-plant effects on larval survival of salicin-using leaf beetle Chrysomela aenicollis Schaeffer (Coleoptera: Chrysomelidae). Oecologia 94, 342—353. Rank, N. E., Ko¨pf, A., Julkunen-Tiitto, R., Tahvanainen, J., 1998. Larval survival for a salicylate-using leaf beetle: Does the host plant chemistry matter? Ecology 179, 618—631. Rank, N. E., Smiley, J. T., Ko¨pf, A., 1996. Natural enemies and host plant relationships for leaf beetles (Chrysomelidae) feeding on Salicaceae and Betulaceae. In: Jolivet, P. H., Cox, M. L. (Eds.), Chrysomelidae Biology. Ecological Studies vol. 2. SPB, Amsterdam, pp. 147—172. Rowell-Rahier, M., Pasteels, J. M., 1982. The significance of salicin for a Salix-feeder, Phratora ("Phyllodecta) vitellinae. In: Visser, J. H., Minks, A. K., (Eds.), Proc. 5th Int. Symp. Insect—Plant Relationships. PUDOC, Wageningen, pp. 73—79. Schulz, S., Gross, J., Hilker, M., 1997. Origin of the defensive secretion of the leaf beetle Chrysomela lapponica. Tetrahedron, 53, 9203—9212. Shao, Y., 1991. Phytochemischer Atlas der Schweizer Weiden. Diss. ETH Nr. 9532. 198 p. Soetens, P., 1993. Analyse des relations entre Phratora vitellinae (L.) et Ph. tibialis Suffr. (Coleoptera: Chrysomelidae) et les Salicaceae. The`se de Doctorat, ULB. 212 p. Topp, W., Beracz, P., 1989. Effect of host plant and changing seasonal development on consumption rates, utilization efficiencies and survival of Melasoma 20-punctata, Scop. (Col., Chrysomelidae). J. Appl. Entomol. 107, 261—274.
Host plant influence on the composition of the defensive secretion of Chrysomela vigintipunctata larvae (Coleoptera: Chrysomelidae) Philippe Soetens!,*, Jacques M. Pasteels!, De´sire´ Daloze", Michel Kaisin" aLaboratoire de Biologie Animale et Cellulaire, Faculte´ des Sciences, Universite´ Libre de Bruxelles, Av. F. D. Roosevelt, 50, 1050 Bruxelles, Belgium bLaboratoire de Chimie Bio-Organique, Faculte´ des Sciences, Universite´ Libre de Bruxelles, Av. F. D. Roosevelt, 50, 1050 Bruxelles, Belgium Received 2 December 1997; accepted 8 April 1998
Abstract Phenolic glucosides from willow leaves are used by Chrysomela vigintipunctata larvae as precursors of salicylaldehyde produced in the defensive secretion. When these larvae were fed on different Salix species with high to moderate phenolic glucoside content (S. purpurea, S. myrsinifolia, S. fragilis), their defensive secretion mainly contained salicylaldehyde and only traces of other constituents (benzaldehyde and phenylethanol). The volume of secretions was reduced in larvae fed on host plant with low to very low phenolic glucoside content (S. caprea). Salicylaldehyde remained the predominant constituent in most secretions, but in some, (Z)-3hexen-1-ol, benzaldehyde and phenylethanol, appeared as major constituents besides salicylaldehyde. Quantitative assessments demonstrate that these compounds did not compensate for the reduction in salicylaldehyde as compared with the amount produced when fed on Salix with high phenolic glucoside content. The results are discussed in terms of sequestration ability, defence, and host range observed in nature. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Chrysomela vigintipunctata; Chrysomelidae; Salicaceae; Phenolic glucosides; Salicylaldehyde; Defensive secretion; Host plant influence
1. Introduction Many Chrysomela species and Phratora vitellinae derive their larval defence, salicylaldehyde, from host-plant phenolic glucosides, i.e. salicin (Pasteels et al., 1983) * Corresponding author. 0305-1978/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 8 )00 0 39 -8
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and salicortin (at least in Ph. vitellinae, Soetens, 1993). In Ph. vitellinae, besides salicylaldehyde, no other volatile compound has been reported so far in larval secretions. When fed on leaves of Salix caprea, which do not contain phenolic glucosides or very little amounts (Shao, 1991), no detectable secretion was produced by the larvae (Rowell-Rahier and Pasteels, 1982). On the contrary, in some Chrysomela species, small amounts of benzaldehyde were reported in larval secretions (Matsuda and Sugawara, 1980; Pasteels et al., 1988) and, when fed on S. caprea, the larvae produce an obvious secretion indicating that host-derived salicylaldehyde is not the sole chemical defence. In this paper, we investigate these additional/alternative chemical defences in the larvae of Chrysomela vigintipunctata. This species was selected because it is reported to feed in nature on different willow species containing high to low amounts of phenolic glucosides (Topp and Beracz, 1989; Ko¨pf et al., in press). The larvae were fed with leaves of either Salix myrsinifolia, S. purpurea, S. fragilis or S. caprea; all natural food plants. Concentration of salicin and salicortin in the leaves of these Salix spp. were reported to be high in S. myrsinifolia and S. purpurea, low in S. fragilis and very low or absent in S. caprea (Julkunen—Tiitto, 1986; Julkunen—Tiitto and Tahvanainen, 1989; Shao, 1991). The volume of secretion produced by full-grown third instar larvae was measured. The chemical composition of the secretions, as well as the total amount and concentration of the major constituents in the secretions were determined. Since the amount of phenolic glucosides in leaves varies between individuals of the same species, the phenolic glucoside content in leaves of the willows used in this study was determined when possible. The results are discussed in terms of flexibility in defence and host-affiliation exemplified in the genus Chrysomela.
2. Material and methods 2.1. Biological material Beetles were collected in early spring on S. myrsinifolia (Petite Camargue, Saint Louis, France) and S. fragilis (Rochefort, Belgium). They were reared on their natural host plant in a growth room at 20°C on a 16 : 8 L : D cycle. Egg clusters were placed on fresh leaves of S. myrsinifolia, S. purpurea, S. fragilis and S. caprea and larvae were allowed to develop on these plants. Fresh leaves were provided every 2—3 days. Leaves of S. myrsinifolia (absent in Belgium) were sent from the Petite Camargue and were not available for chemical analysis. The leaves of the other willows were collected around Brussels. Two different shrubs of S. caprea were used in the experiments: one growing on the campus grounds of the Free University of Brussels, the other in Buizingen (20 km South of Brussels). They will be referred to as S. caprea A and B, respectively. Leaves from a single plant were used for all other willow species. From 30 to 60 larvae were reared in each treatment, i.e. on each plant and from beetles of both origins. Secretions of third instar larvae fed on S. myrsinifolia, S. fragilis
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or S. caprea A were collected individually in calibrated glass capillaries, sealed and stored at !30°C until analysis (see quantification of the major secretion components). Pooled secretions of five to ten larvae fed either on S. purpurea or S. caprea B were collected and stored in the same way. Secretions of larvae from each locality were collected separately. 2.2. Chemical analyses 2.2.1. Quantification of phenolic glucosides in host plants The leaves were prepared for analysis according to the method of Lindroth and Hwang (1996): freshly collected leaves were plunged into liquid nitrogen for a few minutes, freeze-dried for 24 h and then stored at !17°C until analyzed. The extraction and quantification of salicin, salicortin, tremulacin and tremuloidin in leaves of S. fragilis, S. purpurea and S. caprea were determined using the method of Meier et al. (1988), slightly modified as follows. A 200—500 mg amount of dried material was extracted in a clipping homogenisator once with 25 ml and then with 40 ml of methanol. The residue was washed with 20 ml of methanol and the solvent was evaporated using a vacuum evaporator, the water-bath being kept in ice/water. The sample was redissolved in 9 ml of methanol—water (7 : 2) and 1 ml of resorcinol solution (internal standard) was added. 0.5 ml of this extract was purified on a Bond Elut C (100 mg) solid-phase extraction column. The cartridge was washed with 18 0.5 ml of methanol—water (7 : 2). HPLC analyses were performed on a Waters LC Module 1 apparatus equipped with a 100]4.6 mm ID Alltech cartridge filled with Spherisorb ODS II (3 lm) as the stationary phase. The injection volume was 10 or 20 ll. The elution gradient used was the same as that of Meier et al. (1988). The quantities of salicin, salicortin, tremulacin and tremuloidin were calculated using reference phenolic glucosides and resorcinol as an internal standard (1 mg ml~1). Salicin and resorcinol are commercially available (SIGMA', respectively, 99.4 and 99.0% purity) and three other compounds (salicortin, tremulacin and tremuloidin, purity '99.0%) were obtained from Dr. Chenault. The percentage of the four phenolic glucosides on a dry weight basis (% D.W.) are reported in Table 1. 2.2.2. GC and GC—MS analyses Secretions collected in capillaries either pooled or from individuals were dissolved into 2 ll hexane. 0.5 or 1 ll of secretion solution were injected in the gas chromatograph. Capillary gas—liquid chromatographic analyses (GC) were performed on a Varian 3700 gas chromatograph with on-column injection, equipped with a 25 m]0.32 mm Carbowax 20M fused-silica column (Rescom) programmed from 50 to 200°C at 7°C min~1, using nitrogen as a carrier gas. The gas chromatography—mass spectrometry (GC—MS) analyses were determined using (a) a Finnigan ion-trap detector (ITD 800), coupled to a Tracor 540 gas chromatograph under the same conditions as the GC analyses, or (b) a Fisons VG AutoSpec coupled to a Fisons GC 8065 gas chromatograph with split injection (injector temperature 220°C), equipped with a 25 m]0.25 mm Carbowax 25M fused-silica column (Rescom) programmed as follows: 50—100°C at 4°C min~1, 100—180°C at 7°C min~1, isothermal
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condition at 180°C during 10 min~1, 180—200°C at 7°C min~1. For the GC—MS analyses, helium was used as the carrier gas. Ion intensities were recorded using either electron impact (EI, 70 eV) or chemical ionization (CI, 70 eV) with isobutane or NH as the reactant gas. Results of the 3 GC—MS analyses of the secretions from larvae reared on S. myrsinifolia, S. fragilis, S. purpurea and S. caprea are reported in Table 2. Identification of individual components based on MS data were confirmed by co-injection in GC with authentic samples obtained from Sigma-Aldrich'. 8-Hydroxylinalool was synthesized following the method described in Behr et al., 1978. 2.2.3. Quantification of the major secretion components The concentrations and the quantities of the major compounds in individual secretions, namely salicylaldehyde, benzaldehyde, 2-phenylethanol and (Z)-3-hexen1-ol were determined by GC and are reported in Table 3. Phenetole was used as an internal standard and calibration factors were determined for each compound using reference material of at least 98% purity from Sigma-Aldrich'. The concentrations given in Table 3 are mean values calculated from analyses of three different secretions of larvae reared on S. myrsinifolia and S. fragilis and of four different secretions of larvae reared on S. caprea. Each sample was analyzed in triplicate.
3. Results 3.1. Phenolic glucosides in leaves of host plants The concentrations of the four major phenolic glucosides in the leaves of the different willows are given in Table 1. Salicin and salicortin, but not tremulacin, are used by the larvae of Ph. vitellinae to derive salicylaldehyde (Soetens, 1993). The amount of salicortin was thus transformed in salicin-equivalent by taking into account their respective mass—weight, and added to the amount of salicin to obtain the total salicin-equivalent. This offers a better basis of comparison between plants than the concentrations of each compound, assuming that C. vigintipunctata derives salicylaldehyde as does Ph. vitellinae.
Table 1 Quantities of phenolic glucosides determined by HPLC in different Salix species (% D.W.)
Salicin Salicortin Tremuloidin Tremulacin Salicin-equivalent
S. myrsinifolia (from Shao, 1991)
S. purpurea
S. fragilis
S. caprea A
S. caprea B
0.46 3.58 0.00 0.00 2.88
0.06 4.97 0.62 3.29 3.41
0.03 0.59 0.03 1.75 0.42
0.004 0.016 0.025 0.061 0.015
0.015 0.064 0.019 0.079 0.058
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As reported in the literature (Shao, 1991) S. purpurea had high contents of phenolic glucosides in its leaves with 3.4 total salicin-equivalent (% D.W.). S. fragilis had a much lower content in phenolic glucosides (0.4 total salicin-equivalent) and S. caprea still less. The two shrubs of S. caprea varied in their content of phenolic glucosides. S. caprea A had the lowest total salicin-equivalent content (0.015, whereas this content reaches 0.058 in S. caprea B). Since S. myrsinifolia was not available in this study, values given in Table 1 are from Shao (1991). 3.2. Volume of the secretions The volume of larval secretion was identical in larvae fed on S. myrsinifolia (0.103 ll $ 0.029; mean $ s.d. n"10) or S. fragilis (0.103 ll $ 0.019, n"10) despite the fact that the total salicin-equivalent content is much higher in the leaves of the first willow than in the leaves of the second. The volume of secretion of larvae fed on S. purpurea was not measured but appeared to be very similar. However, the volume of secretion (0.047 ll $ 0.019, n"20) was reduced in larvae fed on Salix caprea A, with very low salicin-equivalent in its leaves. The volume of secretion also looked reduced in larvae fed on S. caprea B, but was not quantified. 3.3. Qualitative composition of the secretions In Table 2, the compounds identified in the different secretions are listed as well as their relative abundance expressed as percentage of salicylaldehyde, estimated by their relative peak areas in the chromatograms. No obvious differences were observed between larvae of beetles collected in France or Belgium. In all secretions, salicylaldehyde was the major constituent, except for larvae reared on S. caprea. However, the relative abundance of the other constituents varied according to the host plants. In the secretions of larvae reared on S. myrsinifolia, S. fragilis and S. purpurea, salicylaldehyde was the only major constituent, and the others are at best minor compounds (e.g. benzaldehyde in secretions of larvae fed on S. purpurea) or trace constituents (less than 1% of salicylaldehyde). The secretions of larvae fed on S. caprea were more complex and other compounds were either minor constituents (between 1 and 10% of salicylaldehyde) or even major constituents (above 10% of salicylaldehyde). The secretions of larvae fed on the two different S. caprea were quite distinct in the proportion of their constituents. When fed on S. caprea A, but not on S. caprea B, (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol were major constituents besides salicylaldehyde. 3.4. Quantity of salicylaldehyde and other major constituents secreted The higher proportion of some constituents in the secretion of larvae fed on S. caprea A could be due to a decrease in salicylaldehyde production or to an increase in the production of the other constituents, or both. To check these possibilities salicylaldehyde and these constituents were quantified in secretions of individual larvae (Table 3). As expected, the amount and concentration of salicylaldehyde were
Hexanal (E)-2-Hexenal 1-Hexanol (Z)-3-Hexen-1-ol (Z)-2-Hexen-1-ol (E)-3-Hexen-1-ol Benzaldehyde Phenylacetaldehyde Salicylaldehyde Furfuryl alcohol Geraniol Benzylalcohol Phenylethanol Phenol Eugenol 2-Phenoxyethanol Methyl palmitate 8-Hydroxylinalool Methyl stearate Benzoic acid 5(Hydroxymethyl) furfural Hexadecanoic acid # #
0.0061 0.0014 0.0168
#
#
0.0004
#
##
#
0.0704 0.0005
100
0.0606
0.0871 0.0003
0.2355
0.0745 0.1077 0.0003
100
1.6417
0.0024
S. purpurea F (1) Be (2)
#
##
#
S. myrsinifolia F (1) F (2)
tr #
##
#
0.2744
0.0023 0.0288
0.0055
0.2692 0.0166 0.0083
100
0.3809
0.0139
S. fragilis F (1) Be (2)
A and B refer to 2 different bushes of S. caprea. F (France) and Be (Belgium) refer to the origin of the beetles. (1) and (2) refer to the analytical method. (1): ITD 800, tr: trace ((1%), #: minor ((10%), ##: major; (2): VG AutoSpec, proportion calculated as percentage of salicylaldehyde, based on pic area.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Compounds
Table 2 Components of the defensive secretions of C. vigintipunctata larvae fed on different Salix spp.
#
# #
# ## #
##
tr ## tr tr ##
A F (1)
22.48 1.11 100 0.33 0.12 2.33 29.90 7.46 9.25 11.23 1.07 13.92 0.28 1.28 12.60 2.67
2.50 7.17 0.83 38.58
A Be (2)
S. caprea
1.50
0.36 0.02 0.12 6.50
0.01 0.04 2.34 0.06 0.25
1.89 0.02 100
0.36 0.15
B Be (2)
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Table 3 Mean concentrations (C, lg ll~1) ($SE) and quantities (Q, lg/larva) ($SE) of main components determined by GC in the secretions of C. vigintipunctata larvae reared on different Salix species. Host plant
S. myrsinifolia
S. fragilis
S. caprea A
Salicylaldehyde
C Q C Q C Q C Q
403$69 172$7 2.57$0.34 1.10$0.02 1.37$0.36 0.60$0.19 ND ND
0.83$0.35 0.17$0.11 0.81$0.26 0.18$0.14 1.79$0.92 0.37$0.30 1.06$0.23 0.22$0.08
Benzaldehyde 2-Phenylethanol (Z)-3-hexen-1-ol
526$190 188$73 0.04 or NQ 0.02 or NQ NQ NQ ND ND
Note: NQ: too low to be quantified. ND: not detected.
much reduced (by nearly three orders of magnitude) in the secretions of larvae fed on S. caprea A. The production of salicylaldehyde was somewhat lower in larvae fed on S. fragilis which contained small amounts of total salicin-equivalent in its leaves than when fed on S. myrsinifolia containing a higher amount of total salicin-equivalent. However, the difference is not statistically significant (Anova, F"1.11, P"0.351, DF : 4,1). A larger sample should be studied to check for a possible influence of the host plant on the production of salicylaldehyde when the leaves vary in total salicin-equivalent content. In any case, the amount produced was not proportional to the amount of total salicin-equivalent observed in the plants (see Table 1). The amount of (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol in the secretion vary with the food plant. However, these compounds always remain minor constituents compared with salicylaldehyde produced by larvae fed on S. myrsinifolia and S. fragilis.
4. Discussion Chemical defence was much reduced in larvae fed on Salix caprea. Not only was the volume of secretion that was produced about half, but the total amount of secreted volatiles were even more reduced. As expected, it is salicylaldehyde that is considerably reduced in the secretion of these larvae (by three orders of magnitude). However, quite unexpectedly, salicylaldehyde still remained the major constituent in most secretions of larvae fed on S. caprea. This is in sharp contrast with previous results for Phratora vitellinae which did not produce any detectable amounts of secretion when fed on S. caprea (Rowell-Rahier and Pasteels, 1982). Some salicin could be transferred from the females (fed on S. myrsinifolia or S. fragilis) to their eggs and later used by the larvae to produce salicylaldehyde (up to 2 lg/egg when fed on S. purpurea, Pasteels et al., 1986), but this maternal transfer cannot explain the amount of salicylaldehyde
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found in the secretions of 3rd instar larvae since the secretion appeared much reduced after each moult and most of it is probably discarded with the exuvia. We cannot totally exclude the possibility, but it seems doubtful that salicylaldehyde is actually synthesized by the larvae. It seems far more likely that C. vigintipunctata (as well as other Chrysomela species, unpublished results) has a much greater ability than Ph. vitellinae to sequester and transform traces of salicin and salicortin present in S. caprea leaves. When larvae were fed on leaves of S. caprea A, which contain very low levels of salicin-equivalent, salicylaldehyde was only a major constituent among others. Salicylaldehyde was more abundant than (Z)-3-hexen-1-ol, benzaldehyde and phenylethanol in some secretions (Table 2), but not in all (Table 3). However, when fed on S. caprea B containing four times more salicin-equivalent, salicylaldehyde is by far the dominant compound in the secretion. This high sequestering ability could explain why the larvae produced nearly as much salicylaldehyde when fed on S. fragilis as when fed on S. myrsinifolia, although the first plant has much lower amounts of salicin and salicortin in its leaves than the second. Except for benzaldehyde, the other constituents were never reported in Chrysomela species which specialize on Salicaceae. The origin of these compounds remains to be elucidated, but many are probably sequestered from host-plant or derived from host-plant precursors. Indeed some of the identified compounds are frequently encountered in plant leaves: hexanal, (E)-2-hexenal, hexanol, (Z)-3-hexen-1-ol, geraniol, benzylalcohol, phenylethanol, eugenol and 8-hydroxylinalool were reported in extracts of macerated leaves or leaf extracts after glucosidase treatment of S. fragilis (Schulz et al., 1997). Benzaldehyde could be produced by the oxidation of benzylalcohol by an oxidase present in the secretion, as reported for other Chrysomela species (Pasteels et al., 1990). We cannot exclude, however, a more active participation of the insects in the biosynthesis of some compounds. For example, some phenylethanol is derived from phenylalanine in C. lapponica (Schultz et al., 1997) and in C. vigintipunctata (Soetens et al., in prep.). In any case, when fed on S. caprea, the larvae of C. vigintipunctata do not seem to be able to compensate for the loss of salicylaldehyde by an increased neosynthesis. Interestingly, no isobutyric- or 2-methylbutyric acids or esters of these were found in the secretion of C. vigintipunctata. These acids are synthesized by the larvae of C. lapponica, respectively, from valine and isoleucine (Schultz et al., 1997). So far, these isobutyrates and 2-methylbutyrates were observed in the larval secretions of C. interrupta (Blum et al., 1972), C. lapponica (Hilker and Schulz, 1994, and unpublished results) and in C. walshi, C. mainensis and C. knabi (unpublished results). This neosynthesis from amino acids appears to be restricted to members of the Chrysomela interrupta group of species (sensu Brown, 1956). If chemical defence is reduced when the larvae feed on S. caprea, it would be premature to conclude that the risk of predation is increased in all circumstances. Defence against generalist predators is certainly reduced. Salicylaldehyde was shown to be highly effective in deterring ants (Pasteels et al., 1986) or spiders (Palokangas et al., 1992). However, in the field, the level of predation was not correlated with the amount of salicylaldehyde produced by larvae of Ph. vitellinae feeding on different willows (Rank et al., in press). Actually, some predators, e.g. syrphid larvae, which
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specialize in feeding on Chrysomelinae larvae use the larval defensive secretion as a cue to find their prey, (Rank et al., 1996; Ko¨pf et al., 1997). In Chrysomela aenicollis, feeding on willows with low content of phenolic glucoside is correlated with a very low production of salicylaldehyde, possibly as an adaptation to escape intense predation by specialist wasps (Rank, 1994). Flexibility of defence by using a wider range of plant compounds or by the ability to synthesize additional compounds (e.g. isobutyrates and 2-methylbutyrates) could allow leaf beetles to increase their host-range or even to shift on new host-plants, possibly under the selective pressure of specialized natural enemies. Interestingly, several species of the Chrysomela interrupta group in which the ability to synthesize isobutyrates and 2-methylbutyrates evolved, shifted during speciation from Salicaceae to Betulaceae (Brown, 1956).
Acknowledgements We are grateful to the ‘‘Communaute´ franiaise de Belgique’’ (ARC 93-3318) for financial support. We thank Dr J. Chenault (Laboratoire de Chimie Bioorganique et Analytique, Orle´ans) for sending to us reference phenolic glucosides. The authors also thank Dr M. Rowell—Rahier for sending us insects and leaves of S. myrsinifolia from Petite Camargue (Saint Louis, France).
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