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Keywords:

  • Between-population variation;
  • formylated phloroglucinol derivatives;
  • host shifts;
  • host specificity;
  • plant–herbivore interactions;
  • plant secondary metabolites;
  • polyphagous herbivores;
  • potential for evolution;
  • sideroxylonal;
  • terpenes;
  • within-population variation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix
  • 1
    By examining variation in the abilities of polyphagous insects to develop on host plants with secondary metabolites that they have never encountered previously, we may be able to gain some insights into the nature of evolution of biochemical mechanisms to process plant secondary metabolites by phytophagous insects.
  • 2
    The present study aimed to examine variation in the ability of gypsy moth larvae Lymantria dispar (Lymantriidae) to complete development on different species of the plant genus Eucalyptus (Myrtaceae). Leaves of at least some Eucalyptus species contain formylated phloroglucinol derivatives. These are secondary metabolites that are evolutionarily unfamiliar to the gypsy moth.
  • 3
    Larvae of gypsy moth showed extremely variable responses in larval performance between Eucalyptus species, between individual trees within host plant species, between moth populations, and between individuals within moth populations.
  • 4
    Larval survivorship was in the range 0–94%, depending on the host. Failure of at least some larvae to complete development on some Eucalyptus species indicates that gypsy moth larvae have a limited ability to process secondary metabolites in eucalypt leaves.
  • 5
    At least some individuals, however, appear to already possess biochemical mechanisms that process the secondary metabolites in leaves of Eucalyptus species, and therefore the abilities of larvae to complete development on phylogenetically and chemically unfamiliar hosts are already present before the gypsy moth encounters these potential hosts.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

There is a wide range in the number of host plant species fed on by herbivorous insects. Some species are strictly monophagous, whereas other species show extreme polyphagy. Extreme examples of monophagy are Tegeticula synthetica (Lepidoptera, Incurvariidae) on Yucca brevifolia (Agavaceae) (Pellmyr et al., 1996), Athrotaxivora tasmanica (McQuillan) (Lepidoptera, Gelechinoidea, tentatively associated with Xyloryctinae) on King William pine Athrotaxis selaginoides (Cuprecaceae) (McQuillan, 1998) and 11 species of leaf beetles Blepharida spp. (Coleoptera, Chrysomelidae), each feeding on only one species of Bursera (Burseraceae) (Becerra, 1997). In contrast, gypsy moth Lymantria dispar (L), (Lepidoptera, Lymantriidae) completes development on more than 650 species (53 families) of plants (Liebhold et al., 1995).

Evolutionary responses by insect herbivores to plant secondary metabolites are considered to play a key role in host shifts (Schultz, 1988; but see also Smiley, 1978; Gilbert, 1979; Bernays & Graham, 1988). It has been hypothesized that polyphagous herbivores are more likely to have evolved the use of generalized biochemical mechanisms, such as mixed-function oxidases (Brattsten, 1979) also known as cytochrome P450s, as well as midgut pH and redox potential (Johnson & Felton, 1996), for processing plant secondary metabolites, whereas monophagous and oligophagous species are more likely to have evolved the use of specialized biochemical mechanisms (Krieger et al., 1971; Mitter & Futuyma, 1979; Gould, 1984; Rose, 1985). There are many studies documenting host shifts by specialists to plant species that have similar secondary metabolites to the original host species (Steinbauer & Wanjura, 2002; Grosman et al., 2005; Murphy & Feeny, 2006; Östrand et al., 2008) or to plant species that are taxonomically (and phylogenetically) related to the original host plant species (Futuyma & McCafferty, 1990; Janz & Nylin, 1998; Janz et al., 2001). In contrast, studies of host shifts to plant species that have evolutionarily unfamiliar secondary metabolites are scarce, although such host shifts must have happened in highly polyphagous species, such as the gypsy moth.

When a population of an insect herbivore encounters a species of plant with evolutionarily unfamiliar secondary metabolites, there are at least five possible outcomes.

  • 1
    No individuals in the population are able to develop on this species of plant because the insect's biochemical mechanisms are unable to process the unfamiliar secondary metabolites (i.e. no host shift).
  • 2
    A small number of individuals in the population are able to develop on the new host, whereas other individuals are unable to develop on the new host.
  • 3
    Most individuals in the population are able to survive and develop on the new host species, although most individuals perform very poorly, whereas some individuals perform well (e.g. the distribution of pupal weights is skewed towards very small pupal weights). This outcome may be observed, for example, when some individuals already have biochemical mechanisms suitable for processing the unfamiliar secondary metabolites, whereas biochemical mechanisms of other individuals are only marginally capable of processing the unfamiliar secondary metabolites.
  • 4
    Most individuals in the population are able to develop on the new host species but not as well as on the original hosts species (e.g. the mean pupal weight on the new host is smaller than that on the original hosts, although the distribution of pupal weights is the same for the new host as for the original hosts). This outcome may be observed, for example, when processing of the unfamiliar secondary metabolites incurs some metabolic costs or when the optimal gut environment for processing the unfamiliar secondary metabolites is slightly different from that for processing familiar secondary metabolites.
  • 5
    Most, if not all, individuals in the population are able to develop on the new host species as well as, or better than, on the normal host species because the insect's generalized biochemical mechanisms are robust enough to be able to process the unfamiliar secondary metabolites.

Studies on specialist leaf beetles have indicated that barriers to host shifts are the result of an absence or paucity of genetic variation influencing larval survival, oviposition and feeding responses (Futuyma et al., 1995). In contrast, a large number of examples of rapid evolution of insecticide resistance (Berenbaum & Zangerl, 1999) and biotypes against naturally resistant individual plants (Glynn & Larsson, 2000) suggest that, within many insect populations, there exists sufficient between-individual variation in biochemical mechanisms (such as allozymes of cytochrome P450s) to process evolutionarily unfamiliar chemicals. Such between-individual variation in biochemical mechanisms should result in between-individual variation in performance of insect herbivores on plant species with evolutionarily unfamiliar secondary metabolites, although other factors may also contribute to between-individual variation in the performance.

We examined between-individual variation in performance of larvae of gypsy moth on the evolutionarily unfamiliar plant genus Eucalyptus (Myrtaceae). As indicators of larval performance, we examined survivorship of larvae and pupal weights. We chose the gypsy moth because it is one of the most polyphagous of all insect herbivores. We chose Eucalyptus because Eucalyptus does not occur in the native distribution range of the gypsy moth, although it has become a popular genus in plantations within the distribution range of gypsy moth in recent decades. Furthermore, some species of Eucalyptus may have secondary metabolites that the gypsy moth has not encountered in its evolutionary history. Moreover, egg masses of gypsy moth have been intercepted on cargo ships and on vehicles from East Asia arriving in New Zealand. Thus, there is a real possibility that the gypsy moth may be found on Eucalyptus trees in Australasia.

Eucalyptus is a large genus with over 700 described species. Four species are found outside of Australia (New Guinea, Ceram, Mindanao and Sulawesi), although the rest of species occur in Australia (Williams & Brooker, 1997). Leaves of Eucalyptus species contain phenolics (Hillis, 1966; Gleadow et al., 2008) and essential oils, which are made up primarily of mono- and sesquiterpenes (Boland et al., 1991). At least some Eucalyptus species have the third class of secondary metabolites collectively known as formylated phloroglucinol derivatives. These are hybrids between phenolics and terpenes/isoprenes (Ghisalberti, 1996). Formylated phloroglucinol derivatives were discovered relatively recently, and their distribution within the genus Eucalyptus has not yet been studied extensively (cf. Eschler et al., 2000). Formylated phloroglucinol derivatives, however, have been found almost exclusively in the genus Eucalyptus, and the only non-Eucalyptus species that are known to contain formylated phloroglucinol derivatives comprise a small number of other myrtaceous plants in Australia and New Zealand (Ghisalberti, 1996). Despite their limited geographical and taxonomic distribution among plants, formylated phloroglucinol derivatives show biological activities against a wide range of organisms: Gram-positive bacteria, HIV-RTase, HeLa cells, photosynthetic electron transport chain in plant cells, marine invertebrates, insects and mammals (Ghisalberti, 1996; Singh & Etoh, 1997; Lawler et al., 1998, 1999a, b; 2000; Steinbauer & Matsuki, 2004).

The original distribution range of the gypsy moth covers much of temperate Eurasia from south-east Siberia, Japan and south-east China to southern Scandinavia and Morocco (Giese & Schneider, 1979). There are two strains of the gypsy moth: European gypsy moth and Asian gypsy moth. European gypsy moth occurs in Europe and northern Africa, and this strain has established itself in northeastern U.S.A. after an accidental introduction (Liebhold et al., 1992). Asian gypsy moth occurs from northern Europe to East Asia, and this strain has also been accidentally introduced to North America (Wallner, 1996). Asian gypsy moth is the strain that has been intercepted in New Zealand.

Host selection by gypsy moth is largely the prerogative of wind dispersed first-instar larvae. Although biochemical mechanisms to process plant secondary metabolites by larvae of Asian gypsy moth have not been studied, larvae of Asian gypsy moth are able to develop on host plants of European gypsy moth (Baranchikov, 1989). Thus, the biochemical mechanisms found in European gypsy moth are likely to be found in Asian gypsy moth.

European gypsy moth larvae are able to develop on host species with many different classes of plant secondary metabolites: alkaloids, coumarins, cyanogenic glycosides, flavonoids, quinones, saponins, phenolic glycosides, tannins and terpenes (Barbosa & Krischik, 1987). Plant species with alkaloids are less likely to be favoured hosts of European gypsy moth, whereas the presence of other classes of secondary metabolites has no effects on host preference (Barbosa & Krischik, 1987). European gypsy moth has so far been shown to have generalized biochemical mechanisms to process a wide range of plant secondary metabolites: mixed function oxidase (Ahmad & Forgash, 1978) and the oxidizing midgut chemical environment (Appel & Maines, 1995), in addition to β-glucosidase and esterase that modify phenolic glycosides (Lindroth & Hemming, 1990).

In the first part of the present study, we examined larval survivorship and pupal weights of the Asian gypsy moth fed on 41 species of Eucalyptus and two species of native hosts, Quercus pubescens and Quercus robur. In the second part of the study, we applied purified sideroxylonal (a formylated phloroglucinol derivative) on leaves of a preferred host of the gypsy moth, Q. robur, and examined consequent growth and consumption rates of larvae of gypsy moth. We used sideroxylonal because this compound had been known to reduce larval survivorship of Mnesampela privata (Lepidoptera, Geometridae) (Steinbauer & Matsuki, 2004; but see also Östrand et al., 2008).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

Gypsy moth

Eggs were obtained from three different sources: a wild population of Asian gypsy moth from south-east Siberia near Khabarovsk (48°36′N, 135°06′E); a laboratory culture of a nondiapausing strain of Asian gypsy moth from the Bavarian State Forest Research Institute, Germany, which was originally collected from Beijing, China (39°55′N, 116°30′E); and a laboratory culture from the USDA, France, which was originally collected from north-east France [Haguenau (Bas-Rhin), Alsace: 48°50′N, 7°39′E] in 1995. Genetic analysis has shown that there are European gypsy moth, Asian gypsy moth, and their hybrids in any single egg mass from the French laboratory culture (Hérard et al., 1997). Eggs were surface sterilized using formaldehyde.

Plants

We used 41 species of Eucalyptus in the present study. Thirty-eight species of Eucalyptus originated from three experimental plots in France (Cessenon, Herault, 43°25′N, 3°05′E, established in 1963; Caneiret, Var: 43°30′N, 6°50′E, established in 1974; and la Mole, Var: 43°10′N, 6°30′E, established in 1984), ≥50-year-old trees at a botanic garden (Antibes, Alpes-Maritimes: 43°35′N, 7°10′E), and 5-year-old trees planted at CSIRO European Laboratory near Montpellier, Herault (43°30′N, 3°50′E). Established plants used in this study showed no abnormal growth. We also used seedlings of six species of Eucalyptus (Appendix I), three of which, Eucalyptus maculata, Eucalyptus grandis and Eucalyptus sideroxylon, were not present in the experimental plots in France. The seedlings were 4–6 months old when they were used in the experiments. As reference points, we also included preferred hosts of gypsy moth, Q. robur and Q. pubescens (Liebhold et al., 1995). Quercus robur was sourced from a nusrasy near Nîmes as approximately 30 small saplings in pots, and Q. pubescens was sourced from several mature trees growing near the CSIRO European laboratory.

We examined the relationships between larval performance and four traits of leaf chemistry: formylated phloroglucinol derivatives; terpenes; cyanogenic glycosides and glaucescense (an indication for a type of leaf surface waxes). Terpenes and leaf surface waxes have been shown to have negative effects on at least some insect herbivores on Eucalyptus species (Morrow & Fox, 1980; Edwards, 1982). Information on terpenes was obtained from Boland et al. (1991) and Li et al. (1995, 1996) and is summarized in Table 1. Glaucescense of leaves were noted during the present study (Table 1). Waxy compound on the surface of leaves gives glaucescens in Eucalyptus. Glaucescense can be identified because waxy compounds readily rub off, revealing the colour of the leaf underneath. Of the 41 species, we have information on formylated phloroglucinol derivatives in 20 species (Ghisalberti, 1996; Eschler et al., 2000; Östrand et al., 2008) and information on cyanogenic glycosides in 38 species (Gleadow et al., 2008) (Table 1).

Table 1.  Summary of Eucalyptus species used in the present study
SeriesaSpeciesaPopulationsNumber of treesLeaf typeGlaucescensTerpenesb% cineolebFPCscCGsd
  1. cEschler et al. (2000) and Ghisalberti (1996) (unless otherwise noted).

  2. fSpecies known to show intra-population variation in terpenes (Boland et al., 1991; Edwards et al., 1993).

  3. hM. Matsuki (unpublished data).

  4. jOne tree had nonglaucous leaves, whereas 11 trees had glaucous leaves.

  5. kE. irbyi = E. gunnii×E. dalrympleana.

  6. Series indicate taxonomic (but not necessarily phylogenetic) affinity. Populations: A, Antibes; Ca, Caneiret; Ce, Cessenon; Cs, CSIRO; M, la Mole; S, Seedlings.

  7. Sources of seedlings are listed in Appendix I.

  8. Leaf type: A, adult leaves; J, juvenile leaves; S, leaves of seedlings.

  9. Glaucescence: ng, nonglaucous leaves; g, glaucous leaves.

  10. Terpenes: Mono, essential oils are made up predominantly of monoterpenes; Sesqui, significant proportion of essential oils is made up of sesquiterpenes; —, no data. % cineole: proportion of cineole in essential oils. FPCs, presence of biologically active formylated phloroglucinol derivatives (simple diformylphloroglucinols such as jesenone, sideroxylonal, which is a dimer of jensenone, and macrocarpals): d, presence of simple diformyulphloroglucinols or sideroxylonal or both; (d), possible presence of simple diformyulphloroglucinols or sideroxylonal or both; m, presence of macrocarpals; (m), possible presence of macrocarpals; np, not present; —, no data. CGs, cyanogenic glycosides; p, present; np, not present; —, no data.

Subgenus Corymbiaa
MaculataeEucalyptus maculataS25aSngSesqui18
Subgenus Eucalyptusa
InsulanaeEucalyptus amygdalinaCa5AngMono70np
InsulanaeEucalyptus cocciferaCa10AngSesqui4enp
FraxinalesEucalyptus delegatensisCa5JngSesqui0.3enpnp
PachyphloiusEucalyptus laevopineaCa5AngMono10np
InsulanaeEucalyptus nitidaCa5AngSesqui1enp
EucalyptusEucalyptus obliquaCa15AngSesquif15(d)np
PaucifloraeEucalyptus paucifloraCa, Ce8AngSesqui9e(d)np
PaucifloraeEucalyptus pauciflora nanaCa5AngSesqui
InsulanaeEucalyptus pulchelaCa1AngSesqui50np
RadiataeEucalyptus radiataCa5AngSesquie0.4enp
RegnantesEucalyptus regnansCa5AngSesqui0.2enpnp
InsulanaeEucalyptus risdoniiCa1JgMono56np
ConsidenianaeEucalyptus seiberiCa5AngSesqui10np
LongitudinalesEucalyptus stellulataCa1AngSesqui8e(d)np
Subgenus Symphyomyrtusa
AcaciiformesEucalyptus acaciiformisCa5AngMono66p
FoveolataeEucalyptus aggregataCa10AngSesqui0np
OrbicularesEucalyptus archeriCa10AngMonog60gnp
RostrataeEucalyptus camaldulensisA1AngMonof84dh, mnp
ArgyrophyllaeEucalyptus cinereaCa, Ce6JgMono78np
OrbicularesEucalyptus cordataCa, Cs6JgMono55np
ViminalesEucalyptus dalrympleanaCs2AngMono78np
OrbicularesEucalyptus glaucascensCa5AgSesqui49np
GlobularesEucalyptus globulusS30SgMono69di, mnp
TransversaeEucalyptus grandisS15SngSesqui6dnp
OrbicularesEucalyptus gunniiCa, Ce, Cs12J/AgjSesquif26np
 Eucalyptus irbyikCa5AgSesqui15
ConfinesEucalyptus kartzoffianaA1AngMono72np
FoveolataeEucalyptus macarthuriiCs1J/AngSesqui0dip
GlobularesEucalyptus maideniiA1AgMono66np
MelliodoraeEucalyptus melliodoraCe, S25aS/AngMonof60d, (m)np
NeglectaeEucalyptus neglectaCa5AngMono68np
AcaciiformesEucalyptus nicholiiCa5AngMono84np
GlobularesEucalyptus nitensA, M, S25aS/AngSesqui30d, (m)np
FoveolataeEucalyptus ovataCa10AngSesqui12gmp
OrbicularesEucalyptus perrineanaCa10AngMono86dnp
GlobularesEucalyptus pseudoglobulusCa5J/AgMono69
HeterophloiaeEucalyptus polyanthemosA1AgMonof60d, (m)p
ViminalesEucalyptus rubidaCa5AgMonof60d, mnp
MelliodoraeEucalyptus sideroxylonS25aSngMonof60dnp
OrbicularesEucalyptus urnigeraCa, Cs16AngMono53np
ViminalesEucalyptus viminalisCs2J/AngMono64mp

There are three features of Eucalyptus that may require clarification: intraspecific variation in leaf chemistry; heterophylly; and subgenera. At least some Eucalyptus species show marked genetically based intraspecific variation in leaf chemistry (Boland et al., 1991; Lawler et al., 2000). Therefore, in the host suitability experiments described below, we used multiple individuals of each species to examine mean larval performance for each host species (except E. camaldulensis, E. kartzoffiana, E. macarthurii, E. polyanthemos and E. risdonii). Furthermore, on E. dalrympleana, E. gunnii, E. nitens and E. pauciflora, we measured larval performance for individual trees and explicitly examined between-tree variation.

Eucalyptus typically shows heterophylly (Boland et al., 1984). There are three types of leaves: seedling, juvenile and adult. Within each species, different leaf types have unique physical (and sometimes chemical) characteristics and are produced at particular stages of tree development. Seedling leaves are typically produced when trees are less than 6 months to 1 year old. Juvenile leaves are typically produced prior to onset of reproduction. Adult leaves are produced by reproductively mature trees. Some species, however, retain juvenile leaves even when the trees are reproductively mature. Thus, we specified the type of leaves used for each species and trees in the present study (Table 1).

Taxonomy and phylogenetic systematics of the genus Eucalyptus is still uncertain. In the present study, we use the most recent treatment by Brooker (2000). Eucalyptus is sometimes divided into several subgenera based on morphological characteristics (Pryor & Johnson, 1971; Brooker, 2000). Brooker (2000) treats Corymbia as a subgenus of Eucalyptus, although some authors treat Corymbia as a genus (Hill & Johnson, 1995; Udovicic et al., 1995; Ladiges & Udovicic, 2000). Two larger and undisputed subgenera are Symphyomyrtus and Eucalyptus (Brooker, 2000). The subgenus Eucalyptus was called Monocalyptus prior to Brooker (2000). The subgenera Eucalyptus and Symphyomyrtus show some ecological differences (Noble, 1989). Species in the subgenus Eucalyptus tend to be less susceptible to insect herbivores than species in the subgenus Symphyomyrtus (Stone et al., 1998; but see also Wotherspoon, 1998). Moreover, formylated phloroglucinol derivatives appear to be less common or absent in species in the subgenus Eucalyptus compared with those in the subgenus Smyphyomyrtus (Eschler et al., 2000).

We conducted two types of experiments: (i) host suitability experiments and (ii) a sideroxylonal paint experiment. All experiments were conducted in the quarantine facility at CSIRO European Laboratory near Montpellier, France.

Host suitability experiments

Four host suitability experiments were conducted using three populations of the gypsy moth: the nondiapausing Chinese population (experiments 1 and 3); the Siberian population (experiment 2); and the French population (experiment 4). In experiment 1, larvae were given old leaves of mature trees throughout the trial because new leaves were not available at this stage. In all other experiments, larvae were given a mixture of old and new leaves. New and old leaves here refer to age of leaves, and they should not be confused with the type of leaves (i.e. seedling, juvenile or adult leaves) described earlier. Old leaves are those produced in the previous season (>1 year old), whereas new leaves are leaves from the current season's growth (<4 month old).

The four experiments had the same randomized block design. Because of the availability of suitable leaves on different trees; however, not all trees or species were used in all four experiments. In the quarantine glasshouse, we placed five benches placed perpendicular to the long axis (approximately east–west) of the glasshouse, and we used the benches as blocks. Thus, in each experiment, there were five blocks. There was one replicate of each host species (experimental unit) in each block. An experimental unit consisted of a host plant and ten larvae on that plant (see below for details). Thus, ten larvae on each plant were subsamples.

In all host suitability experiments, larvae were kept in opaque plastic drinking cups with experimental host plants. A twig with leaves was placed in a shorter cup (395 mL) with a hole in the bottom. The stem of the twig, wrapped around by a piece of paper towel, was placed through the hole, and the shorter cup was fitted inside of a taller cup (595 mL) with water in the bottom, allowing the stem to be immersed in water. We then placed ten neonate larvae in each cup, and there were five replicate cups per host plant. When larvae became large enough to consume leaves in 24 h, we placed cut twigs in the larger cups without any source of moisture. When larvae were in the fourth instar, we divided the ten larvae in a cup into two groups of five per cup. Ambient temperature during the experiments was 19–24 °C (mean = 22 °C).

Each cup was checked daily for food availability and pupae. Pupae were individually sexed and weighed fresh 5 days after pupation. After 4–5 days, fresh pupal weight stabilized from decreasing pupal weight as a result of water loss (Matsuki et al., 2001). Pupal weight was linearly related to the number of eggs in adult females (Matsuki et al., 2001). According to survivorship to pupation and the distribution of pupal weight, each host was classified into one of the five categories of larval performance described in the Introduction. When there were more than 15 pupae from a host tree, we checked skewness and kurtosis of size distribution of pupae. We examined trees separately because the size distribution of pupae and the mean size of pupae can be markedly different between different host trees within a species. We used a linear mixed model with the restricted maximum likelihood (REML) to examine differences in survivorship between the four experiments and to estimate variance in survivorship between Eucalyptus species and that between trees within Eucalyptus species. The four experiments were used as the fixed factor, and Eucalyptus species and trees were used as random factors. Survivorship was arcsine-transformed for the analysis.

Using chi-square tests of independence of variables, we examined relationships between the ability of larvae of gypsy moth to complete development on various hosts and leaf chemical characteristics of the hosts. We were unable to examine relationships between the five categories of larval performance and leaf chemical characteristics of the hosts because some cells in the contingency tables had very small number of observations. Also, because of small sample sizes, we pooled results from the four experiments. A host species was scored as suitable for completing development if at least one larva from any of the three gypsy moth populations was able to complete development on any one tree belonging to that species. A host species was scored as unsuitable for completing development if no larva was able to complete development on any trees belonging to that species (except for Eucalyptus urnigera: see Results). We used species, rather than trees or subspecies, as the sampling unit in the contingency table analyses to avoid pseudoreplication because of anticipated similarities in leaf chemistry among trees or subspecies within any species. We are aware, however, that our sampling units may not be evolutionarily independent because we have not used phylogenetically independent contrasts (Harvey & Pagel, 1991). Phylogenetic relationships among Eucalyptus species have not yet been fully determined (Steane et al., 1999).

Sideroxylonal paint experiment

We examined larval growth and consumption rates on Q. robur leaves with and without pure sideroxylonal that was topically applied by painting with a small artists brush. The nondiapausing Chinese population was reared on a mixture of Q. robur and Q. pubescens from neonate to the sixth instar, and we used the sixth-instar larvae in this experiment. There were three treatments: (i) leaves without painting (control); (ii) leaves with applied solvent (acetone); and (iii) leaves with applied sideroxylonal (approximately 80 mg/g leaf dry mass). Eighty milligrams of sideroxylonal per gram leaf dry mass represent approximately a two-fold greater sideroxylonal concentration than the maximum sideroxylonal concentration found in leaves of Eucalyptus naturally.

Fresh leaves of Q. robur were individually weighed and placed in clear plastic screw-top vials (250 mL) with moist plaster of Paris to maintain leaf turgor pressure. Approximately equal amounts of leaf material were placed in each vial. Larvae that had resumed feeding after the moult were individually weighed, and one larva was placed in each vial. We left one half of the vials without larvae, and leaves in those vials served as the control when we calculated the amounts of leaf material consumed by the larvae. Each larva was weighed again at the end of the experiment, when some larvae had consumed almost all leaf material. The position of vials was randomized (i.e. completely randomized design). The ambient temperature was kept at 20 °C.

We calculated relative growth rates (RGR) and relative consumption rates (RCR) based on dry mass of leaves and larvae (Gordon, 1968). Dry mass was estimated from a wet mass : dry mass ratio of a subsample of ten larvae. We used analysis of variance (anova) to examine the effects of treatments and Levene's test of homoscedasticisty (Madansky, 1988) to examine possible differences in variation in larval performance among treatments. All statistical analyses were carried out using the genstat statistical package. (GenStat, VSN International Ltd, U.K.)

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

Gypsy moth larvae completed development on 21 of the 41 species of Eucalyptus. Of the five possible categories of larval performance on evolutionarily unfamiliar hosts explained in the Introduction, we found examples of all but the third category (Fig. 1 and Table 2).

image

Figure 1. Examples of different patterns of size distribution of female pupae of gypsy moth developed on different hosts. (a) Chinese population on leaves from one Quercus pubescens tree (native host); (b) Chinese population on leaves from multiple Quercus robur seedlings (native host); (c) Siberian population on leaves from one Eucalyptus gunnii tree (an example of performance category 5); (d) Siberian population on leaves from one Eucalyptus aggregata tree (an example of performance category 4); (e) Siberian population on leaves from multiple Eucalyptus sideroxylon seedlings (another example of performance category 4); and (f) Chinese population on leaves from multiple E. sideroxylon seedlings (an example of performance category 2). For explanation of the performance categories, see Introduction and Table 2. Grey bars show the estimated number of females died before pupation, assuming a 1 : 1 sex ratio.

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Table 2.  Larval performance categories on different Eucalyptus species by populations of gypsy moth from China, Siberia, and France
SpeciesChina (old)China (new)Siberia (new)France (new)
  1. aE. pauciflora nana.

  2. bE. irbyi = E. gunnii×E. dalrympleana.

  3. cSeedlings.

  4. dOne of two trees.

  5. eOne out of 16 trees.

  6. (Old) indicates old leaves from the previous season, and (new) indicates new season's leaves that are less than 4 months old. Larval performance categories are. 1a, no feeding; 1b, feeding but no moulting; 1c, moulting but no pupation; 2, very small number of larvae was able to pupate; 4, the mean pupal weight is smaller than that on preferred hosts, but there is no change in the shape of size distribution (no skewness or kurtosis); 5, size distribution of pupae is similar to that on preferred hosts. —, no data. For examples of categories 2, 4 and 5, see Fig. 1. On some species, larval performance was different on different trees, and more than one performance categories were shown for those species. In categories 2, 4 and 5, % survivorship is shown in parentheses. When two trees were used for a species, % survivorship for each tree is shown. When three or more trees were used for a species, minimum–maximum % survivorship for each species is shown. In categories 1a, 1b, and 1c, % survivorship is 0.

Subgenus Corymbia
Eucalyptus maculata5 (84)5 (84)5 (36)
Subgenus Eucalyptus
Eucalyptus amygdalina2 (8)2 (20)
Eucalyptus coccifera4 (50)
Eucalyptus delegatensis2 (14)
Eucalyptus laevopinea1a
Eucalyptus nitida2 (38)
Eucalyptus obliqua1c1c
Eucalyptus pauciflora1ca, 2 (18)1ca, 2 (12–30)1ca
Eucalyptus pulchela2 (22)
Eucalyptus radiata1c
Eucalyptus regnans1c
Eucalyptus risdonii5 (82)
Eucalyptus seiberi1b
Eucalyptus stellulata4 (48)2 (8)
Subgenus Symphyomyrtus
Eucalyptus acaciiformis1a1a
Eucalyptus aggregata1c4 (76)
Eucalyptus archeri1c
Eucalyptus camaldulensis2 (52)
Eucalyptus cinerea1a1a1a
Eucalyptus cordata1a1a
Eucalyptus dalrympleana2 (32, 50)
Eucalyptus glaucascens1a
Eucalyptus globulus1a1a1a
Eucalyptus grandis1c2 (22)1c
Eucalyptus gunnii5 (38)5 (8–86)5 (74–94)5 (68)
Eucalyptus irbyib5 (58)5 (24)
Eucalyptus kartzoffiana1a
Eucalyptus macarthurii2 (66)
Eucalyptus maidenii1b
Eucalyptus melliodora1c2 (12)2 (2)1c
Eucalyptus neglecta1c
Eucalyptus nicholii1c
Eucalyptus nitens2c (2, 4)1cc, 2 (2–44)2c (10, 22)2c (2, 2)
Eucalyptus ovata1c
Eucalyptus perrineana1c, 2d (4)
Eucalyptus polyanthemos1a
Eucalyptus pseudoglobulus1a1a
Eucalyptus rubida1a
Eucalyptus sideroxylon2 (20)4 (70)2 (2)
Eucalyptus urnigera1c1c, 5e (82)
Eucalyptus viminalis1a
Quercus robur(77)(56)(60)
Quercus pubsecens(90)(4)

Larval survivorship was less than 50% on ten species and greater than 75% on five species (Eucalyptus aggregata, E. gunnii, E. maculata, E. risdonii and E. urnigera). The maximum survivorship was 94% (on an E. gunnii tree). Neonate larvae failed to initiate feeding on 11 host species (category 1a in Table 2), failed moult on two host species (category 1b in Table 2) and failed to pupate on seven species (category 1c in Table 2).

The mean pupal weight of females on the best host species (E. gunnii) was 10.7-fold greater than that on the poorest host species (E. nitens). Where there were more than 15 pupae from a tree, the size distribution of female pupae tended to be skewed right (more larger individuals than smaller ones: negative skewness) as the mean pupal weight. In contrast, there was no clear trend in males (Fig. 2b). There was no evidence that strong positive skewness was associated with small mean pupal weight (an indication for the third category of larval performance on evolutionarily unfamiliar hosts). Kurtosis of the size distribution of pupae was not different from zero in pupae from any trees. Individual pupal weights ranged from 121 mg on E. dalrymapleana to 2555 mg on E. gunnii in females and from 38 mg on E. grandis to 953 mg on E. gunnii in males. Larval performance also varied markedly among host trees within species. The mean pupal weight of females on the best E. gunnii tree was 3.7-fold greater than that on the worst E. gunnii tree. On E. aggregata, E. melliodora, E. nitens, E. perrineana, E. pauciflora and E. urnigera, larvae failed to complete development on some trees (Table 2).

image

Figure 2. Relationships between the mean pupal weight and skewness of pupal weight in (a) female and (b) male gypsy moth. Each symbol represents a tree. Different symbols indicate different populations of the gypsy moth. Small symbols are Quercus trees, and large symbols are Eucalyptus trees.

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On poor hosts, most larvae died within 2 weeks. One larva out of 50 on E. archeri and one larva out of 100 on E. ovata, however, survived to the sixth instar. Only one larva out of 150 survived to pupation on seedlings of E. melliodora, and six larvae out of 50 survived to pupation on leaves from a mature tree of E. melliodora. In contrast, 127 out of 150 larvae on the best E. gunnii tree and 102 out of 150 larvae on seedlings of E. maculata survived to pupation.

Overall survivorship was the highest in the Siberian population and the lowest in the French population (REML: F3,53.5 = 6.7; P < 0.001). Estimated variance in survivorship between Eucalyptus species using REML was approximately 2.5-fold greater than estimated vairance in survivorship between trees within Eucalyptus species. Different populations of gypsy moth performed similarly on good hosts such as E. maculata and E. gunnii (Table 2). On poor hosts, however, there was between-population variation in larval performance (Table 2). The population of gypsy moth from south-east Siberia was able to complete development on E. grandis, whereas populations from north-east France and China failed to develop on E. grandis. Similarly, the larval performance of the Siberian population on E. nitens seedlings and E. sideroxylon was better than that of populations from north-east France or China.

Eucalyptus species that are known to have formylated phloroglucinol derivatives tended to support poor larval performance (bold-faced species in Table 2). Only two out of the 41 host species tested (E. delegatensis and E. regnans), however, are known to have no formylated phloroglucinol derivatives (Table 3). Larval performance on these two species was also poor (Table 2). Although the mean survivorship for a tree or for a species was different between the four experiments, at least some gypsy moth larvae were able to complete development on seven species with formylated phloroglucinol derivatives. Whether or not larvae can complete development on species with formylated phloroglucinol derivatives for the same host species was generally consistent across experiments.

Table 3.  Relationships between larval development of the gypsy moth and traits of Eucalyptus species, with the number of species in each category shown
Formylated phloroglucinol derivatives (no statistical test was performed because there were only two host species definitely known not to have formylated phloroglucinol derivatives)
 PresentAbsent
Pupation71
No pupation51
Subgenera (χ2 = 2.9, P = 0.09, d.f. = 1)
 EucalyptusSymphyomyrtus
Pupation811
No pupation516
Glaucescence (χ2 = 2.2, P = 0.14, d.f. = 1)
 GlaucousNonglaucous
Pupation317
No pupation615
Terpene composition (χ2 = 8.1, P = 0.004, d.f. = 1)
 Mostly monoterpenesSome sesquiterpenes
Pupation713
No pupation156
Presence/absence of cyanogenic glycosides (no statistical test was performed because there were only five host species with cyanogenic glycosides)
 PresentAbsent
Pupation117
No pupation416

Because formylated phloroglucinol derivatives are hypothesized to be less common in species within the subgenus Eucalyptus than those within the subgenus Symphyomyrtus (Eschler et al., 2000), we also examined whether there was a relationship between the subgeneric grouping and larval development. There was, however, no clear difference in larval ability to complete development on the hosts that belong to the subgenus Eucalyptus or Symphyomyrtus (Table 3). Similarly, there was no clear difference in larval ability to complete development on the hosts with glaucous leaves or nonglaucous leaves (Table 3). Larvae of gypsy moth showed a tendency not to complete development on host species whose essential oils were dominated by monoterpenes (Table 3). In these analyses, E. urnigera was classified as unsuitable for larval development, although larvae performed well on one out of 16 trees (Table 2). We assumed that this one tree had different leaf chemistry from other trees.

Of the 41 species of Eucalyptus used in this study, leaves of E. acaciiformis, E. macarthuri, E. ovata, E. viminalis and E. polyamthemos are known to contain cyanogenic glycosides (Table 1). Larvae were able to complete development on E. macarthuri but were unable to complete development on four other species with cyanogenic glycosides. Larvae also failed to complete development on 16 Eucalyptus species that do not contain cyanogenic glycosides (Table 3).

Purified sideroxylonal decreased mean growth rates (anova: F2,21 = 7.3; P = 0.004) and consumption rates (anova: F2,21 = 9.7; P = 0.001) on Q. robur (Fig. 3). Larvae of gypsy moth responded individualistically to painted sideroxylonal. The performance of some larvae was not affected by painted sideroxylonal, whereas that of other larvae was negatively affected. There was no sign of heteroscedasticity in all three parameters examined (Levene's tests: P = 0.66 for RGR and P = 0.79 for RCR). Thus, variation in larval performance was not different between larvae that fed on a leaf with or a leaf without painted sideroxylonal.

image

Figure 3. Effects of painted sideroxylonal on (a) relative consumption rates (RCR) and (b) relative growth rates (RGR). Data are the mean + SE. n = 6–9 larvae.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

The results obtained in the present study provide some insights into the nature of evolution of host shifts with respect to the ability of larvae to process evolutionarily unfamiliar plants and plant secondary metabolites. First, the ability of larvae to complete development on phylogenetically and chemically unfamiliar hosts (i.e. Eucalyptus) is already present in at least a small proportion of individuals within populations even before the gypsy moth has encountered these potential hosts. We found that at least some larvae of gypsy moth were able to complete development on approximately 50% of Eucalyptus species tested, including species with evolutionarily unfamiliar plant secondary metabolites (i.e. formylated phloroglucinol derivatives).

Second, however, the lower survivorship and smaller pupae on most Eucalyptus species compared with those on the two species of deciduous oaks indicate that there appear to be some barriers for the gypsy moth to include most Eucalyptus species as suitable hosts. Exceptions are E. gunnii, E. irbyi, E. maculata and E. risdonii (Matsuki et al., 2001; present study). Larval growth rates can be correlated with leaf nitrogen, leaf toughness, leaf water content, pubescence or epicuticular wax (Scriber & Slansky, 1981; Matsuki & MacLean, 1994; Steinbauer & Matsuki, 2004). It is likely, however, that these Eucalyptus species lack (or contain only very low concentrations of) secondary metabolites that cause low survivorship and small pupal weights on other Eucalyptus species (Table 3).

Third, although the focus of the present study was to examine the ability of larvae to develop on new host plants, the results obtained indicated that even for an extremely polyphagous species such as gypsy moth, pre-ingestive host discrimination appears to play a role in neonate survivorship. Thus, host shifts in phytophagous insects may require evolutionary changes in host recognition by neonate larvae in addition to the ability of larvae to develop on new host plant species and female oviposition choice. Of the 11 species of hosts on which larvae died without feeding, eight species (E. acciiformis, E. cinerea, E. cordata, E. globulus, E. kartzoffiana, E. polyanthemos, E. rubida and E. viminalis) are known to have high cineole content.

We examined phenotypic variation, rather than additive genetic variation, in the ability of larvae to develop on leaves of Eucalyptus species. Thus, we are unable to confirm that there is a potential for host shifts as a result of changes in gene frequency. However, this is the first study to document (phenotypic or genetic) variation in the ability of larvae of a generalist insect herbivore to develop on evolutionarily unfamiliar hosts. The findings of this study contrast with the responses of specialist insect herbivores. Larvae and adult females of specialist insect herbivores failed to recognize evolutionarily unfamiliar plants as potential hosts (Futuyma et al., 1995).

Some formylated phloroglucinol derivatives (particularly sideroxylonal) have negative effects on various organisms and biochemical reactions. The presence of formylated phloroglucinol derivatives alone does not, however, appear to be an effective barrier against the gypsy moth including Eucalyptus species as its hosts. Some larvae were able to complete development on Eucalyptus species with formylated phloroglucinol derivatives, although many Eucalyptus species with formylated phloroglucinol derivatives tended to be very poor hosts of the gypsy moth (Table 2). A population of gypsy moth in Morocco has been observed to feed on E. camaldulensis in plantations when their normal hosts (Quercus spp.) have been exhausted during outbreaks (Fraval, 1989). Eucalyptus camaldulensis has been shown to contain formylated phloroglucinol derivatives (Table 1). Moreover, when purified sideroxylonal was painted on leaves of Q. robur, larvae of the gypsy moth showed the same degree of variation in larval performance compared with larvae on Q. robur without painted sideroxylonal. Thus, sideroxylonal did not reduce performance of larvae to uniformly low levels.

At least in marsupial folivores, aldehyde groups on formylated phloroglucinol derivatives cause negative effects on feeding (Lawler et al., 1999a). In the insect gut, aldehyde groups can be modified by oxidation or binding with amino groups (M. Lacey, personal communication). High pH environments (pH 11) have been shown to increase rates of binding between aldehyde and amino groups (W. J. Foley, personal communication). Larvae of European gypsy moth have mixed function oxidases (= cytochrome P450s) (Ahmad & Forgash, 1978), and the midgut pH is approximately 8.5 and is independent of host plant species (Appel & Maines, 1995). Both mixed function oxidases and a high midgut pH have been hypothesized to be examples of generalized biochemical mechanisms for processing plant secondary metabolites (Mitter & Futuyma, 1979; Appel, 1994). Some groups of mixed function oxidases demonstrate allozyme variation, and some examples of rapid evolution of insecticide resistance have been attributed to selection for particular mixed function oxidase allozymes (Berenbaum & Zangerl, 1999). Between-individual variation in mixed function oxidases and/or the midgut pH may have resulted in the observed variation in larvae of gypsy moth to complete development on some Eucalyptus species.

It was our intention to simulate potential interactions between the gypsy moth and new host species when they encounter for the first time. Thus, it is perhaps worthwhile to compare the results obtained in the present study with those of studies examining interactions between European gypsy moth and its preferred hosts. Responses of European gypsy moth larvae to phenolic glycosides in Populus tremuloides (Salicaceae) have been studied intensively (Lindroth & Hemming, 1990; Hemming & Lindroth, 1995; Hwang & Lindroth, 1997; Osier et al., 2000). Phenolic glycosides are found in many species within the plant family Salicaceae (e.g. Salix and Populus). Salicaceous plants are distributed widely in the temperate zone in the Northern Hemisphere, and many species support the development of larvae of European gypsy moth (Liebhold et al., 1995). Therefore, it is perhaps not unreasonable to assume a long history of association between European gypsy moth and phenolic glycosides.

High concentrations of tremulacin, a phenolic glycoside, have been shown to reduce larval growth rates and pupal weight of European gypsy moth (Lindroth & Hemming, 1990; Osier et al., 2000). Moreover, there is genetic variation in response of larvae of European gypsy moth to phenolic glycosides, possibly as a result of variation in esterase activity (Rossiter, 1987; Lindroth & Weisbrod, 1991). Thus, despite a presumed long history of association with phenolic glycosides, it appears that the selection pressure by phenolic glycosides on biochemical mechanisms of European gypsy moth larvae has not been strong enough (relative to other selection pressures) to entirely overcome the negative effects of phenolic glycosides by evolution of effective detoxification mechanisms.

At the same time, however, the negative effects of phenolic glycosides on performance of European gypsy moth larvae appear to be much less than the negative effects of secondary metabolites in leaves of many Eucalyptus species on larvae of the gypsy moth used in the present study (i.e. mostly Asian gypsy moth). The mean pupal weight of female European gypsy moth on different clones of P. tremuloides was reported to be in the approximate range 1.1–1.6 g in a laboratory study (Osier et al., 2000). These values are more or less equivalent to the mean pupal weight of females on Quercus robur and Q. pubescens in the present study (1.2 g) and are approximately two-fold greater than the mean pupal weight on most Eucalyptus species. Therefore, we hypothesize that the selection pressure by phenolic glycosides has been strong enough to reduce variation in larval development, possibly equivalent to the variation that we observed in the present study.

In the present study, we illustrated the potential outcomes of host shifts by an insect herbivore using gypsy moth (an extremely polyphagous species) and Eucalyptus species. We showed that there exists between-individual variation in larval survivorship and pupal weight of the gypsy moth on evolutionarily unfamiliar hosts, 41 species of Eucalyptus. The ability of gypsy moth larvae to complete development on phylogenetically and chemically unfamiliar hosts are already present even before the gypsy moth encounters these potential hosts. In Brazil, some native leaf beetles, weevils, and scarab beetles have shown host shifts to Eucalyptus species in plantations (Anjos & Majer, 2003). Some of these beetles are specialists on native Myrtaceous species but other species are generalists on non-Myrtaceous species (Anjos & Majer, 2003). Therefore, the findings of the present study may apply to other generalist herbivores.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

We thank J. Vitou, C. Espiau, Y. Mas, T. Thomann, J.-L. Gornes, M. Jourdan, C. Ducatillon, J.-N. Marien, F. Hérard, B. Webber, H. Scheel, M. Lacey, M. Michie, G. Farrell, and W. Foley for discussions or assistance. INRA, and AFOCEL granted permission to collect foliage. Comments by M. Steinbauer, A. D. Loch, J. Bain, A. Sheppard and three anonymous reviewers improved the manuscript. B. Eschler provided the purified sideroxylonal. Gypsy moth eggs were provided by G. Lobinger (Bavarian State Forest Research Institute, Germany), G. Yurchenko (Russian Far East Forest Research Institute, Russian Republic) and F. Hérard (USDA, Montpellier, France). Financial support was provided by Ministry of Agriculture and Forestry, New Zealand and Agriculture, Fisheries and Forestry–Australia.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Appendix

Appendix I

Sources of seedlings

Seeds were obtained from Australian Tree Seed Centre, Canberra, Australia.

Eucalyptus maculata: seedlot 19308; Kioloa, New South Wales.

Eucalyptus globulus: seedlot 18886; Lorne, Victoria; population know to be susceptible to at least some insect herbivores in Australia.

Eucalyptus globulus: seedlot 18723; Geeveston, Tasmania: population known to be resistant to at least some insect herbivores in Australia.

Eucalyptus nitens: seedlot 16752; Thomson Valley Road, Victoria.

Eucalyptus nitens: seedlot 18310; SSO APPM, Tasmania.

Eucalyptus grandis: seedlot 13883; Urunga, New South Wales.

Eucalyptus melliodora: seedlot 15939; 33 km north east of Peak Hill, New South Wales.

Eucalyptus sideroxylon: seedlot 15094; 33 km north east of Parkes, New South Wales.