Teija Ruuhola

Natural host-plant quality affects immune defence of an insect herbivore

We examined the effect of natural host‐plant quality on immune resistance in the autumnal moth, Epirrita autumnata (Borkhausen) (Lepidoptera: Geometridae). The division of mountain birches [Betula pubescens ssp. czerepanovii (Orlova) Hämet‐ahti (Betulaceae)] into two categories, high‐ and low‐quality food for larvae, was based on previous years’ results on the relative growth rate of the autumnal moth on the trees selected. The strength of the immune defence of autumnal moths was determined by measuring their encapsulation rate to exposure to a foreign antigen and the phenoloxidase (PO) activity of the pupal haemolymph. We found that individuals reared as larvae on naturally low‐quality food had a significantly higher encapsulation rate at the pupal stage than individuals reared on high‐quality food. Females also had a higher encapsulation rate than males. Food quality did not have statistically significant effect on PO activity, nor did this response variable show any differences between the sexes. Using half‐sib analyses, we found significant heritable variation in the encapsulation rate; the heritable variation in PO activity was near to significant, although equally strong. Heritability estimates (h2: 0.19–0.27) for immune defence traits were relatively low and only moderate when compared to other studies with insects. We also found a negative genetic correlation between pupal mass and PO activity, but not between PO activity and encapsulation rate. Our results suggest that the quality of food affects immune defence in the autumnal moth. Thus an intricate tritrophic relationship exists between the folivorous insect, the host tree, and the insects natural enemies (e.g., pathogens, parasites, and parasitoids). This study demonstrates that natural variation within a food plant species has an effect on the innate immune system of an herbivorous insect.

DEFOLIATING INSECT IMMUNE DEFENSE INTERACTS WITH INDUCED PLANT DEFENSE DURING A POPULATION OUTBREAK

During population outbreaks, top-down and bottom-up factors are unable to control defoliator numbers. To our knowledge, details of biotic interactions leading to increased population density have not been studied during real population outbreaks. We experimentally assessed the strength of plant defenses and of insect immunocompetence, assumed to contribute to active insect resistance against parasitoids and pathogens, in the geometrid Epirrita autumnata during a steep increase in population density. We demonstrated rapid (same-season) induced resistance in the foliage of its host, mountain birch. The response was systemic, spreading throughout the tree, and retarded larval growth rate by approximately 10%. On the other hand, no direct delayed carry-over effects were found in the next season in larval growth rate, mortality, or pupal mass. Larval damage to a tree during the previous year, however, significantly (by approximately 13%) accelerated the advance of the immune response (measured as melanization of an implant inserted into the pupal hemocoel). The encapsulation rate correlated positively with larval mortality in trees in which larvae had been introduced the previous year, but not in control trees. Both of these observations suggest that induced plant defense was associated with an increased insect immunocompetence during the population increase.

Differences in Host Use Efficiency of Larvae of a Generalist Moth, Operophtera brumata on Three Chemically Divergent Salix Species

The food selection, growth, and fecundity of insect herbivores are largely dictated by the chemical composition and nutritive values of plant foliage. We studied the host-use efficiency of larvae of the generalist moth, Operophtera brumata (Lepidoptera: Geometridae) on three chemically divergent but nutritively similar willows (Salix spp.). The 4th instars were able to use the salicylate-free leaves of S. phylicifolia efficiently. Growth was slightly reduced on S. pentandra, which contained a moderate level of acetylated salicylates. The high concentration of salicylates found in the leaves of S. myrsinifolia seemed to provide efficient protection against non-specialized O. brumata. We also studied assimilation of nutrients and degradation of salicylates and other secondary compounds in the digestive tract of O. brumata larvae. Neither the assimilation of nitrogen nor of carbon were affected by secondary chemicals of ingested food. Salicylates were shown to be degraded to salicin and catechol, while further degradation of salicin to saligenin was rather slow. In an artificial diet experiment, we showed that two degradation products of salicylates, catechol and saligenin markedly reduced the growth of the larvae. Neither salicin nor chlorogenic acid affected larval growth. We conclude that salicylates reduced the growth of the generalist winter moth mainly by feeding deterrence caused by 6-hydroxy-2-cyclohexenone and catechol. Compared to the deleterious effects of salicylates the effects of other secondary compounds were minor.

Foliar Phenolics are Differently Associated with Epirrita autumnata Growth and Immunocompetence

The quality of available food may affect insect herbivores directly (via growth and survivorship) and/or indirectly (by modifying insect vulnerability to parasitoids and pathogens). We examined the relationship between different phenolic compounds, belonging to various phenolic groups, in Betula pubescens spp. czerepanovii (mountain birch) foliage and the larval performance of the geometrid Epirrita autumnata (autumnal moth). Direct effects on insect performance were described by pupal weight, developmental rate, and survivorship; indirect effects were described by the encapsulation rate of an implant inserted into the insect hemocoel, a commonly used way to describe insect immune defense. We found profound differences in the effects of different phenolic categories: several individual hydrolyzable tannins were associated positively with larval performance but negatively with level of immune defense, whereas flavonoid glycosides were inversely related to larval survival but showed no association with the larvae immune defense.

Trade-off between synthesis of salicylates and growth of micropropagated Salix pentandra

We studied the relationship between biosynthesis of salicylates, the main chemical defenses in willow and growth of Salix pentandra by cultivating plants in the presence of 2-aminoindan-2-phosphonic acid (AIP), a powerful inhibitor of phenylalanine ammonia-lyase (PAL: EC 4.3.1.5.). AIP inhibited efficiently, though not totally, the endogenous synthesis of salicylates. This inhibition markedly increased plant growth. Exogenous application of the precursors of salicylates, benzoic acid (BA), salicylic acid (SA), and helicin, increased the levels of several individual salicylates, but decreased the growth of plants cultivated in the presence of AIP. These results suggest a trade-off between plant growth and the synthesis of salicylates in S. pentandra. Phenylalanine, which accumulated in response to inhibitor treatment, but was decreased by precursor treatments, may be a common and limiting precursor for both plant growth and salicylate synthesis. The biosynthesis of salicin is suggested to proceed mainly via benzoyl-glucose, an intermediate in the synthesis of salicylic acid. Salicin is the most obvious precursor of more substituted salicylates, salicortin, acetylsalicortin, and tremulacin. In addition, we found that the salicylate pools of mature plant parts of S. pentandra were not subject to turnover, implying that the maintenance of salicylates does not demand high resources of plants, although their initial construction is costly.

In Vitro Degradation of Willow Salicylates

Salicylates are defensive compounds against a great variety of generalist herbivores. Salicortin and its derivatives are labile compounds that degrade immediately when cell compartmentalization is ruptured, producing a 6-hydroxy-2-cyclohexenone (6-HCH) moiety that is a strong antifeeding cue. We studied the In Vitro degradation of willow salicylates in the presence and absence of foliar enzymes at acidic, neutral, and alkaline pHs. Higher substituted salicylates were degraded in the absence of foliar enzymes at alkaline pH and in the presence of foliar enzymes at all three pHs. Salicin and its diglucoside, on the other hand, were degraded only in the presence of foliar enzymes at acidic pH, probably by β-glucosidase activity. The main degradation products of higher substituted salicylates were salicin, 6-HCH, and catechol in both the absence and presence of enzymes, suggesting that the production of 6-HCH and catechol do not necessarily demand enzymatic activity. We propose that the degradation of salicylates begins with the cleavage of a 1-hydroxy-6-oxo-2-cyclohexen-1-carbonyl moiety by foliar esterases and/or alkaline condition. This moiety is decarboxylated in nonenzymatic reaction to an anion of 2-hydroxy-3-cyclohexenone, which is tautomerized to the enol form and further to the keto form, 6-HCH. Hydroxyketone can be also oxidized to catechol, a substrate of polyphenol oxidases.

Effects of host-plant shift on immune and other key life-history traits of an eruptive Geometrid, Epirrita autumnata (Borkhausen)

Abstract 1. Population density of Epirrita autumnata (Lepidoptera: Geometridae) reaches outbreak densities regularly in northernmost Scandinavia. During these outbreak years, the most abundant host species, the mountain birch (Betula pubescens ssp. czerepanovii), is regularly exhausted, although larvae may rescue themselves from starvation by using alternative host species.

Foliar oxidases as mediators of the rapidly induced resistance of mountain birch against Epirrita autumnata

Induced resistance of the mountain birch against its main defoliator Epirrita autumnata is a well-characterized phenomenon. The causal mechanism for this induced deterioration, however, has not been unequivocally explained, and no individual compound or group of traditional defensive compounds has been shown to explain the phenomenon. Phenolic compounds are the main secondary metabolites in mountain birch leaves, and the biological activity of phenolics usually depends on their oxidation. In this study, we found that the activity of polyphenoloxidases (PPOs), enzymes that oxidize o-diphenols to o-diquinones, was induced in trees with introduced larvae, and bioassays showed that both growth and consumption rates of larvae were reduced in damaged trees. PPO activity was negatively associated with both larval growth and consumption rates in trees with bagged larvae, but not in control trees. Our results suggest that the oxidation of phenolics by PPOs may be a causal explanation for the rapidly induced resistance of mountain birch against E. autumnata. This finding also helps to explain why correlations between insect performance and phenolics (without measuring indices explaining their oxidation) may not produce consistent results.

Effects of elevated ultraviolet-B radiation on a plant–herbivore interaction

Enhanced ultraviolet-B (UV-B) radiation may have multiple effects on both plants and animals and affect plant–herbivore interactions directly and indirectly by inducing changes in host plant quality. In this study, we examined combined effects of UV-B and herbivory on the defence of the mountain birch (Betula pubescens ssp. czerepanovii) and also the effects of enhanced UV-B radiation on a geometrid with an outbreak cycle: the autumnal moth (Epirrita autumnata). We established an experiment mimicking ozone depletion of 30% (a relevant level when simulating ozone depletion above Northern Lapland). Both arctic species responded only slightly to the enhanced level of UV-B radiation, which may indicate that these species are already adapted to a broader range of UV-B radiation. UV-B exposure slightly induced the accumulation of myricetin glycosides but had no significant effect on the contents of quercetin or kaempferol derivatives. Mountain birch seedlings responded more efficiently to herbivory wounding than to enhanced UV-B exposure. Herbivory induced the activities of foliar oxidases that had earlier been shown to impair both feeding and growth of moth larvae. In contrast, the contents of foliar phenolics did not show the same response in different clones, except for a decrease in the contents of tannin precursors. The induction of foliar phenoloxidase activities is a specific defence response of mountain birches against insect herbivory. To conclude, our results do not support the hypothesis that the outbreak cycle of the autumnal moth can be explained by the cycles of solar activity and UV-B.

Effects of Overproduction of Condensed Tannins and Elevated Temperature on Chemical and Ecological Traits of Genetically Modified Hybrid Aspens (Populus tremula × P. tremuloides)

Gene transfer techniques offer new possibilities to study regulation of phenolic pathways and the defensive role of phenolics. Hybrid aspen lines (Populus tremula × tremuloides) that overexpress the PtMYB134 transcription factor were used to study the effects of condensed tannin production on plant physiology and plant defenses. The MYB134 protein activates all the known genes of the biosynthetic pathway for condensed tannins (CTs), so overexpression of MYB134 was expected to increase CT concentration in all tissues of the plants. Two out of three MYB134 overexpression lines (46 and 54) accumulated high levels of CTs and (+)-catechin, with a concomitant decrease in the levels of salicylates, but one transgenic line, MYB 61, failed to overproduce CTs. The concentrations of phenolic compounds generally were lower in the aspen leaves grown under elevated temperature than in those grown under ambient temperature. A specialist leaf beetle, Phratora vitellinae (Coleoptera: Chrysomelidae), was chosen to examine how over-expression of MYB134 and elevated temperature affect the food choice of a beetle adapted to feed on leaves rich in salicylates but containing little CT. Specialist beetles preferred the leaves grown at ambient temperatures possibly because these leaves had higher concentrations of salicylates, which are feeding stimulants. Beetles also preferred MYB line 61, which contained a normal level of CT but a slightly elevated level of salicylates. Our results show that transgenic plants are powerful tools, but that enhancing one secondary pathway may lead to unexpected effects on other pathways, and thus impact characteristics such as plant resistance against herbivores, especially under changing climatic conditions.