Ecological Eiitomology (1994) 19, 344-348 Root disturbance of common ash, Fraxinus excelsior (Oleaceae), leads to reduced foliar toughness and increased feeding by a folivorous weevil, Stereonychus fraxini (Coleoptera, Cu rcuI ionidae) ANDREW FOGG0,1T2MARTIN R. SPEIGHT' and J E A N - C L A U D E G REG O I R E2 'Department of Zoology, University of Oxford, U. K., and 'Laboratoire de Biologie Animale et Cellulaire, Universitt Libre de Bruxelles, Belgium Abstract. 1. Bioassays were carried out to examine differences in the feeding of a folivorous weevil, Stereonychus fraxini De Geer, on leaves from ash saplings with undamaged roots, and those with damaged roots. Beetles ate significantly more of the leaves of root-damaged saplings in a choice experiment. 2. A separate study of the effect of root damage on leaf toughness was carried out on a second group of ash saplings. Saplings with damaged roots had less tough leaves than control saplings. Decreased leaf toughness in hypothesized as a mechanism to explain the preference of weevils for leaves from damaged trees. 3. Both chemical and physical changes may occur in the tissues of ash trees in response to environmental stress such as drought and root damage. 4. Root damage caused by agricultural disturbance is hypothesized as a mechanism making mature ash trees in hedgerow ecosystems more susceptible to insect herbivores. Key words. Fraxinus excelsior, insect herbivory, foliar toughness, feeding preference, Stereonychus fraxini. Introduction such as proteins with toughness influencing compounds such as lignins and phenolics (Swain, 1079; Scalbert & The toughness of leaf tissue has been shown by several Haslarn, 1987). The effects of changes in toughness upon studies to affect its value as a food resource to herbivorous the feeding and growth rates, fecundities, and survivorship insects. Toughness may be arbitrarily defined in this of insects have been studied in several taxa (Raupp, 1985). context as a measure of the amount of force required to and several authors have demonstrated the potential of cut or tear the tissue in a fashion consistent with the leaf toughness as a significant factor limiting herbivore feeding processes of chewing insects. Choong et al. (1992) exploitation of a resource (Feeny, 1970; Connor, 1988). discuss mechanisms of rneauring leaf toughness, and In the main, studies have concentrated upon leaf age as conclude that 'fracture toughness', as measured by pen- a determinant of toughness, with older leaves having been etrometry, represents a useful predictor of herbivore shown to be tougher in several studies (Feeny, 1970; preference for plant tissues. Raupp (1985) hypothesizes Schultz et al., 1982; Raupp, 1985; Kearsley & Whitham, several mechanisms by which the toughness of leaves may 1989). However, leaf toughness is also known to increase reducc their suitability as food; these include age-related in response to both natural and artificial damage of foliage deposition of indigestible compounds such as cellulose (Coley, 1983) and may represent one of a suite of responses and lignin (Rausher, 1981), dilution of nutrients with to such forms of stress. Changes in foliar toughness affect- indigestible bulk (Rausher, 1081; Coley, 1983), and physico- ing the population dynamics of insect herbivores may be of chemical effects of the complexing of valuable molecules increased importance when insects are suspected of playing a role in the death or decline of trees. Such is the case in Corrcspndcnce: Dr Andrcw Foggo, Dcpartmcnt of Zoology, the instance of ash dieback in Great Britain. South Parks Road, Oxford OX1 3PS. The population of common ash (Fraxinus exvelsior L.) 344 Stress, foliar toughness, and herbivory in ash 345 growing in the hedgerows surrounding fields in central and two groups. Each tree chosen was separated from other eastern Britain has been undergoing a gradual dieback members of the groups by at least 3m. The first group since the early 1960s (Peace, 1962). The dieback affects were labelled as controls and left undisturbed. The second mature, non-aged individuals, and is characterized by the group had their roots disturbed according to the protocol gradual death of branch tips, failure of terminal buds to described by Foggo & Speight (1993). flush, and a clumping of foliage and general reduction in Bioassay techniques. Three weeks after the manipu- shoot numbers. Trees may lose over 90% of the crown and lations were carried out, bioassays commenced. At least survive, often through the process of epicormic sprouting ten replicates were set up at any one time. For each assay, (Hull & Gibbs, 1991). Research into the aetiology of the two leaf termini consisting of three folioles were used, one condition has shown strong links with environmental dis- from a control tree and one from a disturbed tree. The turbance such as ploughing, ditching, and compaction as a leaves of F.excelsior are composite, with a terminal foliole result of proximity to rights of way (Pawsey, 1983; Hull & and four to six pairs of opposed folioles. Termini were Gibbs, 1991). whereas water stress possibly linked to severed from the leaf by cutting just above the second soil type has also been hypothesized as a causal factor foliole pair to leave a triplet of folioles with a short stem. (Hull & Gibbs, 1991). Some studies (Peace, 1962; Pawsey, Each pair of termini for use in a single bioassay were 1983) have suggested that herbivorous insects may play chosen from the same height within trees of similar size, a role as secondary stressing agents, as has shown to be and leaves of similar sizes were used throughout. Several the case in several other dieback scenarios (Mackay et al., leaf termini were picked from each tree. Immediately after 1984; Hosking & Kershaw, 1985; Landsberg, 1988). In being picked, the folioles were placed in a plastic tub particular, the ash bud moth, Prays fraxinella Bjerk. (Lep., containing moist tissue paper, separated from each other Yponomeutidae), was singled out as a candidate for such a by more tissue. Control and treatment leaves were col- role (Pawsey, 1983; Foggo, 1992). lected in separate boxes. In view of the relationship of dieback with environ- Each bioassay replicate was carried out in a rectangular mental disturbance, and the initial observations of workers PVC box measuring 15 x 10 x 7cm with punctures in the that insect damage appeared to be higher in trees showing lid for ventilation. One control leaf terminus was placed at signs of dieback, the role of root damage as a stressing one end of the box; the paired treatment terminus was agent was singled out for study. In this paper we describe placed at the opposite end of the box. Leaves were pro- studies linking apparent induced changes in leaf toughness vided with water throughout the tests by sealing the cut with increased herbivore exploitation of the leaves of stem in a small vial of water. One pair (male and female) disturbed trees by a folivorous weevil, Stereonychus fraxini of recently emerged (within 36h) S.fraxini adults were De Geer. We also hypothesize a role for such changes then added to the box which was placed in a constant in the herbivore-related stress experienced by trees under- temperature chamber at 22"C, 16 h/8 h LD for 72 h. Pairs going dieback in the hedgerow ecosystem of the British of animals were used in this way because numbers of agricultural countryside. individuals of each sex available were inadequate for a single sex study. At the end of this time, leaves were removed from the boxes and refrigerated prior to analysis, Methods and the animals returned to breeding cultures. Measuring feeding preference. Preference was expressed Feeding preference bioassays. To test the hypothesis that as the difference between the absolute areas of the folioles disturbance of the roots of ash trees might influence the in each group offered which was consumed during the feeding of herbivorous insects, a bioassay was set up trial. Weevil feeding creates holes in the leaf surface rather using the folivorous weevil S.fraxini. This species is found than at the perimeter of the leaf; thus the initial area of throughout mainland Europe (Miklos, 1976; Lemperibre leaf offered could be determined after the trial using the & Malphettes, 1983), but not in the British Isles. This undisturbed perimeter as a guide. A photometric technique species was chosen for feeding tests for a variety of reasons. was used to determine leaf areas, with folioles placed Larvae could be collected from the field at an early age under a sheet of clear perspex on a bright, diffusely lit and laboratory reared on material of the same provenance background to allow a monochrome image of the leaf to to preclude effects of larval feeding influencing adult be captured using a CCIR format video camera (Minitron) choice. Freshly emerged adults could thus be easily obtained and the VFG-512 Provision 1.0 image grabbing system and introduced immediately into experimental conditions. (Visionetics). Areas of black pixels corresponding to leaf Experimental manipulations. The aim of the bioassay tissue could be determined using the grabbing software, was to test for differences in weevil feeding between leaves and holes in the leaf image caused by the weevil feeding originating from undisturbed and disturbed trees. Because electronically 'filled in'. The area of black pixels was of the difficulty of experimentally manipulating mature then redetermined and the total leaf area eaten for each trees, saplings were chosen to supply the leaves. A large terminus was thus calculated as: group of 2-3m tall specimens growing in the For& de x(Black areas post reconstruction - Black areas pre- Soignes, south of Brussels, were chosen for the exper- reconstruction) iment, and manipulations were camed out in late April 1991. Thirty saplings were randomly assigned to each of This system was calibrated prior to use by cutting out 346 A.