ORIGINAL ARTICLE Fredrik Schlyter Elisabeth Marling Jan Lo¨fqvist A new microassay for antifeedants in Hylobius pine weevils (Coleoptera) Published online: 23 March 2004 Ó Springer-Verlag 2004 Abstract A micro-feeding assay was developed for test- ing small amounts of synthetics and extracts as antifee- dants for Hylobius pine weevils. We devised a simple setup consisting of 5·5-mm pieces of thin layer chro- matography cellulose plates used in pairs for a bioassay involving choice. After application of 1.5 ll solutions and evaporation of the solvent, each plate received a 5 ll aliquot of 1 M sucrose as a feeding stimulant. The test period was 4 h at 25 °C with 6 days of starvation before the test, compared to just the 1 day sufficient for twig tests. Deprivation of water for 1 day at 65% RH before the test was critical because it increased feeding but not mortality. The combination of starvation and thirst reduced the relative variance (coefficient of variation) to below 50%. There was a good correlation of antifeedant activity in the microassay with the activity in a standard twig test. The microassay needs only several mg for a test series, whereas the twig test needs grams. Keywords Pine weevils Antifeedants Microassay Introduction In general, feeding tests for herbivores are done either on natural host material or on homogeneous artificial materials (Koul 1993; Hare 1998). Artificial diets for Coleoptera are, however, not common (Singh and Moore 1985). They have mostly been used with stored product pests (Nawrot et al. 1991; Daniewski et al. 1993; Alonso et al. 1994; Watson and Barson 1996; Huang et al. 1997; Liu and Ho 1999; Mohan and Fields 2002). Leaf disks of host plants have been used for some coleoptera (Gonzales et al. 1995; Park et al. 2000). Metcalf et al. (1980) described feeding stimulation by direct testing of curcurbitacins separated on TLC (thin layer chromatography) plates. Escoubas et al. (1992) described a feeding assay for insect larvae, including direct testing of chromatographic separations on TLC plates for antifeedant compounds. The first method used by us to find antifeedants for control of the important forest pest Hylobius abietis (Weslien 1998) relied on host material (Klepzig and Schlyter 1999). This test on pine twigs was based on the work of Salom et al. (1994), who showed the antifeedant activity of several compounds for the American species Hylobius pales. Problems associated with the twig test (Klepzig and Schlyter 1999) are the variability of the host material, the quantification of large areas of partly consumed bark, and the long time needed (>24 h) for consumption of a large proportion of the bark. The variation in any living host material before and after cutting it to a size suitable for testing is a general con- cern (Hare 1998). The large amount of chemical material needed for testing was, however, our major concern. In this paper, we describe the crucial factors (starva- tion and thirst) for our TLC plate micro-feeding assay for adult pine weevils and compare it to the pine twig test. Materials and methods In all experiments, except where otherwise noted, the animals were tested individually in 9-cm-diameter, standard, disposable Petri dishes in an environmental chamber at +25 °C, 65% RH and 3,000 lx, 20:4 L:D. After collection in the field in early summer, the weevil adults were stored in the dark at +8 °C for a maximum of 1 year with small diameter branches and twigs of pine, ad lib. Before testing, they were given a minimum 6-day ‘warming up’ period under test conditions. In regular tests, after the development of the bioassay, beetles were left starving with a supply of wetted towel paper for 5 days, after which they were deprived of water (dehydrated) for 1 day. Pilot test conditions for substrate conditions, stimuli, test environment etc are summarised in Table 1. The theoretical volume of the 0.1-mm-thick cellulose covering the 5·5-mm plates (Merck #1.0552) is 2.5 mm 3 , but a 1.5 ll (1.5 mm 3 ) drop of solvent was sufficient to wet a plate fully when F. Schlyter (&) E. Marling J. Lo¨fqvist Chemical Ecology, Department of Crop Sciences, Swedish Agricultural University, P.O. Box 44, SE-230 53 Alnarp, Sweden E-mail: fredrik.schlyter@vv.slu.se J Pest Sci (2004) 77: 191–195 DOI 10.1007/s10340-004-0050-9