Incorporation of Quercus rubra foliage into artificial diet alters development of a fungal pathogen of Lymantria dispar

Entomol. exp. appL 68: 265-267, 1993. 9 1993 Kluwer Academic Publishers. Printed in Belgium.

265

Incorporation of Quercus rubra foliage into artificial diet alters development of a fungal pathogen of Lymantria dispar Ann E. Hajek & J. Alan A. Renwick

Boyce Thompson Institute, Tower Road, Ithaca, NY 14853-1801, USA Accepted: February 2, 1993"

Key words: bioassay, plant phenolics, host plant, Entomophaga maimaiga, fungi

Introduction

Our knowledge of the effects of foliage and/or phytochemicals on insects has often been based on results from studies in which these materials were incorporated into artificial diet (AD) (Berenbaum, 1986). The validity of this technique has been questioned; results from studies using incorporation of plant materials into AD usually demonstrate digestibility reduction but when allelochemicals have been administered externally on leaf material, no digestibility reduction is reported (see Berenbaum, 1986). This argument suggests that although effects can be shown with AD + plant materials, this effect may be artificial. In this study, gypsy moth, Lymantria dispar (L.), larvae were fed AD, red oak (RO = Quercus rubra L.) foliage, or AD + RO foliage; then larvae were infected with a fungal pathogen, Entomophaga maimaiga Humber, Shimazu & Soper and disease development was evaluated. We report inhibition of E. maimaiga development in larvae eating foliage-incorporated AD, although E. maimaiga developed normally in larvae eating AD or RO alone.

Materials and methods

Laboratory colony gypsy moth larvae were obtained as eggs from the USDA, APHIS, Methods

Development Center at Otis Air National Guard Base, Massachusetts, U.S.A. Larvae were reared on high wheat germ diet (Bell et al., 1981) with a wheat germ-casein base in 236.6 ml plastic cups with paper lids. 'Wild' larvae were field-collected as eggs, surface-sterilized in 3.7?/0 formalin for 1 h to kill surface contaminants, and then maintained at 4 ~ until needed. RO foliage was collected in Allegheny hardwood forests near Ithaca, New York. Larvae fed foliage alone were provided bouquets of RO foliage in ventilated plastic boxes after conidial inoculation (see Hajek, 1989a).

Foliage incorporation into artificial diet. AD was made using standard procedures but 10 g finely ground, lyophilized leaves minus petioles were added before AD gelled. Laboratory colony larvae were fed either AD or AD + RO. This experiment was replicated three times for AD + 6-7 June foliage, and twice for AD + 29-30 June foliage. When foliage was collected 6-7 June, third instars were abundant and 29-30 June, fifth instars were predominant. Wild larvae were reared on sleeved branches of RO in the field. Three parallel bioassays, each using larvae from three sleeves on three RO trees, were conducted in the laboratory between 8-28 June. Solvent extracts of RO foliage were made and, after removal of solvent by drying, the extracted material was added to AD. Acetone and methanol were specifically used as solvents based on

266 their use for tannin extraction (Hagerman, 1988). RO leaves collected 30 June were boiled in 100 ~o ethanol for 5 min. and cooled on ice. When cool, the extract was homogenized for 2 min, filtered through glass wool, and then evaporated in vacuo. The residue was washed into hexane and the leaf debris remaining in the glass wool filter was rinsed with diethyl ether. Hexane and ether extracts were combined and evaporated. The remaining defatted extract was shaken with water and filtered to provide a water soluble extract. Dried ethanol extract was also further extracted using 70 ~o acetone and this dried acetone extract was incorporated into AD. Extractions were also made by boiling RO foliage collected 28 August in 50~o methanol using the same procedures. For all extracts, the dry weight equivalent of 25 g of leaves was extracted to make 1 liter of AD.

Infection methodology. All larvae were reared on a treatment food for 6-7 days prior to infection. Larvae were challenged with E. maimaiga using a standard method of immersion in a suspension of 1 x 105 conidia/ml (Soper etal., 1988). Isolate A R S E F 3110 from the U S D A , Agricultural Research Service Collection of Entomopathogenic Fungi, Ithaca, New York was used to infect twenty-five larvae per treatment; fourth instars were used for bioassays using whole leaves and third instars were used for bioassays using solvent extracts of foliage.

Results

Entomophaga maimaiga development in larvae eating AD containing lyophilized, ground RO collected 6-7 June and 29-30 June differed from larvae fed AD alone (Table 1); mortality was equivalent across treatments, but few cadavers of larvae that had eaten RO-incorporated AD sporulated, and time from conidial inoculation until larval death was generally longer for larvae eating RO + AD. For wild larvae eating RO alone, neither mortality nor sporulation decreased between 8-28 June (t tests; P > 0.05). A similar pattern of results was found when laboratory colony larvae were fed AD + blended, entire leaves collected 20 July-5 Aug.; for larvae fed AD + RO, disease incubation was longer and E. maimaiga rarely produced spores in comparison with lab colony larvae fed AD or RO alone (chi-squared tests; P < 0.05). After larval death, fungal growth was frequently evident within bodies of larvae that had eaten AD + RO, but fungal structures were abnormal in appearance, containing abundant large lipid droplets. Fourth instars fed 7 June and 30 June RO + AD increased in weight more slowly over seven days than larvae eating only AD (7 June RO foliage + AD = 0.20 g; 30 June RO foliage + AD = 0.21 g; AD alone = 0.72 g) (t tests; P