Detoxification of host plant phenolic aglycones by the spruce budworm

Abstract

This study examines the post-ingestive fate of two host-plant derived small-molecule phenolics (the acetophenones piceol and pungenol) that have previously been shown to be toxic to the outbreaking forest pest, spruce budworm (Choristoneura fumiferana). We test first whether these compounds are transformed during passage through the midgut, and second whether the budworm upregulates activity of the detoxification enzyme glutathione-s-transferase (GST) in response to feeding on these compounds. Insects were reared on either foliage or artificial diet to the fourth instar, when they were transferred individually to one of two treatment diets, either control or phenolic-laced, for approximately 10 days, after which midguts were dissected out and used for Bradford soluble protein and GST enzyme activity analysis. Frass was collected and subjected to HPLC-DAD-MS. HPLC showed that the acetophenones do not autoxidize under midgut pH conditions, but that glucose- and glutathione- conjugates are present in the frass of insects fed the phenolic-laced diet. GST enzyme activity increases in insects fed the phenolic-laced diet, in both neutral pH and alkaline assays. These data show that the spruce budwom exhibits counter-adaptations to plant phenolics similar to those seen in angiosperm feeders, upregulating an important detoxifying enzyme (GST) and partially conjugating these acetophenones prior to elimination, but that these counter-measures are not totally effective at mitigating toxic effects of the ingested compounds in the context of our artifical-diet based laboratory experiment.

Fig 1. Budworm body mass.

Body mass of sixth instar budworm caterpillars (mean±SE, N = 50) fed on either control- or phenolic-laced artificial diet in the three experimental runs (pretreatment on foliage (2016) or on artificial diet (2016 & 2017)).

Fig 2. Acetophenone incubation chromatograms.

HPLC-DAD chromatograms of the piceol/pungenol mixture incubated at A. neutral and B. alkaline pH. Numbers represent m/z of identified peaks: 137 = piceol, 153 = pungenol, 285, 287 and 303 = putative dimers.

Fig 3. Frass chromatograms.

HPLC-DAD chromatograms of frass from caterpillars fed A. control or B. phenolic-laced artificial diet. Numbers indicate m/z of identified peaks: 137 = piceol, 153 = pungenol, 287 = putative dimer, 299 = tentatively identified as picein, 315 = tentatively identified as pungenin, 425 = tentatively identified as a GSH-piceol conjugate, 441 = tentatively identified as a GSH-pungenol conjugate.

Fig 4. GST activity.

Glutathione-S-transferase enzyme activity of spruce budworm midgut tissue at neutral and alkaline pH, represented per mg soluble protein (mean ± SE, N = 12).