The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole‐larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut‐specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin‐inducible. Computer docking and energy‐minimization of a CYP6AE89 model with all six furanocoumarins showed that 5‐methoxylated bergapten and 8‐methoxylated xanthotoxin had the smallest distances from the heme to the proton‐donor residue in the catalytic I‐helix, and that the 5,8‐dimethoxylated isopimpinellin and bergapten had the smallest energy‐minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono‐methoxylated linear furanocoumarins – bergapten and xanthotoxin – and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant furanocoumarins. A maximum‐likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.
In response to short-day photoperiods in the autumn, the large milkweed bug Oncopeltus fasciatus Dallas enters a reproductive diapause and migrates south to avoid the adverse environmental conditions and food shortages that prevail in the winter. Milkweed bugs are one of only a few temperate insects that undergo long distance migration during diapause, making them a good model for investigating trade-offs associated with migratory diapause. Although enhanced stress tolerance is typical of diapause, it is unclear whether this aspect of diapause would be retained in a species that migrates to more favourable conditions. The present study tests (i) whether diapause enhances thermal tolerance; (ii) whether food shortage, which is required for maximal expression of diapause, influences thermal tolerance during diapause; and (iii) whether the potential changes in stress tolerance are associated with upregulated heat shock protein expression or metabolic adjustments (including cryoprotectant synthesis), or both. Both cold tolerance at −10 ∘ C and heat tolerance at 43 ∘ C are significantly higher in diapausing O. fasciatus, whereas food restriction has no further effect on thermal tolerance. None of the heat shock protein transcripts measured are significantly upregulated in response to diapause, and the experiments also fail to detect any cryoprotectant accumulation during diapause. Thus, although heat shock proteins and cryoprotectants are common mechanisms for enhancing thermal tolerance in many diapausing insects, the results of the present study suggest that alternative mechanisms are responsible in milkweed bugs.
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