Degree of Dietary Specialization on Furanocoumarin-Containing Hostplants in a Newly Invasive Web Building Caterpillar.

The genus Depressaria (Lepidoptera: Depressariidae) mostly comprises specialist herbivores with varying capacity for detoxification of defensive phytochemistry. Depressaria depressana, a Eurasian moth recently introduced into North America, is a family-level specialist of the Apiaceae, whose hosts include more than a dozen species in multiple tribes; Depressaria radiella is a super-specialist of Eurasian origin that feeds exclusively on species in the genera Pastinaca and Heracleum throughout its native and introduced range. In eastern North America, it feeds upon Pastinaca sativa, an invasive European species, and Heracleum maximum, a native species. We determined whether differences in furanocoumarin metabolism exist between D. depressana and two isolated populations of D. radiella, feeding exclusively on either P. sativa or H. maximum. We also compared gravimetric estimates of feeding efficiency to assess D. depressana larval performance on different diets. Both populations of D. radiella metabolized furanocoumarins at a greater rate than D. depressana. Although there was no difference in rates of metabolism of linear furanocoumarins in the two populations of D. radiella, individuals collected from H. maximum metabolized angular furanocoumarins more rapidly. The gravimetric assessments of feeding efficiencies revealed that D. depressana exhibited highest efficiencies consuming Daucus carota; moreover, this species survived to pupation consuming fruits of Zizia aurea, an apiaceous species native to North America. Our preliminary phylogenetic analysis, building on an earlier morphological analysis, incorporates mitochondrial cytochrome oxidase subunit 1 data from the BOLD database and revealed that the presence or absence of furanocoumarins is not a strong predictor of species-level evolution in Depressaria.

A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip.

Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co-localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3-CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α-ketoglutarate-dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p-coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.

Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis.

In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

Testing a vapour-phase model of stomatal responses to humidity.

This study tests two predictions from a recently proposed model for stomatal responses to humidity and temperature. The model is based on water potential equilibrium between the guard cells and the air at the bottom of the stomatal pore and contains three independent variables: gs(0), Z and Θ. gs(0) is the value of stomatal conductance that would occur at saturating humidity and will vary among leaves and with CO2 and light. The value of Z is determined primarily by the resistance to heat transfer from the epidermis to the evaporating site and the value of Θ is determined primarily by the resistance to water vapour diffusion from the evaporating site to the guard cells. This leads to the two predictions that were tested. Firstly, the values of Z and Θ should be constant for leaves of a given species grown under given conditions, although gs(0) should vary among leaves and with light and CO2. And secondly, the ratio of Z to Θ should be higher in leaves having their stomata in crypts because the distance for heat transfer is greater than that for water vapour diffusion. Data from three species, Nerium oleander, Pastinaca sativum and Xanthium strumarium support these two predictions.

Lutein sequestration and furanocoumarin metabolism in parsnip webworms under different ultraviolet light regimes in the montane west.

Both biotic and abiotic selection pressures can contribute to geographic variation in allelochemical production in plants. We examined furanocoumarin production in western North American populations of Heracleum lanatum and Pastinaca sativa that, at different latitudes and altitudes, experience different ultraviolet (UV) light regimes. Total furanocoumarins and linear furanocoumarins of fruits were negatively correlated with UV irradiance, whereas amounts of angular furanocoumarins, which are generally less phototoxic, were not. Another factor potentially influencing furanocoumarin production is the presence of the parsnip webworm Depressaria pastinacella, (Lepidoptera: Oecophoridae), an herbivore that feeds on reproductive structures of both plant species. These insects sequester lutein from their host plants; this carotenoid acts to ameliorate furanocoumarin toxicity. Although the concentration of lutein in fruits did not vary with UV irradiance, lutein sequestration by sixth instars was positively correlated with UV irradiance. Webworm populations are variably infested with the polyembryonic webworm parasitoid Copidosoma sosares Walker (Hymenoptera: Encyrtidae). H. lanatum fruits from populations with webworms parasitized by C. sosares had lower concentrations of furanocoumarins, with the exception of sphondin, than fruits from plants infested with webworms free from parasitism. Lower levels of these furanocoumarins may reduce negative effects on the fitness of this parasitoid. In contrast with the variation in furanocoumarin content, the ability of webworms to metabolize furanocoumarins by cytochrome P450 did not differ significantly among populations from New Mexico to Alberta.

Increase in toxicity of an invasive weed after reassociation with its coevolved herbivore.

The ability of weeds to proliferate into nonindigenous habitats has been attributed to escape from their native natural enemies, allowing reallocation of resources from chemical defense into growth and reproduction. Many invasive weeds, however, eventually encounter their native, coevolved enemies in areas of introduction. Examination of herbarium specimens of an invasive phototoxic European weed, Pastinaca sativa, through 152 years reveals phytochemical shifts coincident in time with the accidental introduction of a major herbivore, the parsnip webworm, Depressaria pastinacella. Plants collected before the introduction of webworms in North America and during the earliest stages of establishment (1850-1889) are lower in toxic furanocoumarins than all plants subsequently collected in North America and lower than European plant samples collected before 1889. Thus, introduction of a major specialist herbivore can increase noxiousness of a species in its area of introduction, illuminating a potential consequence of classical biocontrol programs involving insect herbivores and poisonous weeds.

Genetic variation in primary metabolites of Pastinaca sativa; can herbivores act as selective agents?

Although insect herbivory has been shown to act as a selective agent on plant secondary metabolism, whether primary metabolites contribute to resistance and can respond to selection by herbivores remains untested. In the wild parsnip (Pastinaca sativa), its principal herbivore, Depressaria pastinacella, acts as a selective agent on furanocoumarin resistance factors. In this study, we determined whether webworms can, by causing differential reductions in fitness, act as selective agents on parsnip primary metabolites. Estimates of narrow-sense heritabilities were significantly different from zero for C18 fatty acids in buds and developing fruits, fructose and sorbitol in buds, fructose, myo-inositol, bergapten, and psoralen in fruits. Wild parsnips protected from webworms by insecticide produced 2.5 times as much seed biomass as unsprayed plants; that webworms accounted for this difference in plant fitness was indicated by a significant negative relationship between reproductive effort and an index of webworm damage. Only a handful of metabolites influenced resistance to webworms; these included osthol, sorbitol, and protein in developing fruits as well as previously documented furanocoumarins. Osthol, a coumarinic compound, enhanced resistance, as did protein content, while sorbitol lowered resistance. Other primary metabolites may affect resistance to webworms, but their effect was context-dependent, that is, their effect depended on concentrations of other metabolites (epistasis). Susceptible plant phenotypes were found to have average chemical compositions. Although there was genetic variation in some of the primary metabolites in parsnips, the epistatic nature of their involvement in resistance and the lack of genetic variation in some suggest that selection on them from webworms will be either inconsistent or ineffective.

Impact of folivory on photosynthesis is greater than the sum of its holes.

The effects of herbivores on plant production and fitness may not relate directly to the quantity of biomass removed because folivory may alter photosynthetic rates at a considerable distance from the damaged tissue [Welter, S. C. (1989) in Insect-Plant Interactions, ed. Bernays, E. A. (CRC, Boca Raton), pp. 135-151.]. An impediment to understanding the effects of leaf damage on photosynthesis has been an inability to map photosynthetic function within a single leaf. We developed an instrument for imaging chlorophyll fluorescence and used it to map the effects of caterpillar feeding on whole-leaf photosynthesis in wild parsnip. The adverse effects of caterpillar feeding on photosynthesis were found to extend well beyond the areas of the leaflet in which caterpillars removed tissue. These "indirectly" affected areas remained impaired for at least 3 days after the caterpillars were removed and were six times as large as the area directly damaged by the caterpillars. Although photosynthesis in indirectly affected areas was reduced and not eliminated, these areas accounted for three times as much of the overall reduction in photosynthesis as the area removed by the caterpillars. The size of the indirect effects was positively correlated with defense-related synthesis of furanocoumarins, suggesting that costs of chemical defense may be one factor that accounts for the indirect effects of herbivory on plants.