Different myrosinases activate sequestered glucosinolates in larvae and adults of the horseradish flea beetle.

ß-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous ß-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases.

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the ß-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle's energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

Plants use identical inhibitors to protect their cell wall pectin against microbes and insects.

As fundamentally different as phytopathogenic microbes and herbivorous insects are, they enjoy plant-based diets. Hence, they encounter similar challenges to acquire nutrients. Both microbes and beetles possess polygalacturonases (PGs) that hydrolyze the plant cell wall polysaccharide pectin. Countering these threats, plant proteins inhibit PGs of microbes, thereby lowering their infection rate. Whether PG-inhibiting proteins (PGIPs) play a role in defense against herbivorous beetles is unknown. To investigate the significance of PGIPs in insect-plant interactions, feeding assays with the leaf beetle Phaedon cochleariae on Arabidopsis thaliana pgip mutants were performed. Fitness was increased when larvae were fed on mutant plants compared to wild-type plants. Moreover, PG activity was higher, although PG genes were downregulated in larvae fed on PGIP-deficient plants, strongly suggesting that PGIPs impair PG activity. As low PG activity resulted in delayed larval growth, our data provide the first in vivo correlative evidence that PGIPs act as defense against insects.

Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle.

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the ß-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens.

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.