Roxanne M. Broadway

Are insects resistant to plant proteinase inhibitors

Abstract Six species of Lepidoptera were evaluated for their susceptibility to serine proteinase inhibitors from cabbage. Trypsin and chymotrypsin activity from larval Pieris rapae and Pieris napi were not significantly inhibited (0–18%), in vitro , by cabbage proteinase inhibitors, while the serine proteinase activity in the midguts of larval Plutella xylostella was moderately inhibited (40–50%), and Trichoplusia ni, Lymantria dispar , and Helicoverpa zea were substantially inhibited (55–85%) by cabbage proteinase inhibitors. These results suggest that the growth and development of the latter three species should be reduced following ingestion of these inhibitors. However, chronic ingestion of cabbage proteinase inhibitors only reduced the growth and development of T. ni . This lack of biological activity of the proteinase inhibitors against the other two species was explained by a shift in the relative proportion of digestive enzymes in response to ingestion of proteinase inhibitors. Following ingestion of cabbage proteinase inhibitors, the predominant trypsin-like enzyme(s) in the midgut of larval L. dispar and H. zea were resistant to inhibition by cabbage trypsin inhibitors (13–18% inhibited), while the trypsin(s) in T. ni was moderately susceptible (37% inhibited). These results were confirmed for H. zea and T. ni feeding on proteinase inhibitors in tomato foliage. This is the first demonstration of adaptation to proteinase inhibitors, and has important implications for gene regulation and physiological plasticity.

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors.

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.

Dietary proteinase inhibitors alter complement of midgut proteases

Plant serine proteinase inhibitors (Pls) have the potential to restrict the growth and/or development of herbivorous insects. However, there are limitations to the efficacy of these Pls. An insects susceptibility to a Pl is determined, at least in part, by the relative proportion of proteolytic enzyme activity in the midgut that is suppressed by that inhibitor. Insects adapt to dietary trypsin inhibitor in their host plant by secreting “inhibitor-resistant” trypsin(s). These “inhibitor-resistant” enzyme(s) may be the standard proteinase(s) secreted into the midgut (e.g., Pieris rapae), or may be enhanced following ingestion of proteinase inhibitor (e.g., Helicoverpa zea). In addition, insects may be pre-adapted to specific Pl(s), following adaptation to a Pl from the same family. For example, Pieris rapae is a crucifer specialist that is resistant to cabbage Pl, but is also resistant to Kunitz soybean trypsin inhibitor, a Pl in the same family as cabbage Pl, but from a non-host plant. The ultimate value of this pre-adaptation to herbivory by a species of insect will be determined by the number of different families of Pl in host plant(s) to which the species has adapted, and the distribution of those families among other species of plants. Thus, it is possible that the presence of a plant Pl limits herbivory by insect(s). However, multiple inhibitors, matched to the complement of enzymes in the insects midgut, may be required to enhance this resistance of plants to herbivorous insects.

Characterization and ecological implications of midgut proteolytic activity in larvalPieris rapae andTrichoplusia ni

In their larval luminal midgut fluid,Trichoplusia ni (Lepidoptera: Noctuidae) andPieris rapae (Lepidoptera: Pieridae) contain endopeptidases as their primary proteases. Neither species has detectable exopeptidase activity. Studies using enzyme-specific substrates and inhibitors demonstrate that the endopeptidases are serine proteinases (both trypsinlike and chymotrypsinlike) with histidine at the active site. Optimal pH for the tryptic and chymotryptic activity is 8.5 and 8.0, respectively, forT. ni. and 8.0 and 9.0, respectively, forP. rapae. The efficiency of proteolytic digestion (as measured by the rate of in vitro digestion of a standard protein by the midgut luminal fluid) is positively correlated with the larval dietary protein requirement and is significantly influenced by the ratios of tryptic to chymotryptic activity present in the gut lumen of these two species of Lepidoptera.

Influence of cabbage proteinase inhibitorsin situ on the growth of larvalTrichoplusia ni andPieris rapae.

Trypsin and chymotrypsin inhibitors are proteins that are developmentally regulated in foliage of cabbage plants, appearing at high concentrations in young foliage on mature plants. This temporal and spacial regulation of foliar proteinase inhibitors is synchronized with the appearance and distribution of foliar feeding Lepidoptera. When insects were allowed to select their feeding sites, larvalPieris rapae fed on the young foliage of cabbage plants, while larvalTrichoplusia ni fed on the mature foliage on cabbage plants. LarvalP. rapae that fed on mature plants were significantly smaller than larvae feeding on young plants, while there was no significant difference between larvalT. ni feeding on mature plants and those feeding on young plants. Thus, there was a significant inverse correlation between the level of proteinase inhibitory activity in cabbage foliage and larval growth. WhenP. rapae andT. ni were provided with an artificial diet containing total protein (including significant levels of proteinase inhibitors) that was extracted from cabbage foliage, there was a significant reduction in growth and development of both species of Lepidoptera.

Novel Chitinolytic Enzymes with Biological Activity Against Herbivorous Insects

The soil bacteria, Streptomyces albidoflavus, secretes endochitinases and chitobiosidases that are active over a broad range of pH (4–10). Ingestion of this mixture of chitinolytic enzymes significantly reduced the growth and development of Trichoplusia ni and significantly reduced survival of Myzus persicae, Bemisia argentifolii, and Hypothenemus hampei. Perfusion chromatography was used to separate endochitinases from chitobiosidases. The endochitinases had significantly greater biological activity against Bemisia argentifolii than the chitobiosidases. The utility of chitinolytic enzymes as regulators of populations of herbivorous insects is discussed.

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus

Streptomyces albidoflavus NRRL B‐16746 secreted three types of chitinolytic enzymes: N‐acetyl‐glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4–6; however 55–74% of the chitobiosidase and endochitinase activity was detectable at pH 8–10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8–5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4–10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.

Does host range influence susceptibility of herbivorous insects to non‐host plant proteinase inhibitors?

We examined the influence of proteinase inhibitors on digestive enzymes and development of oriental beetle, Exomala orientalis Waterhouse, European chafer, Rhizotrogus majalis (Razoumowsky), Phyllophaga white grub, Phyllophaga anxia (LeConte), cranberry root grub, Lichnanthe vulpina (Hentz), Japanese beetle, Popillia japonica Newman, Asiatic garden beetle, Maladera castanea (Arrow) (Coleoptera: Scarabaeidae), and the black cutworm, Agrotis ipsilon (Rottemburg) (Lepidoptera: Noctuidae). We demonstrated that all species within our test group had alkaline midguts that contained proteinase activity that could be inhibited, in vitro with serine proteinase inhibitors. Our data suggests that host range may influence the susceptibility to non‐host inhibitors. Chronic ingestion of the serine proteinase inhibitor, Kunitz‐soybean trypsin inhibitor (STI), significantly reduced proteolytic activity in vivo in those species with relatively specialized feeding habits (i.e., cranberry root grub, Japanese beetle, Asiatic garden beetle, and black cutworm). Chronic ingestion of STI also resulted in reduced larval growth and delayed pupation for black cutworm, and elevated larval mortality for Japanese beetle. However, chronic ingestion of STI did not influence larval survival for those species with relatively generalized feeding habits (i.e., oriental beetle, European chafer). Based on these results, we propose mechanistically‐based criteria for selecting proteinase inhibitors for phytochemical defense against herbivorous insects.

Tryptic inhibitory activity in wild and cultivated crucifers

Abstract Tryptic inhibitory activity was detected in all species and cultivars of crucifers examined. When comparing the three groups of crucifers tested [(i) foliage of cultivated crucifers, (ii) foliage of wild crucifers and (iii) storage organs of cultivated crucifers], the foliage of cultivated crucifers contained significantly higher levels of tryptic inhibitory activity than the other two groups.

Purification and partial characterization of trypsin/ chymotrypsin inhibitors from cabbage foliage

Abstract Proteinase inhibitors from cabbage foliage ( Brassica oleracea ) had trypsin and chymotrypsin inhibitory activity that was relatively stable over a broad range of temperatures (0–100°) and pH values (4.5–7.5). The six proteinase inhibitors that were purified by affinity chromatography had M r s that ranged from 9000 to 25 000, and isoelectric points that ranged from 4.5 to 5.0. Separation of these affinity-purified proteins by reverse phase HPLC resulted in 14 unique protein species with trypsin and chymotrypsin inhibitory activity. Based on similarities in the amino acid content, the HPLC-purified inhibitors were arranged into four groups.