Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.     

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K_i values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.     

Molecular biology of wound-inducible proteinase inhibitors in plants

Abstract. The techniques of molecular biology are being employed to investigate at the gene level the systemically mediated, wound-induced accumulation of two defensive proteinase inhibitor proteins in plant leaves. These techniques have added a new dimension to biochemical and physiological studies already underway to understand the mechanism of induction by wounding. The acquisition of cDNAs from the RNAs coding for the two inhibitors facilitated studies of mRNA synthesis in leaves in response to wounding, and provided probes to obtain wound-inducible proteinase inhibitor genes from tomato ( Lycopersicon esculentum ) and potato (Solarium tuberosum) genomes. Successful transformations of tobacco plants with fused genes, containing the 5' and 3' regions of the inhibitor genes with the open reading frame of the chloramphenicol acelyltransferase ( cat ) gene, have provided a wound-inducible chloramphenicol acetyltransferase (CATase) activity with which to seek cis- and transacting elements that regulate wound-inducibility to help to understand the interaction of cytoplasmic and nuclear components of the intracellular communication systems that activate the proteinase inhibitor genes in response to wounding by insect pests.     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.     

Suppression of larval Colorado potato beetle growth and development by digestive proteinase inhibitors

Evidence is presented that the Colorado potato beetle (CPB) (Leptinotarsa decemlineata (Say)) depends, at least partially, on cysteine proteinases for protein digestion. Midgut homogenates of CPB larvae have a mildly acidic pH and exhibit major proteolytic activity in the mildly acidic pH range. This proteolytic activity is activated by reducing agents, is inhibited by E-64 (a specific cysteine proteinase inhibitor), and is not inhibited by serine proteinase inhibitors. In addition, consumption of E-64 treated potato leaves by CPB larvae at rates as low as 0.8 g/cm² of leaf tissue has a deleterious effect on larval growth and development.

---

Résumé Les protéinases intestinales des larves de Leptinotarsa decemlineata sont en partie caractérisées par les pH limitant leur activité, et par l'effet d'inhibiteurs de protéinases de spécificités connues. Les protéinases ont été testées avec la méthémoglobine tritiée comme substrat et nous avons déterminé les taux relatifs de peptides radioactives TCA solubles, libérées, dans des conditions précises, pour des pH compris entre 2,0 et 12,0. Le taux le plus élevé d'activité protéolytique a été observé pour des pH 5,0 à 6,0, bien qu'il y ait eu une activité appréciable aux pH 4,0 et 8,0. Parmi les inhibiteurs examinés, le E-64, inhibiteur protéinase cystéine très sélectif, a été le plus efficace sur l'activité protéinase. La pepstatine, inhibiteur protéinase aspartique, a été actif, mais seulement dans une gamme plus moin de pH. Les inhibiteurs protéinase sérine, ont été pratiquement inactifs sur l'activité protéinase.Chez des larves de doryphores nourries de feuilles de pommes de terre traitées avec différentes concentrations de E-64 ou de pepstatine, la consommation de E-64 a retardé fortement la croissance larvaire et le développement, la pepstatine a provoqué un retard du développement plus limitée.Nos résultats suggèrent que le doryphore dépend, tout au moins partiellement, des protéinases cystéine pour l'assimilation des protéines.

     

Antifeedant and toxic effects of drimanes on Colorado potato beetle larvae

Among the drimane compounds tested, the dialdehydes polygodial and warburganal were the most active as antifeedants against Colorado potato beetle larvae, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), in a dual-choice assay with potato, Solanum tuberosum L., leaf discs. Lactones were less effective. Direct observations showed that decreased feeding on leaf discs treated with polygodial and warburganal was accompanied by increased locomotry activity. Topical application of these two compounds on the insect's cuticle decreased food intake of untreated leaf discs, indicating that besides deterrent effects, toxic properties of these molecules influence feeding behaviour.     

Contact and residual toxicities of 30 plant extracts to Colorado potato beetle larvae

Abstract

Contact and residual toxicities of 30 plant extracts were investigated on third instar larvae of Colorado potato beetle, Leptinotarsa decemlineata. The plant samples were collected during the spring and summer of 2002 and were dried and ground. The plant samples were treated with methanol and the residue was eluted with distilled water containing 10% acetone, resulting in plant extracts. In contact bioassays, the beetle larvae were treated with 40% (w/w) plant extract using a Potter Spray Tower. The insects were incubated at 28±2°C under a 16 h: 8 h photo regime and the mortality was recorded at 24 h intervals for 7 days. The plant extracts exhibited varying toxicity to the larvae ranging from 0 – 91% after 24 h incubation and Artemisia vulgaris, Hedera helix, Humulus lupulus, Lolium temulentum, Rubia tinctoria, Salvia officinalis, Sambucus nigra, Urtica dioica, Verbascum songaricum, and Xanthium strumarium extracts resulted in significantly higher mortality than the control. Generally, prolonged incubation time did not result in an increase in mortality. After 48 h of incubation, 10 plant extracts yielded a significant mortality and H. lupulus extract, the most toxic extract among those tested, caused 99% mortality which is similar the mortality caused by the chemical standard, imidacloprid. In residual assays, potato leaflets were treated with 20% (w/w) plant-extract concentrations using a Potter Spray Tower. Third instar larvae were added to a glass jar to which treated leaflets were transferred before incubation at the temperature and photo regime described above. Mortality was recorded at 24 h intervals for 7 days. Five plant extracts, H. lupulus, L. temulentum, Reseda lutea and Solanum nigrum, induced significantly higher mortalities compared with controls. Chenopodium album extract was the most toxic, killing 34.9% of exposed larvae, followed by L. temulentum, H. lupulus and S. nigrum after 72 h incubation. The results exhibited that certain plant extracts were toxic to the beetle larvae and may have potential for controlling this destructive pest under field conditions.     

Plant volatiles moderate response to aggregation pheromone in Colorado potato beetle

Abstract:  The orientation of the Colorado potato beetle, Leptinotarsa decemlineata , to a male-produced aggregation pheromone, ( S )-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol, a three-component plant attractant blend [comprised of ( Z )-3-hexenyl acetate + (±)-linalool + methyl salicylate], and other potato volatiles (nonanal and 2-phenylethanol) were tested. All compounds were previously shown to be active in coupled gas chromatography/electroantennogram experiments. Both the three-component plant attractant blend and 2-phenylethanol were attractive to adult beetles. While male beetles oriented preferentially to both plant attractants vs. a control, females showed little preference. Combining the plant attractants with the pheromone resulted in sexually dimorphic responses similar to those seen with either plant attractant alone. Addition of nonanal abolished the sexually dimorphic response to the pheromone + 2-phenylethanol blend; the new three-component blend was attractive to both sexes. In both laboratory bioassays and field experiments, a combination of the pheromone + the three-component plant attractant was preferred over the plant attractant alone. Thus, it seems likely that combinations of pheromone + plant volatiles may be the most efficacious for field use.     

Host acceptance behaviour of Colorado potato beetle larvae influenced by potato glandular trichomes

ABSTRACT. In no-choice tests, larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), avoided contact with, and were less frequently observed feeding on, excised foliage of the resistant wild potato Solanum berthaultii Hawkes than on leaves of the cultivated potato, S.tuberosum L. For older larvae, reduced feeding was a consequence of less frequent contact with S.berthaultii leaves. However, first instars appeared unable to penetrate the adhesive barrier presented by glandular trichomes of S. berthaultii , and those that remained on the leaflets fed less often than did first instars on S. tuberosum. Removal of the trichome barrier by wiping leaflets with tissue paper did not attenuate the apparent repellent effect of S. berthaulti foliage, but led to increased incidence of feeding by first instars. Inhibition of larval feeding may therefore rely on a mechanical barrier provided by the glandular trichomes of S.berthaultii.     

Orientation of Colorado potato beetle to natural and synthetic blends of volatiles emitted by potato plants

1 Behavioural responses of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), to volatiles emitted from solanaceous host plants (potato and tomato), a non‐host legume (soybean), and 13 synthetic blends or three individual chemicals emitted by potato plants were investigated in laboratory bioassays. 2 Both male and female CPB were attracted to volatiles emitted by mechanically damaged potato foliage, but not to mechanically damaged tomato foliage; CPB offered a choice between the two damaged solanaceous plants did not show a preference. 3 Among 16 odourous blends or individual chemical components of potato plant emissions tested, six blends were attractive, two were repellent, and eight elicited no preference in laboratory bioassays. Volatile blends containing relatively high amounts of the green leaf volatiles (E)‐2‐hexen‐1‐ol and (Z)‐3‐hexen‐1‐ol, or the sesquiterpene β‐ caryophyllene, were unattractive or repellent. Minimal blends attractive to CPB were comprised of (Z)‐3‐hexenyl acetate (±)‐linalool and methyl salicylate: the combination of all three chemicals elicited sexually dimorphic attraction of males; two component blends comprised of (Z)‐3‐hexenyl acetate and either (±)‐linalool and methyl salicylate attracted both sexes. Individual compounds were inactive. No significant difference was noted between two attractive blends, or an attractive synthetic blend vs. mechanically damaged potato foliage. 4 These results show that CPB are attracted to blends of specific chemicals emitted by their host plants and provide a basis for the use of plant attractants as a component of integrated management of pestiferous populations.     

Characterization and distribution of chymotrypsin-like and other digestive proteases in Colorado potato beetle larvae

Chymotrypsin-like, carboxypeptidase A-like and leucine aminopeptidase-like activities have been detected in the midgut of Colorado potato beetle larvae, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), in addition to the previously identified cathepsin B, D, and H. We have characterized a new chymotrypsin-like activity using the specific substrates N- succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide and N-benzoyl-L-tyrosine p-nitroanilide. This novel proteinase, with a pH optimum of 5.5–6.5, was neither activated by thiol compounds nor inhibited by cysteine proteinase inhibitors. Among several serine proteinase inhibitors tested, PMSF was the most effective. Gelatin-containing SDS-PAGE gels and activity staining after gel electrophoresis indicated that chymotrypsin-like activity was associated with a major band of about 63 Kda and a minor band of about 100 Kda. The major exopeptidases found in the larval midgut extracts were leucine aminopeptidase and carboxypeptidase A. Most endo- and exoproteolytic activities studied were evenly distributed among the midgut sections, indicating that there is no clear regional differentiation in the digestion of proteins. Chymotrypsin and cathepsin B, D, and H were mainly located in the endoperitrophic and ectoperitrophic spaces, with only a small activity associated with the midgut epithelium. In contrast, leucine aminopeptidase was mainly located on the wall tissue, although some activity was distributed between the ecto- and endoperitrophic spaces. The potential roles of Colorado potato beetle digestive chymotrypsin in the proteolytic activation of the δ-endotoxin from Bacillus thuringiensis, and in the use of protease inhibitors to disrupt protein digestion, are discussed. Arch. Insect Biochem. Physiol. 36:181–201, 1997. © 1997 Wiley-Liss, Inc.     

Seasonal occurrence and relative abundance of aphids on potato plants with classical and transgenic characters of resistance to Colorado potato beetle Leptinotarsa decemlineata (Say)

The seasonal abundance and dynamics of potato-infesting aphids were studied in two seasons, on potato Solanum tuberosum L. with various types of transgenic and classical resistance to primary pests, especially the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say). The lines tested were the NewLeaf potato, a transgenic expressing the Bacillus thuringiensis CryIIIA toxin (BTT); NYL 235-4 a parental line derived from S. berthaultii (NYL) with a high density of glandular trichomes; and a transgenic line expressing oryzacystatln I (OCI), a proteinase inhibitor gene from rice; potatoes from two commercial cultivars, Superior and Kennebec, served as controls. Over the two seasons, Macrosiphum euphorbiae (Thomas) and, to a lesser extent, Myzus persicae (Sulzer) were by far the most abundant aphids observed in the experimental plots. M. persicae was observed relatively late in the season, and was most abundant on the Kennebec-OCI potato. Macrosiphum euphorbiae was more regular over seasons, and its highest densities were observed on the NYL 235-4 line. The population of M. euphorbiae showed markedly different patterns on the five lines studied, which was clearly related to potato plant phenology. Density of M. euphorbiae was observed on the NYL 235-4 line, in late July and early August.     

Genetic transformation of potato with nptII-gus marker genes enhances foliage consumption by Colorado potato beetle larvae

Little is known about the effect of transgenic plants containing commonly used marker genes, such as aph(3)II (nptII encoding neomycinphosphotransferase) and uidA (gus encoding -glucuronidase) on insect feeding behaviour. We report here, for the first time, that transgenic potato plants containing only nptII and gus marker genes enhance foliage consumption by the Colorado potato beetle (CPB, Leptinotarsa decemlineata S.). Transformation of potato cultivar Désirée was performed with Agrobacterium tumefaciens. Internode explants were inoculated with different strains of bacteria, carrying either nptII-gus or nptII alone. A total of 180 transgenic and untransformed control plants were grown in the greenhouse for the analysis of food consumption by CPB. For each transformed and untransformed line tested, four bioassays were conducted each consisting of 10 second-instar larvae feeding independently on a 2 cm diameter leaf disc for 20 h. Our data show up to 50% increase of mean foliage consumption on plants transformed with the nptII-gus construct, indicating that transgenic plants containing these marker genes can affect the feeding behaviour of the insects. These results were obtained from the primary regenerants (R₀ lines) as well as from tuber-derived plants (R₁ lines). Further tests with transgenic plants containing the nptII marker gene only, showed no significant difference in feeding when compared to untransformed control plants, allowing us to rule out a direct effect of this marker gene on foliage consumption by the insect larvae. It is suggested that gus protein is involved in the increase of foliage consumption by CPB.     

Behavioral responses of Colorado potato beetle larvae to combinations of temperature and insolation, under field conditions

In short-term field trials at combinations of ambient temperature (°C) and insolation (W·m⁻²), larval Colorado potato beetles (Leptinotarsa decemlineata [Say] [Coleoptera: Chrysomelidae]) were observed after their release on the adaxial surface of leaflets on potato plants (Solanum tuberosum L. Solanaceae). The larvae either began feeding or moved under the leaflet; mean interval from release to expression of these behaviors (2.9±0.05 min [n =358]) was independent of air temperature and insolation. Proportion of larvae moving under the leaflet increased logistically with both air temperature and insolation. A 1 W·m⁻² change in insolation (P) evoked the same effect on this proportion as a 0.0838 °C change in air temperature (T _a ), so the two quantities were combined as T^*=T _a +P·0.0838 °C/(W·m⁻²), which has units of °C. The proportion of larvae moving under the leaflet increased logistically with T^*. In 1-day field trials we monitored air temperature, insolation and proportion of larvae under the leaflet, and compared the latter to predictions from the logistic regression derived from the short-term trials. Consistently more larvae occurred under leaflets than predicted from the logistic regression; this bias diminished as T^* increased until at T^*≥40 °C, observed and predicted proportions were equal. This pattern of deviation from the predictions of the logistic regression is consistent with a thermoregulatory strategy in which larvae move away from hostile conditions, rather than seek optimal conditions.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.     

Disrupting spring colonization of Colorado potato beetle to nonrotated potato fields

Overwintering Colorado potato beetles (Leptinotarsa decemlineata (Say)) were concentrated primarily within woody borders, and mortality was lower in borders than in potato fields. After overwintering, only 15–44% of live beetles were in the potato fields. In experiments with small plots, colonization of fields from woody borders was reduced ∼60% by a trap crop, either treated with adulticide or with beetles collected daily. Such trap crops, or simply pitfall traps to prevent colonization from woody borders, could significantly reduce early-season adult numbers and subsequent larval populations. However, success is dependent on the local densities of overwintered beetles, and the prevalence of arrestment behavior in the case of trap crops.     

Diverse enzymatic specificities of digestive proteases, 'intestains', enable Colorado potato beetle larvae to counteract the potato defence mechanism

In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the Ki values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms.     

Response to host plant odors and aggregation pheromone by larvae of the Colorado potato beetle on a servosphere

Using a servosphere (locomotion compensator), locomotory behavior of Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) larvae was measured in detail in response to pulsed and non-pulsed odors of hostplant and conspecific pheromone. Second instars showed decreased straightness of movement, and all larvae showed decreased speed, in response to potato odor. Change in straightness by 2nd instars was also significantly affected by the interaction of pheromone and pulsing treatments. Fourth instars showed increased positive anemotaxis in response to the combined hostplant and pheromone odors. To our knowledge, this is the first demonstration of larval behavioral responses to adult pheromone in a holometabolous insect.