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.     

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.     

Colorado potato beetles show differential digestive compensatory responses to host plants expressing distinct sets of defense proteins

Herbivorous insects fed plants expressing proteinase inhibitors (PIs) compensate for the loss of digestive proteolytic functions by producing novel proteinases. We assessed here whether such compensatory responses represent a general, non-specific adaptation to defense-related proteins in host plant tissues, or if distinct responses occur depending on the stress exerted on the plant. As a model, growth, development, and digestive proteases of the Colorado potato beetle (Leptinotarsa decemlineata Say) were monitored after feeding larvae with plants pre-treated with either methyl jasmonate or arachidonic acid, two compounds inducing different sets of defense genes in potato. In brief, larvae fed plants treated with jasmonate or arachidonate were negatively affected compared to larvae fed non-treated plants, suggesting the potency of both molecules to induce partial resistance to potato beetles in potato. On the other hand, larvae fed treated plants partially compensated for the presence of defense-related proteins by adapting their digestive proteolytic system, both quantitatively and qualitatively. These compensatory processes varied depending on the treatment, the larvae fed arachidonate-treated plants showing the most dramatic response. Compensation to jasmonate and arachidonate was also influenced by a cysteine PI from rice expressed in the plant, pointing out the possible indirect effects of recombinant defense proteins on naturally-occurring plant-insect interactions. These observations, while showing the potential of jasmonate and arachidonate as inducers of partial resistance to the potato beetle in potato, also suggest that digestive compensation in herbivorous insects is determined, at least in part, by defense-related compounds found in the plant in response to different stress stimuli or as a result of ectopic expression in transgenic plants.     

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.     

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.     

Physiological response of Colorado potato beetle and beet armyworm larvae to depletion of wound-inducible proteinase inhibitors in transgenic potato plants

Larvae of Colorado potato beetle (CPB), Leptinotarsa decemlineata, and beet armyworm (BAW), Spodoptera exigua, reared on potato plants in which wound-induced accumulation of proteinase inhibitors (PIs) was largely reduced through antisense-mediated depletion of a specific lipoxygenase (LOX H3) had significantly larger weight gains than those fed on non-transformed plants. The midgut endoproteolytic activities of CPB larvae fed on non-transformed potato were significantly higher than those from larvae fed on LOX-H3-deficient plants. However, none of these proteolytic activities was inhibited by potato leaf extracts, regardless of the plant that they were fed on. Taken together, these data suggest that CPB, a leaf-feeding specialist of solanaceous plants, is largely adapted to the inducible PIs of potato, though the metabolic cost associated with the hyperproduction of digestive proteases may account for the 14-31% lower weight gain of larvae fed on non-transformed plants. The effect of LOX-H3 depletion on insect performance was more evident with larvae of the polyphagous BAW (52-63% higher weight gain and 73% higher fecundity when reared on LOX-H3-deficient plants). The poorer larval performance of BAW on non-transformed plants may be due to the susceptibility to inhibition by potato leaf tissues of most BAW digestive proteases. Indeed, BAW larvae fed on non-transformed potato showed a significant reduction in most endoproteolytic activities compared to larvae fed on LOX-H3-deficient plants, suggesting a that these insects deal poorly with induced plant defences in potato.     

Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production

Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.     

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.     

Adult Colorado potato beetles, Leptinotarsa decemlineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases

Protease inhibitors have been proposed as potential control molecules that could be engineered into potato plants for developing crops resistant to the Colorado potato beetle, Leptinotarsa decemlineata, a major pest of potato and other Solanaceae. In this study, we examined the effects of feeding young female beetles with foliage from a cultivar of the "Kennebec" potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of larvae and adults of the potato beetle. To evaluate the insect's performance, we collected data over a 16-d postemergence period on survival, diapause incidence, foliage consumption, weight gain, and oviposition of females. Tested individuals were fed untransformed (control) and OCI-transformed foliage at two stages of potato leaf differentiation, corresponding to "low" and "high" levels of OCI expression in leaves of K52. The OCI-expressing foliage did not affect female survival (close to 100%), incidence of diapause (15-30%), relative growth rate (RGR) during postemergence growth (5-9% d(-1)) or maximum weight reached (140-160 mg). Neither did it affect female reproductive fitness as measured by preoviposition time (8-9 d), 16-d fecundity (220-290 eggs), or egg eclosion incidence (86-91%). However, nutritional stress to females feeding on OCI foliage was evident, as reflected in their lower efficiency of conversion of ingested foliage (ECI) during postemergence growth, increased foliage consumed per egg laid (up to 119% more), and adaptation of their digestive proteolytic system to the inhibitory effect of OCI. Interestingly, beetles fed foliage expressing the highest level of OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich foliage apparently as a compensatory response for nutritional stress due to the protease inhibitor in their diet.     

Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say)

Quantitative and qualitative changes in digestive proteolytic activities were monitored in fourth-instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say) subjected to three different leaf diets. Depending on the diet, the larvae exhibited variable growth rates, similar for potato (Solanum tuberosum) and eggplant (Solanum melongena) diets but lower for the tomato (Lycopersicon esculentum) diet. Interestingly, these growth rates were not associated with total protease activity in the midgut. While growth of tomato-fed insects was negligible, midgut protease activity in these insects was 1.5 and 4.2 times higher than that measured for potato- and eggplant-fed insects, respectively. As seen on gelatin-containing polyacrylamide gels, midgut extracts from insects that ingested eggplant leaves contained only a few proteinase forms, while numerous forms were observed in extracts of potato- and tomato-fed larvae. Although several forms were common to the three diets, their relative importance in the insect midgut varied. This diet-related plasticity of the digestive proteolytic system in Colorado potato beetle larvae leads one to question the potential for control approaches based on the inhibition of digestive proteases. Arch. Insect Biochem. Physiol. 36:241–250, 1997. © 1997 Wiley-Liss, Inc.     

新烟碱类杀虫剂拌种对马铃薯甲虫幼虫食物利用和生长发育的影响

为研究马铃薯甲虫Leptinotarsa decemlineata（Say）幼虫取食新烟碱杀虫剂噻虫嗪和吡虫啉处理（有效成分18 g/100 kg种薯拌种）播种90 d后马铃薯叶片的消化吸收和生长发育情况,在室内采用重量法测定了幼虫取食量、体重增加量、食物利用率,近似消化率以及蛹重等营养指标。 结果表明：70%噻虫嗪种子处理可分散粉剂拌种后,马铃薯甲虫幼虫的取食量、体重增加量、食物利用率、食物转化率、近似消化率和相对生长率以及蛹重显著低于10%吡虫啉可湿性粉剂拌种和空白对照（P＜0.05）,发育历期显著长于10%吡虫啉可湿性粉剂拌种和空白对照（P＜0.05）;而10%吡虫啉可湿性粉剂拌种后,马铃薯甲虫幼虫的取食量和3～4龄期的体重增加量与空白对照差异显著（P＜0.05）,蛹重、1～2龄幼虫的体重增长量、食物利用率、食物转化率、近似消化率、相对生长率和生长发育历期与空白对照差异不显著（P＞0.05）。 说明70%噻虫嗪种子处理可分散粉剂拌种对马铃薯甲虫幼虫的取食有显著抑制作用,不能很好地满足其生长发育营养物质的需要,幼虫取食其叶片后,近似消化率和相对生长速率显著降低,生长发育历期显著延长。 这有利于减少当季世代数,降低危害。     

Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.     

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

Influence of fertilization on the Colorado potato beetle, Leptinotarsa decemlineata, in organic potato production

The abundance of the Colorado potato beetle, Leptinotarsa decemlineata (Say), in organically grown potato did not change significantly in response to increasing rates of dehydrated poultry manure. However, peaks of abundance of larvae were shifted forward in time in response to the high rate of organic fertilizer. Tests using excised foliage showed that the shift was not caused by differential larval mortality or longer developmental times. Time allocation to resting, walking, and feeding by adults was similar regardless of fertilizer rate. Adult foliage consumption was unaffected by organic fertilizer rates in no choice tests and significantly affected in few choice tests. A 22% longer larval development time on plants treated with low fertilizer rate than on plants with high rate was the most significant effect. Even though maximum plant height, canopy, biomass, and yield were significantly smaller in the organic than in conventional plots, the suitability of the plants was not affected except for reduced feeding by summer beetles. Summer adults spent less time feeding and consumed two to five times less foliage on organic potato than on inorganically fertilized and conventionally produced plants. The overall influence of fertilizer on Colorado potato beetle populations was limited and therefore can only play a secondary role in management strategies for organic potato. Avoidance of excessive organic fertilizer that promotes short larval development time and extension of the period over which large Colorado potato beetle larvae are present should be recommended.     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Colorado potato beetle manipulates plant defenses in local and systemic leaves

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle’s performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.     

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.     

Proteolytic profile in the larval midgut of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Chilo suppressalis is a key constraint on production of rice. The current research was conducted to study the types of digestive proteases in the larval midgut of C. suppressalis. It was found that activity of total digestive proteases increased from the first to the fifth larval instars, which showed different nutritional requirements. Four types of proteinases and two types of exopeptidase were identified so that their activities from the highest to the lowest activities is trypsin‐like, chymotrypsin‐like and elastase for proteinases, and amino and carboxypeptidases for exopeptidases. Meanwhile, just one type of cysteine protease, cathepsin D, was determined in the fourth and fifth instar larvae. The optimal pH for activity of total protease was found to be pH 9–10 and optimal temperature was observed to be 35–40°C, where there was the highest proteolytic activity. Some specific inhibitors of proteases including PMSF, TLCK, TPCK, DTT, E‐64, cystatin, phenanthroline and EDTA were used to confirm the types of proteases in the midgut of C. suppressalis.     

Bioassay determination of the distribution of imidacloprid in potato plants: implications to resistance development

Soil-applied imidacloprid exhibits exceptional efficacy as a systemic insecticide against the Colorado potato beetle, Leptinotarsa decemlineata (Say). An uneven distribution of the chemical within potato plants could result in differential concentrations, which may allow for discrimination between genotypes of varying susceptibility. In this study, susceptible and tolerant larvae were fed leaves from the lower, middle, and upper canopy of treated and untreated plants to characterize within-plant distribution of imidacloprid at 4, 6, 8, 10, 12, and 14 wk after planting. Significant differences in larval mortality and development indicated that the concentration of imidacloprid was unevenly distributed in the potato foliage during 6-14 wk after planting. The concentration of imidacloprid was lowest in the younger tissues of the upper leaves and highest in the older, lower leaves. At 6 wk, a time when the postdiapause beetles are colonizing potato fields, the lower concentration in upper leaves was toxic to susceptible larvae but did not kill a substantial portion of the tolerant larvae. Results suggest that higher concentrations of imidacloprid in the lower canopy leaves may act as a toxic barrier to colonizing susceptible beetles but may allow more tolerant individuals to reach the upper canopy with lower concentrations. Possible scenarios of how different concentrations of the systemic insecticide could influence the rate of resistance development are discussed.