Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

Host plant defenses of black (Solanum nigrum L.) and red nightshade ( Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say).

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in-vitro glutathione S-transferase (GST) inhibition activity, we used insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat-body homogenate. In-vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis-hex-3-enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.

Overexpression of a cytochrome P450 and a UDP-glycosyltransferase is associated with imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata.

Current control of insect pests relies on chemical insecticides, however, insecticide resistance development by pests is a growing concern in pest management. The main mechanisms for insecticide resistance typically involve elevated activity of detoxifying enzymes and xenobiotic transporters that break-down and excrete insecticide molecules. In this study, we investigated the molecular mechanisms of imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), an insect pest notorious for its capacity to develop insecticide resistance rapidly. We compared the transcriptome profiles of imidacloprid-resistant and sensitive beetle strains and identified 102 differentially expressed transcripts encoding detoxifying enzymes and xenobiotic transporters. Of these, 74 were up-regulated and 28 were down-regulated in the resistant strain. We then used RNA interference to knock down the transcript levels of seven up-regulated genes in the resistant beetles. Ingestion of double-stranded RNA successfully knocked down the expression of the genes for three cytochrome P450s (CYP6BQ15, CYP4Q3 and CYP4Q7), one ATP binding cassette (ABC) transporter (ABC-G), one esterase (EST1), and two UDP-glycosyltransferases (UGT1 and UGT2). Further, we demonstrated that silencing of CYP4Q3 and UGT2 significantly increased susceptibility of resistant beetles to imidacloprid, indicating that overexpression of these two genes contributes to imidacloprid resistance in this resistant strain.

Developing an in vivo toxicity assay for RNAi risk assessment in honey bees, Apis mellifera L.

Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments.

Potential of the Lectin/Inhibitor Isolated from Crataeva tapia Bark (CrataBL) for Controlling Callosobruchus maculatus Larva Development.

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato.

Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.

Knocking down a putative Δ(1) -pyrroline-5-carboxylate dehydrogenase gene by RNA interference inhibits flight and causes adult lethality in the Colorado potato beetle Leptinotarsa decemlineata (Say).

BACKGROUND: Leptinotarsa decemlineata is an able disperser by flight. Novel control strategies must be explored to control the damage and inhibit the dispersal efficiently. Proline is a major energy substrate during flight. Δ-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyses the second step of proline degradation for the production of ATP. RESULTS: A full-length Ldp5cdh cDNA was cloned. Ldp5cdh was ubiquitously expressed in the eggs, the first through fourth larval instars, wandering larvae, pupae and adults. In the adults, Ldp5cdh mRNA was widely distributed in thorax muscles, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion, fat body and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdp5cdh1 and dsLdp5cdh2) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of dsLdp5cdh1 and dsLdp5cdh2 successfully silenced Ldp5cdh, significantly increased the contents of proline, arginine and alanine, but strongly decreased the contents of asparate, asparagine, glutamate and glutamine in the haemolymph. Moreover, knocking down Ldp5cdh significantly reduced ATP content, decreased flight speed, shortened flight distance and increased adult mortality. CONCLUSIONS: It seems that identified Ldp5cdh encodes a functional P5CDh enzyme, and Ldp5cdh may serve as a potential target for dsRNA-based pesticide for controlling the damage and dispersal of L. decemlineata adults. © 2014 Society of Chemical Industry.

A putative Δ1-pyrroline-5-carboxylate synthetase involved in the biosynthesis of proline and arginine in Leptinotarsa decemlineata.

Delta 1-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the conversion of glutamate (Glu) to Glu semialdehyde (GSA). GSA spontaneously cyclizes to form P5C. P5C is then reduced to proline (Pro) or is converted to ornithine, the intermediate for arginine (Arg) biosynthesis. In the present study, a full-length Ldp5cs complementary DNA was cloned from the Colorado potato beetle Leptinotarsa decemlineata, a notorious insect defoliator of potato in most potato-growing regions of the world. Ldp5cs encodes a 792-amino-acid protein which shares high identity to homologues from other insect species. Quantitative reverse transcription polymerase chain reaction revealed that Ldp5cs was ubiquitously expressed in the eggs, first to fourth-instar larvae, wandering larvae, pupae and sexually mature adults. In the adults, Ldp5cs mRNA levels were higher in the fat body, foregut, midgut and hindgut, moderate in the ventral ganglion, lower in the thorax muscles, epidermis and Malpighian tubules. Two double-stranded RNAs (dsRNAs) (dsLdp5cs1 and dsLdp5cs2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of dsLdp5cs1 or dsLdp5cs2 successfully silenced Ldp5cs, significantly reduced the contents of Pro and Arg in the hemolymph, decreased flight speed and shortened flight distance of the resulting adults. Furthermore, knocking down Ldp5cs significantly increased adult mortality. Thus, our results suggest that identified Ldp5cs encodes a functional P5CS enzyme that is involved in the biosynthesis of Pro and Arg in L. decemlineata.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles.

Plant cell walls are the largest reservoir of organic carbon on earth. To breach and utilize this carbohydrate-rich protective barrier, microbes secrete plant cell wall degrading enzymes (PCWDEs) targeting pectin, cellulose and hemicelluloses. There is a growing body of evidence that genomes of some herbivorous insects also encode PCWDEs, raising questions about their evolutionary origins and functions. Among herbivorous beetles, pectin-degrading polygalacturonases (PGs) are found in the diverse superfamilies Chrysomeloidea (leaf beetles, long-horn beetles) and Curculionoidea (weevils). Here our aim was to test whether these arose from a common ancestor of beetles or via horizontal gene transfer (HGT), and whether PGs kept their ancestral function in degrading pectin or evolved novel functions. Transcriptome data derived from 10 beetle species were screened for PG-encoding sequences and used for phylogenetic comparisons with their bacterial, fungal and plant counterparts. These analyses revealed a large family of PG-encoding genes of Chrysomeloidea and Curculionoidea sharing a common ancestor, most similar to PG genes of ascomycete fungi. In addition, 50 PGs from beetle digestive systems were heterologously expressed and functionally characterized, showing a set of lineage-specific consecutively pectin-degrading enzymes, as well as conserved but enzymatically inactive PG proteins. The evidence indicates that a PG gene was horizontally transferred ∼200 million years ago from an ascomycete fungus to a common ancestor of Chrysomeloidea and Curculionoidea. This has been followed by independent duplications in these two lineages, as well as independent replacement in two sublineages of Chrysomeloidea by two other subsequent HGTs. This origin, leading to subsequent functional diversification of the PG gene family within its new hosts, was a key event promoting the evolution of herbivory in these beetles.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

Pre-invasion history and demography shape the genetic variation in the insecticide resistance-related acetylcholinesterase 2 gene in the invasive Colorado potato beetle.

BACKGROUND: Invasive pest species offers a unique opportunity to study the effects of genetic architecture, demography and selection on patterns of genetic variability. Invasive Colorado potato beetle (Leptinotarsa decemlineata) populations have experienced a rapid range expansion and intense selection by insecticides. By comparing native and invasive beetle populations, we studied the origins of organophosphate (OP) resistance-associated mutations in the acetylcholinesterase 2 (AChE2) gene, and the role of selection and demography on its genetic variability. RESULTS: Analysis of three Mexican, two US and five European populations yielded a total of 49 haplotypes. Contrary to the expectations all genetic variability was associated with a point mutation linked to insecticide resistance (S291G), this mutation was found in 100% of Mexican, 95% of US and 71% of European beetle sequences analysed. Only two susceptible haplotypes, genetically very differentiated, were found, one in US and one in Europe. The genetic variability at the AChE2 gene was compared with two other genes not directly affected by insecticide selection, diapause protein 1 and juvenile hormone esterase. All three genes showed reduction in genetic variability indicative of a population bottleneck associated with the invasion. CONCLUSIONS: Stochastic effects during invasion explain most of the observed patterns of genetic variability at the three genes investigated. The high frequency of the S291G mutation in the AChE2 gene among native populations suggests this mutation is the ancestral state and thus, either a pre-adaptation of the beetle for OP resistance or the AChE2 is not the major gene conferring OP resistance. The long historical association with host plant alkaloids together with recombination may have contributed to the high genetic variation at this locus. The genetic diversity in the AChE2 locus of the European beetles, in turn, strongly reflects founder effects followed by rapid invasion. Our results suggest that despite the long history of insecticide use in this species, demographic events together with pre-invasion history have been strongly influential in shaping the genetic diversity of the AChE2 gene in the invasive beetle populations.

Oligocarbonate molecular transporters: oligomerization-based syntheses and cell-penetrating studies.

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-side chain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one-step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains.

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.

Insecticidal effects of extracts of seven plant species on larval development, alpha-amylase activity and offspring production of Tribolium castaneum (Herbst) (Insecta: Coleoptera: Tenebrionidae).

Bioinsecticidal effects of methanol extracts from seven plant species on Tribolium castaneum were investigated. Centaurium erythraea, Peganum harmala, Ajuga iva, Aristolochia baetica, Pteridium aquilinum and Raphanus raphanistrum extracts inhibit growth of larvae. C. erythraea was the most toxic with 63% mortality 10 days after treatment, followed by P. harmala with 58%. C. erythraea and P. aquilinum reduce the emergence rate respectively of 66% and 19%. The duration of larval period was shortened by Launaea arborescens, P. aquilinum and A. iva extracts, whereas R. raphanistrum and P. harmala extracts extend the larval period when compared to the control. Extracts of C. erythraea, P. harmala, A. iva and A. baetica inhibited F1 progeny production. Larvae possess three alpha-amylase isoforms as determined by SDS-PAGE. Larvae fed on treated diet had lower alpha-amylase activity than larvae feed on untreated diet. C. erythraea and P. harmala are the most potent extracts. These plant extracts could be useful to reduce seed damage caused by this pest species.

Catalase from the white-spotted flower chafer, Protaetia brevitarsis: cDNA sequence, expression, and functional characterization.

Catalase, which is one of the key enzymes of the cellular antioxidant defense system, prevents free hydroxyl radical formation by breaking down hydrogen peroxide into oxygen and water. Here, we show the cloning and characterization of a catalase gene in a coleopteran insect. This gene was isolated by searching the white-spotted flower chafer Protaetia brevitarsis cDNA library, and the gene itself encodes a protein of 505 amino acids in length, named PbCat. PbCat shows high similarities to the insect catalase genes known to date. The recombinant PbCat, which is expressed as a 56-kDa polypeptide in baculovirus-infected insect Sf9 cells, shows the highest activity at 30 degrees C and pH 7.0. Northern and Western blot analyses revealed the presence of PbCat in all tissues examined, showing its ubiquitous expression. P. brevitarsis larvae in which H(2)O(2) was overloaded, showed a marked up-regulation in PbCat expression. Moreover, P. brevitarsis larvae showed an apparent increase in PbCat expression even after a wounding through injection. These results indicate that PbCat is up-regulated after wounding and oxidative pressure induced by H(2)O(2), reflecting an important role of PbCat in H(2)O(2) scavenging.

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.