Entomotoxic Effects Research Articles

Effects ofRhizoctonia solaniAgglutinins on Intermediary Metabolism ofPieris brassicae(Lepidoptera: Pieridae)

Effects of two purified agglutinins from Rhizoctonia solani Kuhen (Rhizoctonia solani agglutinin) were determined on larval intermediary metabolism of Pieris brassicae L. Two concentrations of Rhizoctonia solani agglutinin (RSA; 1 and 2 mg ml−1) significantly decreased activities of both alanine and aspartate aminotransferases, but no significant activity was observed on γ-glutamyl transferase. However, activity of aldolase decreased in the treated larvae by both concentrations of RSAs, but lactate dehydrogenase showed the highest activity when the larvae fed on 2 mg ml−1 of RSAs. Activities of acid- and alkaline phosphatases significantly decreased followed by feeding on the diets containing RSAs. Similar results were observed in the amounts of high-density lipoprotein and low-density lipoprotein. The amounts of protein and triacylglycerol significantly decreased in the treated larvae, while glycogen significantly increased in the larvae fed on RSAs-containing diets. Gene expression analyses of glycerol-3-phosphate in the control and treated larvae revealed significant expression in the larvae fed on RSAI in comparison with other treatments. These results demonstrated intervening effects of RSAs on intermediary metabolism of P. brassicae larvae, further confirming their entomotoxic effects.

Molecular Mechanism Underlying the Entomotoxic Effect of Colocasia esculenta Tuber Agglutinin against Dysdercus cingulatus

Colocasia esculenta tuber agglutinin (CEA), a mannose binding lectin, exhibits insecticidal efficacy against different hemipteran pests. Dysdercus cingulatus, red cotton bug (RCB), has also shown significant susceptibility to CEA intoxication. However, the molecular basis behind such entomotoxicity of CEA has not been addressed adequately. The present study elucidates the mechanism of insecticidal efficacy of CEA against RCB. Confocal and scanning electron microscopic analyses documented CEA binding to insect midgut tissue, resulting in an alteration of perimicrovillar membrane (PMM) morphology. Internalization of CEA into insect haemolymph and ovary was documented by western blotting analyses. Ligand blot followed by mass spectrometric identification revealed the cognate binding partners of CEA as actin, ATPase and cytochrome P450. Deglycosylation and mannose inhibition assays indicated the interaction to probably be mannose mediated. Bioinformatic identification of putative glycosylation or mannosylation sites in the binding partners further supports the sugar mediated interaction. Correlating entomotoxicity of CEA with immune histological and binding assays to the insect gut contributes to a better understanding of the insecticidal potential of CEA and endorses its future biotechnological application.

Elderberries: a source of ribosome-inactivating proteins with lectin activity.

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

Effects of a Lectin from Polygonum Persicaria L. on Pieris Brassicae L. (Lepidoptera: Pieridae)

Abstract In the current study, the entomotoxic effects of a lectin, extracted from Polygonum persicaria, were determined on Pieris brassicae larvae. The purified molecule significantly decreased larval survival, weight, and mass protein. Different concentrations of P. persicaria agglutinin (PPA) (0.5, 1 and 2 mg/ml) significantly decreased nutritional indices including: the efficiency of conversion of ingested food (ECI), the efficiency of conversion of digested food (ECD), and approximate digestibility (AD) in a dose-dependent manner. There was a significant decrease in larval α-amylase activity after larval fed on PPA. The decreased activity was found in both the biochemical assessment and gel electrophoresis; sharpness of one isozyme decreased and another completely disappeared. Meanwhile, PPA significantly decreased activities of glucosidases mainly β-glucosidase. Fed of P. brassicae larvae on different concentrations of PPA, significantly decreased activity of TAG-lipase in both the biochemical assessment and gel electrophoresis. It was found that alkaline phosphatase (ALP) and acid phosphatase (ACP) were also affected by PPA but concentrations of 1 and 2 mg/ml showed statistical differences compared to the control. General and specific proteolytic activities statistically decreased after larvae fed on different concentrations of PPA. Results of our study could bring attention to this molecule, showing it to be a safe and efficient control of the pest worldwide.

Synthetic fusion-protein containing domains of Bt Cry1Ac and Allium sativum lectin (ASAL) conferred enhanced insecticidal activity against major lepidopteran pests

Different transgenic crop plants, developed with δ-endotoxins of Bacillus thuringiensis (Bt) and mannose-specific plant lectins, exhibited significant protection against chewing and sucking insects. In the present study, a synthetic gene (cry-asal) encoding the fusion-protein having 488 amino acids, comprising DI and DII domains from Bt Cry1Ac and Allium sativum agglutinin (ASAL), was cloned and expressed in Escherichia coli. Ligand blot analysis disclosed that the fusion-protein could bind to more number of receptors of brush border membrane vesicle (BBMV) proteins of Helicoverpa armigera. Artificial diet bioassays revealed that 0.025μg/g and 0.50μg/g of fusion-protein were sufficient to cause 100% mortality in Pectinophora gossypiella and H. armigera insects, respectively. As compared to Cry1Ac, the fusion-protein showed enhanced (8-fold and 30-fold) insecticidal activity against two major lepidopteran pests. Binding of fusion-protein to the additional receptors in the midgut cells of insects is attributable to its enhanced entomotoxic effect. The synthetic gene, first of its kind, appears promising and might serve as a potential candidate for engineering crop plants against major insect pests.

Structure–function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease

BackgroundUreases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation. MethodsAiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61–74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1–44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45–93), Jbtx C-ter. Results1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure–function analysis. Major conclusionsThe N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. General significanceJbtx represents a new type of insecticidal and membrane-active peptide.

Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects.

Plant Ureases and Related Peptides: Understanding Their Entomotoxic Properties

Recently, ureases were included in the arsenal of plant defense proteins, alongside many other proteins with biotechnological potential such as insecticides. Isoforms of Canavalia ensiformis urease (canatoxin—CNTX and jack bean urease—JBURE-I) are toxic to insects of different orders. This toxicity is due in part to the release of a 10 kDa peptide from the native protein, by cathepsin-like enzymes present in the insect digestive tract. The entomotoxic peptide, Jaburetox-2Ec, exhibits potent insecticidal activity against several insects, including many resistant to the native ureases. JBURE-I and Jaburetox-2Ec cause major alterations of post-feeding physiological processes in insects, which contribute to, or can be the cause of, their entomotoxic effect. An overview of the current knowledge on plant urease processing and mechanisms of action in insects is presented in this review.

Jasmonate response of the Nicotiana tabacum agglutinin promoter in Arabidopsis thaliana

NICTABA is a carbohydrate-binding protein (also called lectin) that is expressed in several Nicotiana species after treatment with jasmonates and insect herbivory. Analyses with tobacco lines overexpressing the NICTABA gene as well as lines with reduced lectin expression have shown the entomotoxic effect of NICTABA against Lepidopteran larvae, suggesting a role of the lectin in plant defense. Until now, little is known with respect to the upstream regulatory mechanisms that are controlling the expression of inducible plant lectins. Using Arabidopsis thaliana plants stably expressing a promoter-β-glucuronidase (GUS) fusion construct, it was shown that jasmonate treatment influenced the NICTABA promoter activity. A strong GUS staining pattern was detected in very young tissues (the apical and root meristems, the cotyledons and the first true leaves), but the promoter activity decreased when plants were getting older. NICTABA was also expressed at low concentrations in tobacco roots and expression levels increased after cold treatment. The data presented confirm a jasmonate-dependent response of the promoter sequence of the tobacco lectin gene in Arabidopsis. These new jasmonate-responsive tobacco promoter sequences can be used as new tools in the study of jasmonate signalling related to plant development and defense.

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E 2 (PGE 2) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE 2 increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

Nicotiana tabacum agglutinin is active against Lepidopteran pest insects

A jasmonate-inducible lectin called Nicotiana tabacum agglutinin or NICTABA was found in tobacco (Nicotiana tabacum cv Samsun) leaves. Since NICTABA expression is also induced after insect herbivory, a role in the defence response of tobacco was suggested. In this report, a detailed analysis was made of the entomotoxic properties of NICTABA using different transgenic approaches. First, purified NICTABA was shown to be strongly resistant to proteolytic degradation by enzymes present in the Lepidopteran midgut. To address the question of whether NICTABA is also active against Lepidopteran larvae, transgenic N. tabacum plants that silence endogenous NICTABA expression were constructed using RNA interference. Feeding experiments with these transgenic N. tabacum plants demonstrated that silencing of NICTABA expression enhances the larval performance of the generalist pest insect Spodoptera littoralis. In a second transgenic approach, NICTABA was ectopically expressed in the wild diploid tobacco Nicotiana attenuata, a species that lacks a functional NICTABA gene. When these transgenic N. attenuata plants were used in feeding experiments with S. littoralis larvae, a clear reduction in mass gain and significantly slower development were observed. In addition, feeding experiments with the Solanaceae specialist, Manduca sexta, provided further evidence that NICTABA exerts clear entomotoxic effects on Lepidopteran larvae.

Entomotoxic effects of fungal lectin from Rhizoctonia solani towards Spodoptera littoralis

The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival of an economically important caterpillar in agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for feeding experiments with cotton leafworm. Rearing of insects on a diet containing different concentrations of RSA exerted a strong effect on the larval weight gain. This effect was visible at the lowest concentration of 0.1 % RSA at day 8 and day 11. Interestingly with 1 % RSA, there was a dramatic reduction in larval weight of 89 % at the end of L6 which was followed by a high mortality rate of 82 % in the treated larvae. Furthermore, the other developmental stages of pupation and adult formation were also affected. In addition, the data demonstrated that the combination of RSA with Bt toxin yielded synergistic effects. For instance, 0.03 % RSA + 0.005 % Bt toxin caused reduced growth rate and higher mortalities. These findings suggest that RSA is an interesting tool that can be used for bioengineering insect resistance in important agronomical crops.

Invitro effect of Canavalia ensiformis urease and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules

Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease–JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests.

BackgroundRice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests.ResultsAllium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects.ConclusionIn planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper.

Separation of jack bean (Canavalia ensiformis) urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity

In this work we described the separation of two isoforms of urease from jack bean seeds, the “classical” jack bean urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)–Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains 1 eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators.

Separation of jack bean ( Canavalia ensiformis) urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity

In this work we described the separation of two isoforms of urease from jack bean seeds, the “classical” jack bean urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)–Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains 1 eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators.