The mechanisms of action of insecticidal lectins from snowdrop (GNA) and jackbean (concanavalin a) ontomato moth larvae

Artificial diet bioassays were carried out to investigate the impact of GNA and Con A upon the development of L. oleracea larvae. GNA, at 2 % of total dietary protein, exerted a significantly detrimental effect upon larval development, growth and consumption, with little effect upon survival. Con A was shown to be the more toxic of the 2 lectins. When tested at concentrations of 2.0 %, 0.2 % and 0.02 %, Con A caused a significant decrease in survival and larval development, and caused greater reductions in larval growth and consumption compared to GNA. The potential for GNA and Con A to exert insecticidal effects via binding to the brush border membrane (BBM) and peritrophic membrane (PM) of L. oleracea larvae was investigated. Con A, which specifically binds a-D-mannopyranoside and a-D- glucopyranoside residues, was shown to bind in vitro to the majority of BBM and PM proteins. In contrast GNA, which exhibits strict specificity for a(l,3) and a(l,6)-linked D-mannose residues, bound to only 5 BBMV and 2 PM proteins. In agreement, higher levels of Con A, compared to GNA, were shown to accumulate in larval gut tissue after feeding the proteins in vivo. Despite this both lectins were shown to have a similar ability to disrupt the digestive capacity of the larval midgut. GNA and Con A stimulated similar short term elevations in BBM enzyme and soluble trypsin activities and a long-term reduction in a-glucosidase activity. Increases in levels of trypsin activity in faecal material collected from lectin-fed larvae suggested that the proteins may act by disrupting mechanisms of enzyme recycling. Aminopeptidase, an abundant and avidly binding BBM protein (120 kDa), was identified as a major Con A binding species in L. oleracea. A 98 kDa GNA-binding BBM protein was purified and amino acid sequence data was obtained from digest polypeptides allowing oligonucleotide primers to be designed. Subsequent attempts to amplify (by PCR and RT-PCR) fragments containing coding sequence corresponding to the 98 kDa protein were unsuccessful. This was attributed to oligonucleotide degeneracy together with the low abundance and relatively large size of the protein. The potential for GNA and Con A to exert systemic effects upon I. oleracea was demonstrated by the detection of both lectins in the haemolymph of larvae exposed to experimental diets. GNA was detected in haemolymph of larvae exposed to experimental diet for just 2 hours. In contrast, no Con A was detectable in haemolymph extracted from larvae fed for 24 hours, although it was present in the haemolymph after 5 days of exposure to the diet. That GNA and Con A may act directly upon organs other than the insect gut was indicated by the detection of lectins in vivo in malpighian tubule and fat body tissue extracts. A significant reduction in haemocyte abundance in haemolymph samples extracted from lectin-fed larvae suggested that both GNA and Con A may also act by disrupting the immune system of L. oleracea

Which Insect Species and Why?

This chapter describes the common features determining the suitability of insects for small- and industrial-scale farming, the main insect species currently being produced on a large scale for feed production and other potential candidate species. Natural consumption of insects by animals and which insects are suitable for which animal feed is also briefly discussed

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies

Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC 50) derived from dose-response artificial diet assays of 27, 20 and 52 μM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies

Life cycle cost assessment of insect based feed production in West Africa

While there is a growing body of research investigating the technical feasibility and nutritional properties of insect based feeds (IBFs), thus far little attention has been devoted to gauge the economic implications of implementation. This study has investigated the economic performance of ex-ante modelled IBF production systems operating in the geographical context of West Africa. A Life Cycle Cost (LCC) analysis of recently published life cycle inventory (LCI) data served as a basis to analyse and compare the economic performances of IBF production systems using Musca domestica and Hermetia illucens reared on different substrates. To gauge the application potential of IBF in West Africa, estimated breakeven sale prices of IBFs were benchmarked against the customary market prices of conventional feeds. The results show that the economic performance of IBF production in West Africa is largely determined by the costs attributed to labour and the procurement of rearing substrates, attesting economic advantages to the production of M. domestica larvae by measure of breakeven price (1.28–1.74 EUR/kg IBF) and LCC (1.72–1.99 EUR/kg IBF). A comparison of the breakeven sale prices of IBF with market prices of conventional feeds suggest that IBF has potential to substitute imported fishmeal, but findings offer no support for conjectured economic advantages over plant based feeds

A Systematic Study of RNAi Effects and dsRNA Stability in Tribolium castaneum and Acyrthosiphon pisum, Following Injection and Ingestion of Analogous dsRNAs

RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle (Tribolium castaneum) and recalcitrant pea aphid (Acyrthosiphon pisum) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E (VTE) and inhibitor of apoptosis (IAP) genes. Injection and ingestion of VTE and IAP dsRNAs resulted in up to 100% mortality of T. castaneum larvae and sustained suppression (>80%) of transcript levels. In A. pisum, injection of VTE but not IAP dsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) of VTE transcript levels. Feeding aphids on VTE dsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability in T. castaneum larvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency

The insecticidal activity of recombinant nemertide toxin α-1 from Lineus longissimus towards pests and beneficial species

The nemertide toxins from the phylum Nemertea are a little researched family of neurotoxins with potential for development as biopesticides. Here we report the recombinant production of nemertide α-1 (α-1), a 65-residue inhibitor cystine knot (ICK) peptide from Lineus longissimus, known to target insect voltage-gated sodium channels. The insecticidal activity of α-1 was assessed and compared with the well characterised ICK venom peptide, ω-atracotoxin/hexatoxin-Hv1a (Hv1a). α-1 elicited potent spastic paralysis when injected into cabbage moth (Mamestra brassicae) larvae; conferring an ED50 3.90 μg/larva (10.30 nmol/g larva), followed by mortality (60% within 48 h after 10 μg injection). By comparison, injection of M. brassicae larvae with recombinant Hv1a produced short-lived flaccid paralysis with an ED50 over 6 times greater than that of α-1 at 26.20 μg/larva (64.70 nmol/g larva). Oral toxicity of α-1 was demonstrated against two aphid species (Myzus persicae and Acyrthosiphon pisum), with respective LC50 values of 0.35 and 0.14 mg/mL, some 6-fold lower than those derived for recombinant Hv1a. When delivered orally to M. brassicae larvae, α-1 caused both paralysis (ED50 11.93 μg/larva, 31.5 nmol/g larva) and mortality. This contrasts with the lack of oral activity of Hv1a, which when fed to M. brassicae larvae had no effect on feeding or survival. Hv1a has previously been shown to be non-toxic by injection to the beneficial honeybee (Apis mellifera). By contrast, rapid paralysis and 100% mortality was observed following injection of α-1 (31.6 nmol/g insect). These results demonstrate the great potential of naturally occurring non-venomous peptides, such as α-1, for development as novel effective biopesticides, but equally highlights the importance of understanding the phyletic specificity of a given toxin at an early stage in the quest to discover and develop safe and sustainable pesticides

Insecticidal effects of dsRNA targeting the Diap1 gene in dipteran pests

The Drosophila melanogaster (fruit fly) gene Diap1 encodes a protein referred to as DIAP1 (DrosophilaInhibitor of Apoptosis Protein 1) that acts to supress apoptosis in “normal” cells in the fly. In this study we investigate the use of RNA interference (RNAi) to control two dipteran pests, Musca domestica and Delia radicum, by disrupting the control of apoptosis. Larval injections of 125–500 ng of Diap1 dsRNA resulted in dose-dependent mortality which was shown to be attributable to down-regulation of target mRNA. Insects injected with Diap1 dsRNA have approx. 1.5-2-fold higher levels of caspase activity than controls 24 hours post injection, providing biochemical evidence that inhibition of apoptotic activity by the Diap1 gene product has been decreased. By contrast adults were insensitive to injected dsRNA. Oral delivery failed to induce RNAi effects and we suggest this is attributable to degradation of ingested dsRNA by intra and extracellular RNAses. Non-target effects were demonstrated via mortality and down-regulation of Diap1 mRNA levels in M. domestica larvae injected with D. radicum Diap1 dsRNA, despite the absence of 21 bp identical sequence regions in the dsRNA. Here we show that identical 15 bp regions in dsRNA are sufficient to trigger non-target RNAi effects

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion () and snowdrop lectin shows oral toxicity to target insects-2

<p><b>Copyright information:</b></p><p>Taken from "A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion () and snowdrop lectin shows oral toxicity to target insects"</p><p>BMC Biotechnology 2006;6():18-18.</p><p>Published online 16 Mar 2006</p><p>PMCID:PMC1459149.</p><p>Copyright © 2006 Trung et al; licensee BioMed Central Ltd.</p>d saline (PBS; n = 40). GNA was injected at a dose of 20 μg which equates to approx. 500 μg GNA/g insect (n = 20); ButaIT/GNA was injected at two different concentrations; 4 μg which equates to approx. 100 μg fusion protein or 30 μg toxin/g insect (n = 25), and 20 μg which equates to approx. 450 μg of fusion protein or 135 μg toxin/g insect (n = 10)