Enhanced Activity and Stability of an Acetyl Xylan Esterase in Hydrophilic Alcohols through Site-Directed Mutagenesis.

Current demands for the development of suitable biocatalysts showing high process performance is stimulated by the need to replace current chemical synthesis with cleaner alternatives. A drawback to the use of biocatalysts for unique applications is their low performance in industrial conditions. Hence, enzymes with improved performance are needed to achieve innovative and sustainable biocatalysis. In this study, we report the improved performance of an engineered acetyl xylan esterase (BaAXE) in a hydrophilic organic solvent. The structure of BaAXE was partitioned into a substrate-binding region and a solvent-affecting region. Using a rational design approach, charged residues were introduced at protein surfaces in the solvent-affecting region. Two sites present in the solvent-affecting region, A12D and Q143E, were selected for site-directed mutagenesis, which generated the mutants MUT12, MUT143 and MUT12-143. The mutants MUT12 and MUT143 reported lower Km (0.29 mM and 0.27 mM, respectively) compared to the wildtype (0.41 mM). The performance of the mutants in organic solvents was assessed after enzyme incubation in various strengths of alcohols. The mutants showed improved activity and stability compared to the wild type in low strengths of ethanol and methanol. However, the activity of MUT143 was lost in 40% methanol while MUT12 and MUT12-143 retained over 70% residual activity in this environment. Computational analysis links the improved performance of MUT12 and MUT12-143 to novel intermolecular interactions that are absent in MUT143. This work supports the rationale for protein engineering to augment the characteristics of wild-type proteins and provides more insight into the role of charged residues in conferring stability.

Characterisation of a Novel Acetyl Xylan Esterase (BaAXE) Screened from the Gut Microbiota of the Common Black Slug (Arion ater).

Acetyl xylan esterases (AXEs) are enzymes capable of hydrolysing the acetyl bonds in acetylated xylan, allowing for enhanced activity of backbone-depolymerizing enzymes. Bioprospecting novel AXE is essential in designing enzyme cocktails with desired characteristics targeting the complete breakdown of lignocellulose. In this article, we report the characterisation of a novel AXE identified as Gene_id_40363 in the metagenomic library analysed from the gut microbiota of the common black slug. The conserved domain description was identified with an NCBI BLASTp search using the translated nucleotide sequence as a query. The activity of the recombinant enzyme was tested on various synthetic substrates and acetylated substrates. The protein sequence matched the conserved domain described as putative hydrolase and aligned closely to an uncharacterized esterase from Buttiauxella agrestis, hence the designation as BaAXE. BaAXE showed low sequence similarity among characterized CE family proteins with an available 3D structure. BaAXE was active on 4-nitrophenyl acetate, reporting a specific activity of 78.12 U/mg and a Km value of 0.43 mM. The enzyme showed optimal activity at 40 °C and pH 8 and showed high thermal stability, retaining over 40% activity after 2 h of incubation from 40 °C to 100 °C. BaAXE hydrolysed acetyl bonds, releasing acetic acid from acetylated xylan and ß-D-glucose pentaacetate. BaAXE has great potential for biotechnological applications harnessing its unique characteristics. In addition, this proves the possibility of bioprospecting novel enzymes from understudied environments.

Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One.

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and ß-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.

Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

Circular and linear: a tale of aptamer selection for the activation of SIRT1 to induce death in cancer cells.

It is a challenge to select the right target to treat conditions without affecting non-diseased cells. Cancer belongs to the top 10 causes of death in the world and it remains difficult to treat. Amongst cancer emerging targets, silent information regulator 1 (SIRT1) - a histone deacetylase - has shown many roles in cancer, ageing and metabolism. Here we report novel SIRT1 ligands that bind and modulate the activity of SIRT1 within cells and enhance its enzymatic activity. We developed a modified aptamer capable of binding to and forming a complex with SIRT1. Our ligands are aptamers, they can be made of DNA or RNA oligonucleotides, their binding domain can recognise a target with very high affinity and specificity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to develop circular and linear aptamers selectively binding to SIRT1. Cellular consequences of the interaction were monitored by fluorescence microscopy, cell viability assay, stability and enzymatic assays. Our results indicate that from our pool of aptamers, circular AC3 penetrates cancerous cells and is recruited to modulate the SIRT1 activity. This modulation of SIRT1 resulted in anticancer activity on different cancer cell lines. Furthermore, this modified aptamer showed no toxicity on one non-cancerous cell line and was stable in human plasma. We have demonstrated that aptamers are efficient tools for localisation of internal cell targets, and in this particular case, anticancer activity through modulation of SIRT1.

Metagenomic Analysis of the Gut Microbiome of the Common Black Slug Arion ater in Search of Novel Lignocellulose Degrading Enzymes.

Some eukaryotes are able to gain access to well-protected carbon sources in plant biomass by exploiting microorganisms in the environment or harbored in their digestive system. One is the land pulmonate Arion ater, which takes advantage of a gut microbial consortium that can break down the widely available, but difficult to digest, carbohydrate polymers in lignocellulose, enabling them to digest a broad range of fresh and partially degraded plant material efficiently. This ability is considered one of the major factors that have enabled A. ater to become one of the most widespread plant pest species in Western Europe and North America. Using metagenomic techniques we have characterized the bacterial diversity and functional capability of the gut microbiome of this notorious agricultural pest. Analysis of gut metagenomic community sequences identified abundant populations of known lignocellulose-degrading bacteria, along with well-characterized bacterial plant pathogens. This also revealed a repertoire of more than 3,383 carbohydrate active enzymes (CAZymes) including multiple enzymes associated with lignin degradation, demonstrating a microbial consortium capable of degradation of all components of lignocellulose. This would allow A. ater to make extensive use of plant biomass as a source of nutrients through exploitation of the enzymatic capabilities of the gut microbial consortia. From this metagenome assembly we also demonstrate the successful amplification of multiple predicted gene sequences from metagenomic DNA subjected to whole genome amplification and expression of functional proteins, facilitating the low cost acquisition and biochemical testing of the many thousands of novel genes identified in metagenomics studies. These findings demonstrate the importance of studying Gastropod microbial communities. Firstly, with respect to understanding links between feeding and evolutionary success and, secondly, as sources of novel enzymes with biotechnological potential, such as, CAZYmes that could be used in the production of biofuel.

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

Characterization of cellulolytic activity in the gut of the terrestrial land slug Arion ater: Biochemical identification of targets for intensive study.

The level of cellulolytic activity in different areas of the gut of the terrestrial slug Arion ater was assayed at different temperatures and pH values. To do this, crude gut proteins were isolated and assayed using modified dinitrosalicylic acid reducing sugar assay. Crude protein samples were also separated and cellulolytic activity identified using in gel CMC zymography and esculin hydrate activity gel assays. pH and temperature profiling revealed optimum cellulolytic activity between pH5.0 and 6.0 for different gut regions and retention of up to 90% of activity at temperatures up to 50°C. Zymograms and activity gels revealed multiple endoglucanase and ß-glucosidase enzymes. To further investigate the source of this cellulolytic activity bacterial isolates from the gut were tested for endoglucanase and ß-glucosidase activity using growth plate assays. 12 cellulolytic microbes were identified using 16S rDNA gene sequencing. These include members of the genera Buttiauxella, Enterobacter, Citrobacter, Serratia and Klebsiella. Gut metagenomic DNA was then subjected to PCR, targeting a 400bp region of the 16SrDNA gene which was subsequently separated and individuals identified using DGGE. This identified members of the genera Citrobacter, Serratia, Pectobacterium, Acinetobacter, Mycoplasma, Pantoea and Erwinia. In summary, multiple glycoside hydrolase enzymes active over a broad range of temperature and pH values in a relatively under studied organism were detected, indicating that the gut of A. ater is a viable target for intensive study to identify novel carbohydrate active enzymes that may be used in the biofuel industry.

Characterization of four midgut aminopeptidase N isozymes from Ostrinia furnacalis strains with different susceptibilities to Bacillus thuringiensis.

The full-length cDNA of four Ofapn aminopeptidases were cloned and sequenced from susceptible and resistant Ostrinia furnacalis strains. Four sequences were identified as APN because they shared the common structural features with APN from Lepidoptera, including the signal peptide, GPI anchor signal, the zinc binding/gluzincin motif HEX2HX18E and the gluzincin aminopeptidase motif GAMEN. Compared with APN sequences from the susceptible strain, there were 9, 5, 10 and 12 amino acid variations in the deduced protein sequences from the resistant strain. There were also differences in mRNA expression of the four Ofapn genes between resistant and susceptible O. furnacalis strains.

A proteomic approach to study the mechanism of tolerance to Bt toxins in Ostrinia furnacalis larvae selected for resistance to Cry1Ab.

A Cry1Ab-resistant population of Asian corn borer (ACB-AbR) exhibiting approximately 100 times greater resistance to activated Cry1Ab than a susceptible population (Ostrinia furnacalis; ACB-BtS), was previously shown to exhibit high levels of cross-resistance to Cry1Ah (131-fold), but no cross-resistance to Cry1Ie. It was suggested that the proposed mechanism of resistance was due to the alteration of specific receptors for Cry toxins in the midgut brush border membrane. In the present study a proteomic-based approach was used to identify proteins from brush border membrane vesicles (isolated from both resistant and susceptible Ostrinia furnacalis larvae) interacting with biotinylated Cry1Ab, Cry1Ah, and Cry1Ie. 2D-Electrophoresis in combination with ligand blots were employed and putative protein identities obtained using MALDI-ToF/ToF mass spectrometry. The V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as interacting with the Cry toxins tested in the ACB-AbR and ACB-BtS larvae. The biotinylated Cry toxins showed markedly stronger interactions with proteins in the resistant compared to the susceptible larvae, suggesting an up-regulation of the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins in the resistant (ACB-AbR) larvae. Interestingly, Cry1Ie interactions with the V-type proton ATPase catalytic subunit A in the ACB-BtS larvae appeared to be absent.

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome.

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.

Characterisation of adult green lacewing (Chrysoperla carnea) digestive physiology: impact of a cysteine protease inhibitor and a synthetic pyrethroid.

BACKGROUND: In spite of concern regarding potential non-target effects of GM crops, few studies have compared GM pest control with conventional methods. The impacts of cypermethrin and oilseed rape expressing oryzacystatin-1 (OC-1) were compared in this study on the predator Chrysoperla carnea (Stephens). RESULTS: Adults fed purified rOC-1 showed a subtle shift in digestive protease profile, with an increasing reliance on serine proteases (chymotrypsin), increase in aspartic proteases and a slight reduction in elastase activity. Although there were no effects on mortality, onset of oviposition was delayed; however, once egg production commenced, egg laying and hatching success rates were comparable with those of controls. Oryzacystatin-1 expressed in pollen showed no detrimental effects. Cypermethrin had no effect on mortality owing to high levels of non-specific esterase activity resulting in partial breakdown of the insecticide. In spite of this, there was a significant delay in onset of oviposition and a significant reduction in egg production and viability. CONCLUSION: This study demonstrates the potential for pest management to impact on predators, but importantly it highlights the ability of the predator to detoxify/respond to treatments with different modes of action. In this case, exposure to an insecticide carried a greater fitness cost than exposure to a protease inhibitor expressed in transgenic crops.

Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis.

The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase.

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis.

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.

Characterisation of midgut digestive proteases from the maize stem borer Busseola fusca.

BACKGROUND: Insect damage is a major constraint on maize production. Control of Busseola fusca (Fuller) in sub-Saharan Africa is relatively ineffective; the major larval digestive enzymes were characterised with a view to developing future control strategies. RESULTS: Using BODIPY-FL Casein, maximal activity was at pH 9.5, with six protease forms visualised by gelatin-PAGE. Synthetic substrates and diagnostic inhibitors demonstrated the presence of serine proteases. Chymostatin was a potent inhibitor of general proteolysis (90%), providing strong evidence for the presence of chymotrypsin; it also caused significant inhibition (>95%) with SA(2)PFpNA as substrate. The I(50) values for chymostatin with casein and SA(2)PFpNA were 0.0075 microM and 0.06 microM respectively. Z-Phe-Arg-pNA activity was inhibited by chymostatin and TLCK (50 and 30% respectively), suggesting the presence of trypsin-like activity. BApNA hydrolysis was also strongly inhibited by chymostatin and TLCK (92 and 75%), suggesting trypsin activity, while SBBI, PMSF, pepstatin and E-64 had no significant effect. Interestingly, SBBI (I(50) = 0.39 microM) and SBTI both inhibited general proteolysis by approximately 70%, suggesting that SBBI's dual inhibitory role makes this inhibitor a potentially useful candidate for expression in maize for control of B. fusca. CONCLUSION: These results provide a basis for the rational design of insect-resistant transgenic maize expressing protease inhibitors.

Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera: Coccinellidae).

The present study investigated prey-mediated effects of two maize varieties expressing a truncated Cry1Ab, Compa CB (event Bt176) and DKC7565 (event MON810), on the biology of the ladybird Stethorus punctillum. Although immuno-assays demonstrated the presence of Cry1Ab in both prey and predator collected from commercial maize-growing fields, neither transgenic variety had any negative effects on survival of the predator, nor on the developmental time through to adulthood. Furthermore, no subsequent effects on ladybird fecundity were observed. As a prerequisite to studying the interaction of ladybird proteases with Cry1Ab, proteases were characterised using a range of natural and synthetic substrates with diagnostic inhibitors. These results demonstrated that this predator utilises both serine and cysteine proteases for digestion. In vitro studies demonstrated that T. urticae were not able to process or hydrolyze Cry1Ab, suggesting that the toxin passes through the prey to the third trophic level undegraded, thus presumably retaining its insecticidal properties. In contrast, S. punctillum was able to activate the 130 kDa protoxin into the 65 kDa fragment; a fragment of similar size was also obtained with bovine trypsin, which is known to cleave the protoxin to the active form. Thus, despite a potential hazard to the ladybird of Bt-expressing maize (since the predator was both exposed to, and able to proteolytically cleave the toxin, at least in vitro), no deleterious effects were observed.

Bitrophic and tritrophic effects of Bt Cry3A transgenic potato on beneficial, non-target, beetles.

Insect-resistant transgenic plants have been suggested to have unpredictable effects on the biodiversity of the agro-ecosystem, including potential effects on insect natural enemies, beneficial in control of crop pests. Whilst carnivorous as adults, many of these predators may also consume plant tissues, in particular plant pollen and nectar. Coleoptera are important in terms of agro-ecological research not only because of the large number of species in this order, but also because of their role as biological control agents. Thus any detrimental impact on this group of insects would be highly undesirable. The effects of potato expressing the coleopteran-specific Bacillus thuringiensis delta-endotoxin Cry3A (Bt Cry3A) on the ladybird beetle Harmonia axyridis and the carabid beetle Nebria brevicollis were investigated via the bitrophic interaction of the adult ladybird with potato flowers and the tritrophic interaction of the carabid consuming a non-target potato pest. Immunoassays confirmed accumulation of the transgene product in potato leaves and floral tissues (at levels of up to 0.01% (pollen) and 0.0285% (anthers) of total soluble protein). Despite H. axyridis and N. brevicollis belonging to the targeted insect order, no significant effects upon survival or overall body mass change of either beetle were observed. Furthermore, Bt Cry3A had no detrimental effects on reproductive fitness of either beetle species, either in terms of fecundity or subsequent egg viability. Behavioural analysis revealed no significant impact of Bt Cry3A on beetle activity or locomoter behaviour. Ligand blots indicate that this is due to either the absence of Bt-binding sites in brush border membrane vesicles (BBMV) isolated from Nebria brevicollis, or in the case of Harmonia axyridis, the binding did not functionally lead to behavioural or physical effects.

Prey-mediated effects of transgenic canola on a beneficial, non-target, carabid beetle.

Transgenic plants producing insecticidal proteins from Bacillus thuringiensis (Bt) can control some major insect pests and reduce reliance on sprayed insecticides. However, large scale adoption of this technology has raised concerns about potential negative effects, including evolution of pest resistance to Bt toxins, transgene flow from Bt crops to other plants, and harm to non-target beneficial organisms. Furthermore, concern has also been expressed over the effects this technology may have on biodiversity in general. Ecologically relevant risk assessment is therefore required (Risk = Hazard x Exposure). Transgenic plants that produce Bt toxins to kill insect pests could harm beneficial predators. This might occur directly by transmission of toxin via prey, or indirectly by toxin-induced reduction in prey quality (Hazard). To test these hypotheses, we determined the effects of Bt-producing canola on a predatory ground beetle (Pterostichus madidus) fed larvae of diamondback moth (Plutella xylostella) that were either susceptible or resistant to the Bt toxin. Survival, weight gain, and adult reproductive fitness did not differ between beetles fed prey reared on Bt-producing plants and those fed prey from control plants. Furthermore, while Bt-resistant prey was shown to deliver high levels of toxin to the beetle when they were consumed, no significant impact upon the beetle was observed. Subsequent investigation showed that in choice tests (Exposure), starved and partially satiated female beetles avoided Bt-fed susceptible prey, but not Bt-fed resistant prey. However, in the rare cases when starved females initially selected Bt-fed susceptible prey, they rapidly rejected them after beginning to feed. This prey type was shown to provide sufficient nutrition to support reproduction in the bioassay suggesting that Bt-fed susceptible prey is acceptable in the absence of alternative prey, however adults possess a discrimination ability based on prey quality. These results suggest that the direct effects of Bt-producing canola on predator life history was minimal, and that predators' behavioural preferences may mitigate negative indirect effects of reduced quality of prey caused by consumption of Bt-producing plants. The results presented here therefore suggest that cultivation of Bt canola may lead to conservation of non-target predatory and scavenging organisms beneficial in pest control, such as carabids, and may therefore provide more sustainable agricultural systems than current practices. In addition, minimal impacts on beneficial carabids in agro-ecosystems suggest that Bt canola crops are likely to be compatible with integrated pest management (IPM) systems.

Comparing the impact of conventional pesticide and use of a transgenic pest-resistant crop on the beneficial carabid beetle Pterostichus melanarius.

The potential impact of a chemical pesticide control method has been compared with that of transgenic plants expressing a protease inhibitor conferring insect resistance by utilising a tritrophic system comprising the crop plant Brassica napus (L.) (Oilseed rape), the pest mollusc Deroceras reticulatum (Müller) and the predatory carabid beetle Pterostichus melanarius (Illiger). Cypermethrin, as the most widely used pesticide in UK oilseed rape (OSR) cultivation, was selected as the conventional treatment. OSR expressing a cysteine protease inhibitor, oryzacystatin-1 (OC-1), was the transgenic comparator. In feeding trials, D. reticulatum showed no significant long-term effects on measured life history parameters (survival, weight gain, food consumption) as a result of exposure to either the cypermethrin or OC-1 treatment. However, D. reticulatum was able to respond to the presence of the dietary inhibitor by producing two novel proteases following exposure to OC-1-expressing OSR. Similarly, P. melanarius showed no detectable alterations in mortality, weight gain or food consumption when feeding on D. reticulatum previously fed either pesticide-contaminated or GM plant material. Furthermore, as with the slug, a novel form of protease, approximately M(r) 27 kDa, was induced in the carabid in response to feeding on slugs fed OC-1-expressing OSR.