Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity.

Conotoxins are a diverse collection of more than 50,000 peptides produced by predatory marine snails of the genus Conus in order to immobilize their prey. Many conotoxins modulate the activity of ion channels, and show high specificity to their targets; as a result, some have valuable pharmaceutical applications. However, obtaining active peptide is difficult and to date has only been achieved though natural collection, chemical synthesis, or the use of prokaryotic expression systems, which often have the disadvantage of requiring subsequent steps to correctly fold the peptide. This paper reports the production of a conotoxin, TxVIA from Conus textile, as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host. The presence of the pro-peptide was found to be necessary for the expression of biologically active conotoxin. We also show that TxVIA is not, as previously reported, mollusc-specific, but also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae. In contrast, recombinant TxVIA was not found to be molluscicidal to the grey field slug Deroceras reticulatum.

Insect-resistant biotech crops and their impacts on beneficial arthropods.

With a projected population of 10 billion by 2050, an immediate priority for agriculture is to achieve increased crop yields in a sustainable and cost-effective way. The concept of using a transgenic approach was realized in the mid-1990s with the commercial introduction of genetically modified (GM) crops. By 2010, the global value of the seed alone was US $11.2 billion, with commercial biotech maize, soya bean grain and cotton valued at approximately US $150 billion. In recent years, it has become evident that insect-resistant crops expressing δ-endotoxin genes from Bacillus thuringiensis have made a significant beneficial impact on global agriculture, not least in terms of pest reduction and improved quality. However, because of the potential for pest populations to evolve resistance, and owing to lack of effective control of homopteran pests, alternative strategies are being developed. Some of these are based on Bacillus spp. or other insect pathogens, while others are based on the use of plant- and animal-derived genes. However, if such approaches are to play a useful role in crop protection, it is desirable that they do not have a negative impact on beneficial organisms at higher trophic levels thus affecting the functioning of the agro-ecosystem. This widely held concern over the ecological impacts of GM crops has led to the extensive examination of the potential effects of a range of transgene proteins on non-target and beneficial insects. The findings to date with respect to both commercial and experimental GM crops expressing anti-insect genes are discussed here, with particular emphasis on insect predators and parasitoids.

A venom metalloproteinase from the parasitic wasp Eulophus pennicornis is toxic towards its host, tomato moth (Lacanobia oleracae).

Three genes encoding clan MB metalloproteinases (EpMP1-3) were identified from venom glands of the ectoparasitic wasp Eulophus pennicornis. The derived amino acid sequences predict mature proteins of approximately 46 kDa, with a novel two-domain structure comprising a C-terminal reprolysin domain, and an N-terminal domain of unknown function. EpMP3 expressed as a recombinant protein in Pichia pastoris had gelatinase activity, which was inhibited by EDTA. Injection of recombinant EpMP3 into fifth instar Lacanobia oleracea (host) larvae resulted in partial insect mortality associated with the moult to sixth instar, with surviving insects showing retarded development and growth. EpMP3 is expressed specifically in venom glands. These results suggest that EpMP3 is a functional component of Eulophus venom, which is able to manipulate host development.

Effects of Galanthus nivalis agglutinin (GNA) expressed in tomato leaves on larvae of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) and the effect of GNA on the development of the endoparasitoid Meteorus gyrator (Hymenoptera: Braconidae).

The effect of ingestion of transgenic tomato leaves expressing the plant lectin Galanthus nivalis agglutinin (GNA) on development of larvae of Lacanobia oleracea (Linnaeus) was studied under laboratory conditions. When L. oleracea larvae were fed on tomato line 14.1H, expressing approximately 2.0% GNA, significant increases in the mean larval weight and in the amount of food consumed were found. This resulted in an overall reduction in the mean development time to the pupal stage of approximately 7 days. A significant increase in the percentage survival to the adult moth was also recorded when newly hatched larvae were reared on transgenic tomato leaves (72%) compared to larvae reared on untransformed leaves (40%). The effects of ingestion of GNA by L. oleracea larvae, via artificial diet or the leaves of transgenic tomato or potato plants, on the subsequent development of its solitary endoparasitoid Meteorus gyrator (Thunberg) was also studied. No significant effects on the life cycle parameters of M. gyrator developing in L. oleracea fed on GNA-containing diets were observed. Experiments with transgenic potato plants indicated that the stadium of the host larvae at parasitism had a greater influence on M. gyrator development than the presence of GNA. Potential GNA-binding glycoproteins were detected in the gut and body tissues of larval M. gyrator. Despite detection in host tissues, GNA could not be detected in adult M. gyrator and therefore it is likely that at the time of pupation M. gyrator are able to void the GNA in the meconial pellet.

Influence of host size on the clutch size and developmental success of the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae) [corrected] attacking larvae of the tomato moth Lacanobia oleracea (L.) (Lepidoptera: Noctuidae).

The relationship between clutch size, host size and progeny survival in the gregarious ectoparasitoid Eulophus pennicornis was investigated in a number of scenarios. When naive parasitoids were exposed singly to Lacanobia oleracea hosts, clutch size was strongly correlated with the size of the host. However, survival of parasitoid offspring was negatively affected by the size of the host such that, in larger hosts, greater wasp larval and pupal mortality was recorded. As a result, no gain in realised fecundity was achieved through parasitizing L. oleracea larvae of mass >0.4 g over hosts of mass between 0.2-0.3 g. When exposed to populations of mixed stadium hosts (larvae in the fourth, fifth and sixth instars) during the entire lifespan of the wasp, host size and clutch size were correlated in early ovipositions (first three ovipositions). However, as the wasps aged, the relationship was much less apparent. When the parasitoid was restricted to foraging upon populations of sixth instar hosts only, no relationship between host size and clutch size was apparent. Exposure of the parasitoid to mixed and fixed stadium host populations showed that final (sixth) stadium hosts were the most frequently parasitized (ca. 96% of parasitized hosts) and that the average numbers of eggs laid per wasp, and the number of hosts parasitized, was significantly lower when the parasitoid was provided with fourth or fifth instar hosts only. The results indicate that the reproductive success of E. pennicornis does not increase with increasing host size or greater resource availability above a certain threshold, and that the physiological status of the host at the time of parasitism is the governing factor determining oviposition decisions and parasitoid survival. We conclude that E. pennicornis has been selected to preferentially utilize those hosts that maximize progeny survival and to adapt clutch size to the size of such hosts. We hypothesize that the major driver leading to the evolution of this strategy is the ability of the parasitoid to physiologically regulate the host.

Interactions between the solitary endoparasitoid, Meteorus gyrator (Hymenoptera: Braconidae) and its host, Lacanobia oleracea (Lepidoptera: Noctuidae), infected with the entomopathogenic microsporidium, Vairimorpha necatrix (Microspora: Microsporidia).

Infection of Lacanobia oleracea (Linnaeus) larvae with the microsporidium Vairimorpha necatrix (Kramer) resulted in significant effects on the survival and development of the braconid parasitoid, Meteorus gyrator (Thunberg). Female M. gyrator did not show any avoidance of V. necatrix-infected hosts when they were selecting hosts for oviposition. When parasitism occurred at the same time as infection by the pathogen, or up to four days later, no significant detrimental effects on the parasitoid were observed. However, when parasitism occurred six to eight days after infection, a greater proportion (12.5-14%) of hosts died before parasitoid larvae egressed. Successful eclosion of adult wasps was also reduced. When parasitism and infection were concurrent, parasitoid larval development was significantly faster in infected hosts, and cocoons were significantly heavier. However, as the time interval between infection and parasitism increased, parasitoid larval development was significantly extended by up to two days, and the cocoons formed were significantly (c. 20%) smaller. Vairimorpha necatrix spores were ingested by the developing parasitoid larvae, accumulated in the occluded midgut, and were excreted in the meconium upon pupation.

Effects of three vertebrate hormones on the growth, development, and reproduction of the tomato moth, Lacanobia oleracea L. (Lepidoptera: Noctuidae).

In recent years, concern has been growing that numerous manmade chemicals entering the environment are capable of mimicking endogenous hormones in wildlife. In an attempt to define and evaluate the possible impact of endocrine-disrupting substances (EDS) on insects, three vertebrate hormones were tested for their effects on growth, development, and reproduction of the tomato moth. Lacanobia oleracea. Dietary administration of estrogen or thyroxine caused a significant increase in the length of the third, fourth, fifth, and sixth larval stadia (p < 0.001). The mean time for development of the pupa, however, was not significantly different between treatments. Relative to the control groups, a significant (p < 0.05) reduction in the mean weights of fifth- and sixth-instar larvae was also observed when larvae were exposed to estrogen or thyroxine and in pupae derived from insects exposed to thyroxine or testosterone (p < 0.001). Despite this, the number of larvae that survived to adulthood was not affected by any of the treatments; neither was the pupal sex ratio affected. However, exposure of larvae to testosterone significantly (p < 0.05) increased the number of deformed pupae. In addition, the reproductive potential of adults derived from the testosterone treatment was markedly reduced. Exposure of L. oleracea larvae to this steroid caused a highly significant (p < 0.001) decrease in egg production. coupled with a significant (p < 0.05) decrease in egg viability. The physiological effects observed in L. oleracea and their possible causes are presented in this paper, and the likely impact of EDS and their effects on terrestrial invertebrates are discussed.

Effect of dietary cowpea trypsin inhibitor (CpTI) on the growth and development of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) and on the success of the gregarious ectoparasitoid Eulophus pennicornis (Hymenoptera: Eulophidae).

Cowpea trypsin inhibitor (CpTI) was shown to have a deleterious effect on the growth and development of larvae of the tomato moth, Lacanobia oleracea, when incorporated in artificial diet (2.0% of soluble protein) and expressed in transgenic potato leaf (up to 1.0% of soluble protein). The effect of CpTI on parasitism of L oleracea by the ectoparasitoid Eulophus pennicornis was investigated. The parasitic success of the wasp was reduced by the presence of CpTI in the diet of the host and, in the case of transgenic potato leaves expressing the transgene protein, was collated with the length of time the host fed on the diet prior to parasitism. In all cases the proportion of hosts parasitised when fed CpTI-containing diets was reduced when compared with controls, although these differences were only significant when hosts were fed from the third instar on the transgenic potato leaves. Parasitoid progeny that developed on L oleracea reared on CpTI-containing diets, however, were not adversely affected. These results show that, whilst expression of CpTI in transgenic potato plants confers resistance to the lepidopterous pest L oleracea, adverse effects on the ability of the ectoparasitoid E pennicornis to parasitise this moth species successfully may also occur. These results are discussed in relation to the potential impact of transgenic crops on beneficial biological control agents.

Transgenic GNA expressing potato plants augment the beneficial biocontrol of Lacanobia oleracea (Lepidoptera; Noctuidae) by the parasitoid Eulophus pennicornis (Hymenoptera; Eulophidae).

The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F2 progeny produced by parasitoids derived from hosts fed on GNA expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.

The biology of Meteorus gyrator (Hymenoptera: Braconidae), a solitary endoparasitoid of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae).

There is a need to identify potential biological control agents for use against lepidopterous pests in greenhouses. The solitary endoparasitoid Meteorus gyrator (Thunberg) attacks a range of macrolepidopterous larvae, including those of some important horticultural pest species. Laboratory trials designed to investigate the biology of M. gyrator on larvae of the tomato moth, Lacanobia oleracea Linnaeus, reveal that this parasitoid is capable of parasitizing all larval stages of its host, third instars being parasitized most frequently. Each female parasitoid lives for up to 40 days (at 25 degrees C), ovipositing into an average of 78 hosts. Preadult development is rapid ( approximately 2 weeks), and the sex ratio of offspring is 1:1. Parasitism by M. gyrator suppresses the growth of both early and late host instars, and there is a concomitant reduction in the amount of food consumed (overall feeding reduction over a 12 day period is 68%). Our results indicate that inoculative releases of M. gyrator could provide effective biological control of L. oleracea and other noctuid pests of greenhouses.

Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition.

Eukaryote ribosomal translation is terminated when release factor eRF1, in a complex with eRF3, binds to one of the three stop codons. The tertiary structure and dimensions of eRF1 are similar to that of a tRNA, supporting the hypothesis that release factors may act as molecular mimics of tRNAs. To identify the yeast eRF1 stop codon recognition domain (analogous to a tRNA anticodon), a genetic screen was performed to select for mutants with disabled recognition of only one of the three stop codons. Nine out of ten mutations isolated map to conserved residues within the eRF1 N-terminal domain 1. A subset of these mutants, although wild-type for ribosome and eRF3 interaction, differ in their respective abilities to recognize each of the three stop codons, indicating codon-specific discrimination defects. Five of six of these stop codon-specific mutants define yeast domain 1 residues (I32, M48, V68, L123, and H129) that locate at three pockets on the eRF1 domain 1 molecular surface into which a stop codon can be modeled. The genetic screen results and the mutant phenotypes are therefore consistent with a role for domain 1 in stop codon recognition; the topology of this eRF1 domain, together with eRF1-stop codon complex modeling further supports the proposal that this domain may represent the site of stop codon binding itself.

The effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on Eulophus pennicornis (Hymenoptera: Eulophidae), a parasitoid of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae).

Snowdrop lectin (Galanthus nivalis agglutinin, GNA) has previously been shown to confer significant levels of protection against the lepidopteran pest Lacanobia oleracea when expressed in transgenic potato. The effect of GNA on the parasitism of L. oleracea by the gregarious ectoparasitoid Eulophus pennicornis was investigated. Maize-based, and potato leaf-based diets containing GNA, and excised transgenic potato leaves expressing GNA, were fed to L. oleracea larvae from the beginning of either the third or fourth larval instar. Lacanobia oleracea larvae were individually exposed to single mated adult female E. pennicornis parasitoids from the fifth instar onwards.The success of the wasp was not reduced by the presence of GNA in any of the diets, or by the length of feeding of the host prior to parasitism. However, the mean number of wasps that developed on L. oleracea reared from the third instar on the GNA-containing maize diet was significantly higher than on the controls (20.6 and 9.3 adults/host respectively). In all other cases differences were not significant. Eulophus pennicornis progeny that developed on L. oleracea reared on GNA-containing diets showed little or no alteration in size, longevity, egg load and fecundity when compared with wasps that had developed on hosts fed the respective control diets.The results suggest that expression of GNA in transgenic crops to confer resistance to lepidopteran pests will not adversely affect the ability of the ectoparasitoid E. pennicornis to utilise the pest species as a host.