Barbara Manachini

A mathematical model of exposure of non- target Lepidoptera to Bt-maize pollen expressing Cry1Ab within Europe

Genetically modified (GM) maize MON810 expresses a Cry1Ab insecticidal protein, derived from Bacillus thuringiensis (Bt), toxic to lepidopteran target pests such as Ostrinia nubilalis. An environmental risk to non-target Lepidoptera from this GM crop is exposure to harmful amounts of Bt-containing pollen deposited on host plants in or near MON810 fields. An 11-parameter mathematical model analysed exposure of larvae of three non-target species: the butterflies Inachis io (L.), Vanessa atalanta (L.) and moth Plutella xylostella (L.), in 11 representative maize cultivation regions in four European countries. A mortality–dose relationship was integrated with a dose–distance relationship to estimate mortality both within the maize MON810 crop and within the field margin at varying distances from the crop edge. Mortality estimates were adjusted to allow for physical effects; the lack of temporal coincidence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed; and seven further parameters concerned with maize agronomy and host-plant ecology. Sublethal effects were estimated and allowance made for aggregated pollen deposition. Estimated environmental impact was low: in all regions, the calculated mortality rate for worst-case scenarios was less than one individual in every 1572 for the butterflies and one in 392 for the moth.

Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (~50%), granulocytes (~35%), prohemocytes (~8%), oenocytes (~4%) and spherulocytes (~3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (~12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.

Study of the aminopeptidase N gene family in the lepidopterans Ostrinia nubilalis (Hübner) and Bombyx mori (L.): Sequences, mapping and expression

Aminopeptidases N (APNs) are a class of ectoenzymes present in lepidopteran larvae midguts, involved in the Bacillus thuringiensis (Bt) toxins mode of action. In the present work, seven aminopeptidases have been cloned from the midgut of Ostrinia nubilalis, the major Lepidopteran corn pest in the temperate climates. Six sequences were identified as APNs because of the presence of the HEXXH(X)18E and GAMEN motifs, as well as the signal peptide and the GPI-anchor sequences. The remaining sequence did not contain the two cellular targeting signals, indicating it belonged to the puromycin-sensitive aminopeptidase (PSA) family. An in silico analysis allowed us to find orthologous sequences in Bombyx mori. A phylogenetic study of lepidopteran aminopeptidase sequences resulted in their clustering into nine classes. Linkage analysis revealed that the onapn genes as well as all bmapn genes clustered in a single linkage group. O. nubilalis aminopeptidases were expressed in all larval instars. In 5th instar larvae tissues, apns transcripts were found mainly in midguts while apn8 was also highly expressed in Malpighian tubules, and psa showed an ubiquitous expression pattern in O. nubilalis and B. mori. The sequence homology and gene organization of apns suggest a single origin from an ancestral lepidopteran apn gene.

The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

BackgroundThe red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is one of the major pests of palms. The larvae bore into the palm trunk and feed on the palm tender tissues and sap, leading the host tree to death. The gut microbiota of insects plays a remarkable role in the host life and understanding the relationship dynamics between insects and their microbiota may improve the biological control of insect pests. The purpose of this study was to analyse the diversity of the gut microbiota of field-caught RPW larvae sampled in Sicily (Italy).ResultsThe 16S rRNA gene-based Temporal Thermal Gradient Gel Electrophoresis (TTGE) of the gut microbiota of RPW field-trapped larvae revealed low bacterial diversity and stability of the community over seasons and among pools of larvae from different host trees. Pyrosequencing of the 16S rRNA gene V3 region confirmed low complexity and assigned 98% of the 75,564 reads to only three phyla: Proteobacteria (64.7%) Bacteroidetes (23.6%) and Firmicutes (9.6%) and three main families [Enterobacteriaceae (61.5%), Porphyromonadaceae (22.1%) and Streptococcaceae (8.9%)]. More than half of the reads could be classified at the genus level and eight bacterial genera were detected in the larval RPW gut at an abundance ≥1%: Dysgonomonas (21.8%), Lactococcus (8.9%), Salmonella (6.8%), Enterobacter (3.8%), Budvicia (2.8%), Entomoplasma (1.4%), Bacteroides (1.3%) and Comamonas (1%). High abundance of Enterobacteriaceae was also detected by culturing under aerobic conditions. Unexpectedly, acetic acid bacteria (AAB), that are known to establish symbiotic associations with insects relying on sugar-based diets, were not detected.ConclusionsThe RPW gut microbiota is composed mainly of facultative and obligate anaerobic bacteria with a fermentative metabolism. These bacteria are supposedly responsible for palm tissue fermentation in the tunnels where RPW larvae thrive and might have a key role in the insect nutrition, and other functions that need to be investigated.

Towards a landscape scale management of pesticides: ERA using changes in modelled occupancy and abundance to assess long-term population impacts of pesticides.

Pesticides are regulated in Europe and this process includes an environmental risk assessment (ERA) for non-target arthropods (NTA). Traditionally a non-spatial or field trial assessment is used. In this study we exemplify the introduction of a spatial context to the ERA as well as suggest a way in which the results of complex models, necessary for proper inclusion of spatial aspects in the ERA, can be presented and evaluated easily using abundance and occupancy ratios (AOR). We used an agent-based simulation system and an existing model for a widespread carabid beetle (Bembidion lampros), to evaluate the impact of a fictitious highly-toxic pesticide on population density and the distribution of beetles in time and space. Landscape structure and field margin management were evaluated by comparing scenario-based ERAs for the beetle. Source-sink dynamics led to an off-crop impact even when no pesticide was present off-crop. In addition, the impacts increased with multi-year application of the pesticide whereas current ERA considers only maximally one year. These results further indicated a complex interaction between landscape structure and pesticide effect in time, both in-crop and off-crop, indicating the need for NTA ERA to be conducted at landscape- and multi-season temporal-scales. Use of AOR indices to compare ERA outputs facilitated easy comparison of scenarios, allowing simultaneous evaluation of impacts and planning of mitigation measures. The landscape and population ERA approach also demonstrates that there is a potential to change from regulation of a pesticide in isolation, towards the consideration of pesticide management at landscape scales and provision of biodiversity benefits via inclusion and testing of mitigation measures in authorisation procedures.

Biological Responses of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) to Steinernema carpocapsae (Nematoda: Steinernematidae)

ABSTRACT Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) is becoming a serious problem in Mediterranean areas where it is well-adapted, and now is present even in the United States (California). The infestations are primarily in urban areas where chemical control is not advisable and million of Euros are spent to control it. The effects of the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Steinernematidae) on mortality, growth, as well as the immune activity of R. ferrugineus larvae, were investigated. R. ferrugineus mortality exhibited a positive trend with the dosage and duration of exposure to S. carpocapsae. The median lethal dose and median lethal time, important to optimize the treatments, were calculated. S. carpocapsae also had a detrimental effect on R. ferrugineus weight. In vivo and in vitro effects of S. carpocapsae on the phagocytic responses of R. ferrugineus hemocytes also were recorded. S. carpocapsae was not encapsulated by R. ferrugineus hemocytes. After 24 h, the number of hemocytes recorded in treated larvae was reduced. To investigate the defensive abilities of R. ferrugineus humoral and cellular immune systems, specifically against the bacterium Xenorhabdus nematophila (Enterobacteraceae), the minimum inhibitory concentration that inhibits bacterial growth was measured. This is the first time that this technique is applied to entomopathogenic bacteria.

The usefulness of a mathematical model of exposure for environmental risk assessment.

We respond to the Comment of Lang et al . [[1][1]] regarding our mathematical model [[2][2]] of exposure of non-target Lepidoptera to Bt -maize pollen expressing Cry1Ab within Europe. Lang et al . remark on the degree to which the model was subject to uncertainty. Perry et al . [[2][2]] did indeed

Modulation of immune responses of Rhynchophorus ferrugineus （Insecta： Coleoptera）induced by the entomopathogenic nematode Steinernema carpocapsae （Nematoda： Rhabditida）

Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase‐phenoloxidase (proPO) system, involved in melanization of not‐self and hemocytes recognition processes responsible for not‐self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell‐mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body‐surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.

Evaluating resistance to Bt toxin Cry1Ab by F2 screen in European populations of Ostrinia nubilalis (Lepidoptera: Crambidae).

ABSTRACT The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the CrylAb toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the CrylAb toxin. The cost of F2 screen performed in the four countries varied from US

Eco-physiological response of two marine bivalves to acute exposition to commercial Bt-based pesticide

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