Effects of organic and conventional fertiliser treatments on host selection by the aphid parasitoid Diaeretiella rapae

The type and quantity of fertilizer supplied to a crop will differ between organic and conventional farming practices. Altering the type of fertilizer a plant is provided with can influence a plant’s foliar nitrogen levels, as well as the composition and concentration of defence compounds, such as glucosinolates. Many natural enemies of insect herbivores can respond to headspace volatiles emitted by the herbivores’ host plant in response to herbivory. We propose that manipulating fertilizer type may also influence the headspace volatile profiles of plants, and as a result, the tritrophic interactions that occur between plants, their insect pests and those pests’ natural enemies. Here, we investigate a tritrophic system consisting of cabbage plants, Brassica oleracea, a parasitoid, Diaeretiella rapae, and one of its hosts, the specialist cabbage aphid Brevicoryne brassicae. Brassica oleracea plants were provided with either no additional fertilization or one of three types of fertilizer: Nitram (ammonium nitrate), John Innes base or organic chicken manure. We investigated whether these changes would alter the rate of parasitism of aphids on those plants and whether any differences in parasitism could be explained by differences in attractivity of the plants to D. rapae or attack rate of aphids by D. rapae. In free-choice experiments, there were significant differences in the percentage of B. brassicae parasitized by D. rapae between B. oleracea plants grown in different fertilizer treatments. In a series of dual-choice Y-tube olfactometry experiments, D. rapae females discriminated between B. brassicae-infested and undamaged plants, but parasitoids did not discriminate between similarly infested plants grown in different fertilizer treatments. Correspondingly, in attack rate experiments, there were no differences in the rate that D. rapae attacked B. brassicae on B. oleracea plants grown in different fertilizer treatments. These findings are of direct relevance to sustainable and conventional farming practices.

Identifying key knowledge needs for evidence-based conservation of wild insect pollinators: a collaborative cross-sectoral exercise

In response to evidence of insect pollinator declines, organisations in many sectors, including the food and farming industry, are investing in pollinator conservation. They are keen to ensure that their efforts use the best available science. We convened a group of 32 ‘conservation practitioners’ with an active interest in pollinators and 16 insect pollinator scientists. The conservation practitioners include representatives from UK industry (including retail), environmental non-government organisations and nature conservation agencies. We collaboratively developed a long list of 246 knowledge needs relating to conservation of wild insect pollinators in the UK. We refined and selected the most important knowledge needs, through a three-stage process of voting and scoring, including discussions of each need at a workshop. We present the top 35 knowledge needs as scored by conservation practitioners or scientists. We find general agreement in priorities identified by these two groups. The priority knowledge needs will structure ongoing work to make science accessible to practitioners, and help to guide future science policy and funding. Understanding the economic benefits of crop pollination, basic pollinator ecology and impacts of pesticides on wild pollinators emerge strongly as priorities, as well as a need to monitor floral resources in the landscape.

Transgenic cereals: current status and future prospects

This review summarises the history of transgenic (GM) cereals, principally maize, and then focuses on the scientific literature published in the last two years. It describes the production of GM cereals with modified traits, divided into input traits and output traits. The first category includes herbicide tolerance and insect resistance, and resistance to abiotic and biotic stresses; the second includes altered grains for starch, protein or nutrient quality, the use of cereals for the production of high value medical or other products, and the generation of plants with improved efficiency of biofuel production. Using data from field trial and patent databases the review considers the diversity of GM lines being tested for possible future development. It also summarises the dichotomy of response to GM products in various countries, describes the basis for the varied public acceptability of such products, and assesses the development of novel breeding techniques in the light of current GM regulatory procedures.

Effects of organic and conventional fertilizer treatments on host selection by the aphid parasitoid Diaeretiella rapae

The type and quantity of fertilizer supplied to a crop will differ between organic and conventional farming practices. Altering the type of fertilizer a plant is provided with can influence a plant’s foliar nitrogen levels, as well as the composition and concentration of defence compounds, such as glucosinolates. Many natural enemies of insect herbivores can respond to headspace volatiles emitted by the herbivores’ host plant in response to herbivory. We propose that manipulating fertilizer type may also influence the headspace volatile profiles of plants, and as a result, the tritrophic interactions that occur between plants, their insect pests and those pests’ natural enemies. Here, we investigate a tritrophic system consisting of cabbage plants, Brassica oleracea, a parasitoid, Diaeretiella rapae, and one of its hosts, the specialist cabbage aphid Brevicoryne brassicae. Brassica oleracea plants were provided with either no additional fertilization or one of three types of fertilizer: Nitram (ammonium nitrate), John Innes base or organic chicken manure. We investigated whether these changes would alter the rate of parasitism of aphids on those plants and whether any differences in parasitism could be explained by differences in attractivity of the plants to D. rapae or attack rate of aphids by D. rapae. In free-choice experiments, there were significant differences in the percentage of B. brassicae parasitized by D. rapae between B. oleracea plants grown in different fertilizer treatments. In a series of dual-choice Y-tube olfactometry experiments, D. rapae females discriminated between B. brassicae-infested and undamaged plants, but parasitoids did not discriminate between similarly infested plants grown in different fertilizer treatments. Correspondingly, in attack rate experiments, there were no differences in the rate that D. rapae attacked B. brassicae on B. oleracea plants grown in different fertilizer treatments. These findings are of direct relevance to sustainable and conventional farming practices.

EDITOR'S CHOICE: REVIEW: Trait matching of flower visitors and crops predicts fruit set better than trait diversity

Understanding the relationships between trait diversity, species diversity and ecosystem functioning is essential for sustainable management. For functions comprising two trophic levels, trait matching between interacting partners should also drive functioning. However, the predictive ability of trait diversity and matching is unclear for most functions, particularly for crop pollination, where interacting partners did not necessarily co-evolve. World-wide, we collected data on traits of flower visitors and crops, visitation rates to crop flowers per insect species and fruit set in 469 fields of 33 crop systems. Through hierarchical mixed-effects models, we tested whether flower visitor trait diversity and/or trait matching between flower visitors and crops improve the prediction of crop fruit set (functioning) beyond flower visitor species diversity and abundance. Flower visitor trait diversity was positively related to fruit set, but surprisingly did not explain more variation than flower visitor species diversity. The best prediction of fruit set was obtained by matching traits of flower visitors (body size and mouthpart length) and crops (nectar accessibility of flowers) in addition to flower visitor abundance, species richness and species evenness. Fruit set increased with species richness, and more so in assemblages with high evenness, indicating that additional species of flower visitors contribute more to crop pollination when species abundances are similar. Synthesis and applications. Despite contrasting floral traits for crops world-wide, only the abundance of a few pollinator species is commonly managed for greater yield. Our results suggest that the identification and enhancement of pollinator species with traits matching those of the focal crop, as well as the enhancement of pollinator richness and evenness, will increase crop yield beyond current practices. Furthermore, we show that field practitioners can predict and manage agroecosystems for pollination services based on knowledge of just a few traits that are known for a wide range of flower visitor species.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midguit cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap-3) by a new class-II piggyBac-related insect transposon

A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system

Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel.

HOST-MEDIATED INDUCTION OF alpha-AMYLASES BY LARVAE OF THE MEXICAN BEAN WEEVIL Zabrotes subfasciatus (COLEOPTERA: CHRYSOMELIDAE: BRUCHINAE) IS IRREVERSIBLE AND OBSERVED FROM THE INITIATION OF THE FEEDING PERIOD

Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days ( age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes. (C) 2010 Wiley Periodicals, Inc.

Subsite substrate specificity of midgut insect chymotrypsins

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1`, P2, and P3. The binding energy (Delta G(s), calculated from Km values) and the activation energy (Delta G(T)(double dagger), determined from k(cat)/K-m values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1`) vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1` bind the substrate ground state, whereas only S1` binds the transition state, supporting aspects of the present accepted mechanism of catalysis. 2008 Elsevier Ltd. All rights reserved.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

Characterization of a beta-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to beta-glucan-binding proteins

Spodoptera frugiperda beta-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against beta-1,3-glucan (laminarin), but cannot hydrolyze yeast beta-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive beta-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of beta-1,3-glucanases and beta-glucan-binding protein supports the assumption that the beta-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived beta-1,3-glucanases by the loss of an extended N-terminal region and the beta-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells. (C) 2010 Elsevier Ltd. All rights reserved.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.