Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe ( SPINE) consortium have developed and implemented high- throughput ( HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast ( Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for coexpression, selenomethionine labelling ( in all three eukaryotic systems) and control of glycosylation ( for secreted proteins in mammalian cells) are assessed.

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean. (C) 2004 Elsevier Ltd. All rights reserved.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

Effects of seed addition on beetle assemblages during the re-creation of species-rich lowland hay meadows

Species-rich lowland hay meadows are of conservation importance for both plants and invertebrates; however, they have declined in area across Europe as a result of conversion to other land uses and management intensification. The re-creation of these grasslands on ex-arable land provides a valuable approach to increasing the extent and conservation value of this threatened habitat. Over a 3-year period a replicated block design was used to test whether introducing seeds promoted the re-creation of both plant and phytophagous beetle assemblages typical of a target hay meadow. Seeds were harvested from local hay meadows, and applied to experimental plots in the form of either green hay or brush harvesting seeds. Green hay spreading achieved the greatest success in re-creating plant and phytophagous beetle assemblages. While re-creation success increased over time for both taxa, for the phytophagous beetles the greatest increase in re-creation success relative to the establishment year also occurred where green hay was applied. We also considered the phytophagous beetles in terms of functional traits that describe host plant specificity, larval feeding location and dispersal. Phytophagous beetle functional trait composition was most similar to the target hay meadow assemblage where some form of seed addition was used, i.e. hay spreading or brush harvested seeds. This study identified the importance of introducing target plant species as a mechanism to promote the re-creation of phytophagous beetle communities. Seed addition methods (e.g. green hay spreading) are crucial to successful hay meadow re-creation.

Dispersal of soil-dwelling clover root weevil (Sitona lepidus Gyllenhal, Coleoptera: Curculionidae) larvae in mixed plant communities

Insect pests that have a root-feeding larval stage often cause the most sustained damage to plants because their attrition remains largely unseen, preventing early diagnosis and treatment. Characterising movement and dispersal patterns of subterranean insects is inherently difficult due to the difficulty in observing their behaviour. Our understanding of dispersal and movement patterns of soil-dwelling insects is therefore limited compared to above ground insect pests and tends to focus on vertical movements within the soil profile or assessments of coarse movement patterns taken from soil core measurements in the field. The objective of this study was to assess how the dispersal behaviour of the clover root weevil (CRW), Sitona lepidus larvae was affected by differing proportions of host (clover) and non-host (grass) plants under different soil water contents (SWC). This was undertaken in experimental mini-swards that allowed us to control plant community structure and soil water content. CRW larval survival was not affected either by white clover content or planting pattern or SWC in either experiment; however, lower clover composition in the sward resulted in CRW larvae dispersing further from where they hatched. Because survival was the same regardless of clover density, the proportion of infested plants was highest in sward boxes with the fewest clover plants (i.e. the low host plant density). Thus, there is potential for clover plants over a larger area to be colonised when the clover content of the sward is low.

Marine Isotope Stage 9 environments of fluvial deposits at Hackney, north London, UK

Middle Pleistocene deposits at Hackney, north London comprise a thick unit of organic sands and silts occupying a channel near the confluence of the River Thames in south-eastern England and its left-bank tributary the River Lea. They represent a short time interval, perhaps no more than a few years, within a late Middle Pleistocene interglacial. The organic sediments are overlain by unfossiliferous sands and gravels indicating deposition on the floodplain of a braided river under cool or cold climatic conditions. The fossil plant, insect, mollusc and vertebrate remains from the interglacial deposits all indicate climatic conditions with summers warmer than the present in SE England, and winters with a similar thermal climate. The biostratigraphic evidence suggests that the time period represented by the organic unit is part of MIS 9, although the geochronological evidence for such an age is inconclusive. The palaeontological evidence strongly suggests that this temperate stage was warmer than the succeeding temperate stage MIS 7 or the Holocene, and approaching the Ipswichian (MISs 5e) in its warmth. The multidisciplinary description of the Hackney deposits is one of the first to reconstruct terrestrial conditions in Marine Isotope Stage 9 in Western Europe.

Managing maize under pest species competition: is Bt (Bacillus thuringiensis) maize the solution?

Transgenic crops that contain Cry genes from Bacillus thuringiensis (Bt) have been adopted by farmers over the last 17 years. Unlike traditional broad spectrum chemical insecticides, Bt's toxicity spectrum is relatively narrow and selective, which may indirectly benefit secondary insects that may become important pests. The economic damage caused by the rise of secondary pests could offset some or all of the benefits associated with the use of Bt varieties. We develop a bioeconomic model to analyze the interactions between primary and secondary insect populations and the impact of different management options on insecticide use and economic impact over time. Results indicate that some of the benefits associated with the adoption of genetically engineered insect resistant crops may be eroded when taking into account ecological dynamics. It is suggested that secondary pests could easily become key insect pests requiring additional measures - such as insecticide applications or stacked traits – to keep their populations under the economic threshold.

Pollination biology of Syngonanthus elegans (Eriocaulaceae - Poales)

Studies on the pollination biology of Eriocaulaceae are scarce although particularly interesting because of its inclusion in the Poales, a predominantly wind-pollinated order. The pollination biology of Syngonanthus elegans (Bong.) Ruhland was studied during two annual flowering periods to test the hypothesis that insect pollination was its primary pollination system. A field study was carried out, including observations of the morphology and biology of the flowers, insect visits and pollinator behaviour. We also evaluated seed set, seed germination and seedling development for different pollination modes. Although seeds were produced by self-pollination, pollination by small insects contributed most effectively to the reproductive success of S. elegans, resulting in the greatest seed set, with the highest germination percentage and optimum seedling vigour. The. oral resources used by flower visitors were pollen and nectar that was produced by staminate and pistillate flowers. Self-pollination played a minor role and its consequence was inbreeding depression.

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Visitantes florais de Erythrina speciosa Andr. (Leguminosae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Entomofauna visitante das flores de Cassia spectabilis (L.) D C. (Leguminosae)

Realizaram-se estudos sobre sistemas de reprodução de Cassia spectabilis (L.) D C. (Leguminosae) observando-se a diversidade, freqüência e constância dos insetos visitantes em diferentes horários. Também testou-se a influência dos fatores ambientais em relação às visitas. Os resultados de polinização manual sugerem que C. spectabilis é autocompatível, porém, a xenogamia é o sistema de reprodução predominante. As inflorescências foram visitadas por uma grande quantidade de insetos, havendo predominância de abelhas. O horário de maior ocorrência dos insetos nas flores de C. spectabilis foi das 8 às 14 h e de menor ocorrência entre 7 e 8 h e das 17 às 18 h. Quanto ao comportamento dos insetos em relação à flor de C. spectabilis, observou-se que Xylocopa frontalis Olivier, X. suspecta Camargo & Moure, Bombus morio Swederus e Centris scopipes Friese possuem comportamento e morfologia adequados aos polinizadores legítimos; C. similis F., Oxaea flavescens Klug e Epicharis rustica flava Cockerell foram considerados polinizadores ocasionais. Pseudaugochloropsis graminea (F.), Tetragonisca angustula Latreille e A. mellifera L. foram considerados pilhadores. A polinização por vibração é o método usado pelas abelhas para coleta de pólen.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resistência de genótipos de feijão-vagem ao ataque de Zabrotes subfasciatus (Bohemann, 1833) (Coleoptera: Chrysomelidae)

O feijão-vagem (Phaseolus vulgaris L.) pode sofrer ataque de carunchos, como os da espécie Zabrotes subfasciatus (Bohemann, 1833) (Coleoptera: Chrysomelidae), afetando, diretamente, a qualidade das vagens e sementes e deixando-as inviáveis para o consumo e comercialização. A fim de buscar uma alternativa para o controle deste inseto, foram determinados os tipos e graus de resistência, envolvidos em genótipos de feijão-vagem, ao ataque de Z. subfasciatus, em testes com e sem chance de escolha. Os genótipos utilizados foram UEGD7, UEG05, UEG06, UEG11, UEG13, UEG15, UEG18, UEG19 e UEG26, com quatro repetições. No teste com chance de escolha, os parâmetros avaliados foram o número de ovos viáveis, inviáveis e total e a atratividade dos adultos pelos genótipos. No teste sem chance de escolha, foram avaliados o número de ovos viáveis, inviáveis e total, massa seca consumida e massa seca consumida por inseto, massa e longevidade dos adultos, período de ovo a adulto, razão sexual e número e percentagem de insetos emergidos. Concluiu-se que o genótipo UEG05 foi o menos preferido por Z. subfasciatus, para oviposição, em teste com chance de escolha. O UEG13 foi altamente resistente e UEG05, UEG15 e UEG19 foram moderadamente resistentes. O UEG18 foi susceptível e os demais genótipos foram altamente susceptíveis a Z. subfasciatus.

Dietary L-tryptophan alters aggression in juvenile matrinxa Brycon amazonicus

This study evaluated the effect of dietary supplementation with -tryptophan (L-TRP), a serotonin precursor, on the aggressiveness of juvenile matrinx . Fish were kept in individual aquaria for 7 days receiving the diets: D1 (control: 0.47% of TRP), D2 (0.94% of TRP), D3 (1.88% of TRP), and D4 (3.76% of TRP). After this, they were grouped with an intruder fish to establish a resident-intruder relationship during periods of 20 min. Blood cortisol, glucose, chloride, sodium and calcium; hemoglobin, hematocrit, red blood cell count and volume; liver glycogen and lipids were measured. Territoriality had significant effect on the aggressiveness of matrinx (the residents were more aggressive than intruders, < 0.001) and tryptophan significantly affected their behavior. Fish fed with the D2 diet presented a longer latency until the first attack ( = 0.0069) and bit the intruder fewer times ( = 0.0136) during the period of observation, compared to the control group. The frequency of bites and chases after the first attack was not affected by the dietary supplementation of TRP. Physiological variables were not significantly affected by the diet, except for a moderate increase in cortisol level in fish fed with D2 diet after the fight, indicating slight activation of the hypothalamus-pituitary-interrenal axis. The results show that juvenile matrinx have aggressive and territorial behavior and that a diet containing 9.4 g TRP kg(-1) alter their aggressiveness, without affecting the stress-related physiological parameters.