Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications. © 2013 Elsevier Ltd.

Four year multitrophic food web: source food web description, methodology accuracy and species diversity

Food webs have been used in order to understand the trophic relationship among organisms within an ecosystem, however the extension by which sampling efficiency could affect food web responses remain poorly understood. Still, there is a lack of long-term sampling data for many insect groups, mainly related to the interactions between herbivores and their host plants. In the first chapter, I describe a source food web based on the Senegalia tenuifolia plant by identifying the associated insect species and the interactions among them and with this host plant. Furthermore, I check for the data robustness from each trophic level and propose a cost-efficiently methodology. The results from this chapter show that the collected dataset and the methodology presented are a good tool for sample most insect richness of a source food web. In total the food web comprises 27 species belonging to four trophic levels. In the second chapter, I demonstrate the temporal variation in the species richness and abundance from each trophic level, as well as the relationship among distinct trophic levels. Moreover, I investigate the diversity patterns of the second and third trophic level by assessing the contribution of alfa and beta-diversity components along the years. This chapter shows that in our system the parasitoid abundance is regulated by the herbivore abundances. Besides, the species richness and abundances of the trophic levels vary temporally. It also shows that alfa-diversity was the diversity component that most contribute to the herbivore species diversity (2nd trophic level), while the contribution of alfa- and beta-diversity changed along the years for parasitoid diversity (3rd level). Overall, this dissertation describes a source food web and bring insights into some food web challenges related to the sampling effort to gather enough species from all trophic levels. It also discuss the relation among communities associated with distinct trophic levels and their temporal variation and diversity patterns. Finally, this dissertation contributes for the world food web database and in understanding the interactions among its trophic levels and each trophic level pattern along time and space

Caracterização de organossulfurados em diferentes cultivares de alho (Allium sativum L.) e cebola (Allium cepa L.)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Dermatite alérgica à picada de insetos em ovinos no estado do Pará

Foi estudada uma doença em 159 ovinos em 15 propriedades localizadas em sete municípios das mesorregiões nordeste e sudeste do estado do Pará e uma em um município do estado de Roraima. Os ovinos da raça Santa Inês e seus mestiços, de ambos os sexos e com dois a quatro anos de idade, eram os mais acometidos. A doença caracterizava-se por alopecia em diversas regiões do corpo, principalmente ao redor dos olhos; essas lesões evoluíam para eritemas multifocais, pequenas pápulas e crostas. Os animais apresentavam prurido intenso no local das lesões, inquietação, perda de peso e corrimento ocular. Sempre era observada a presença de mosquitos ao redor dos animais. Com o auxílio de um puçá foi realizada a captura de insetos associados às lesões, durante o repasto nos ovinos, para a identificação. Foram identificados insetos dos gêneros Simulium e Hippelates. Realizou-se biópsia da pele lesada de 10 ovinos. As lesões histológicas se caracterizaram por leves infiltrados inflamatórios mononucleares na derme, com presença de eosinófilos. Os estudos epidemiológicos, clínicos e patológicos desses casos, bem como o descarte dos diagnósticos diferenciais, permitiram concluir que se trata de dermatite alérgica por picada de insetos.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

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

THE PARASITOID, Cotesia flavipes (CAMERON) (HYMENOPTERA: BRACONIDAE), INFLUENCES FOOD CONSUMPTION AND UTILIZATION BY LARVAL Diatraea saccharalis (F.) (LEPIDOPTERA: CRAMBIDAE)

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

Diversity and Localization of Bacterial Endosymbionts from Whitefly Species Collected in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Heterorhabditis amazonensis RSC5 (Rhabditida: Heterorhabditidae) movement and host recognition

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The prophenoloxidase system and in vitro interaction of Trypanosoma rangeli with Rhodnius prolixus and Triatoma infestans haemolymph

The presence of the prophenoloxidase (proPO) system in the haemolymph of Rhodnius prolixus and Triatoma infestans and the role played by Trypanosoma rangeli in the in vitro activation of proPO were tested. Both R. prolixus and T. infestans whole blood preparations showed a very active ProPO system. The proPO cascade of the two insect species were differentially activated by microbial-derived extracts: laminarin was a better activator of T. infestans haemolymph than of R. prolixus blood, and lipopolysaccharides from Shigella flexneri or Pseudomonas aeroginosa caused significant proPO activation of T. infestans haemolymph but not of R. prolixus preparations. For the two insect species, neither T. rangeli from culture nor parasite lysates were able to trigger proPO activation. The presence of the parasite in R. prolixus haemolymph/laminarin assays, however, significantly reduced the level of proPO activation to that of spontaneous activating controls. The immobilization of T. rangeli in vitro in haemolymph preparations occurred in both insect species and was dependent on the proPO activation intensity. Our results suggest that the susceptibility of R. prolixus to T. rangeli haemocoel infection may be explained, at least in part, by the suppression of the insect immune defence system i.e., inhibition of proPO in the presence of this protozoan parasite.

Comparing de novo and reference-based transcriptome assembly strategies by applying them to the blood-sucking bug Rhodnius prolixus

High Throughput Sequencing capabilities have made the process of assembling a transcriptome easier, whether or not there is a reference genome. But the quality of a transcriptome assembly must be good enough to capture the most comprehensive catalog of transcripts and their variations, and to carry out further experiments on transcriptomics. There is currently no consensus on which of the many sequencing technologies and assembly tools are the most effective. Many non-model organisms lack a reference genome to guide the transcriptome assembly. One question, therefore, is whether or not a reference-based genome assembly gives better results than de novo assembly. The blood-sucking insect Rhodnius prolixus-a vector for Chagas disease-has a reference genome. It is therefore a good model on which to compare reference-based and de novo transcriptome assemblies. In this study, we compared de novo and reference-based genome assembly strategies using three datasets (454, Illumina, 454 combined with Illumina) and various assembly software. We developed criteria to compare the resulting assemblies: the size distribution and number of transcripts, the proportion of potentially chimeric transcripts, how complete the assembly was (completeness evaluated both through CEGMA software and R. prolixus proteome fraction retrieved). Moreover, we looked for the presence of two chemosensory gene families (Odorant-Binding Proteins and Chemosensory Proteins) to validate the assembly quality. The reference-based assemblies after genome annotation were clearly better than those generated using de novo strategies alone. Reference-based strategies revealed new transcripts, including new isoforms unpredicted by automatic genome annotation. However, a combination of both de novo and reference-based strategies gave the best result, and allowed us to assemble fragmented transcripts.

Subacute toxicity assessment of diflubenzuron, an insect growth regulator, in adult male rats

Difubenzuron (DFB), an insecticide and acaricide insect growth regulator, can be used in agriculture against insect predators and in public health programs, to control insects and vectors, mainly Aedes aegypti larvae. Due to the lack of toxicological assessments of this compound, the objective of the present study was to evaluate the toxicological effects of subacute exposure to the DFB insecticide in adult male rats. Adult male rats were exposed (gavage) to 0, 2, 4, or 8 mg/kg of DFB for 28 days. No clinical signs of toxicity were observed in the DFB-treated animals of the experimental groups. However, there was an increase in serum levels of alanine aminotransferase in the group that received 8 mg/Kg/DFB/day and urea at doses of 4 and 8 mg/Kg/DFB/day, without altering other biochemical or hematological parameters. The subacute exposure to the lowest dose of DFB caused significant decrease in testis weight, daily sperm production, and in number of sperm in the epididymis in relation to the control group. However, no alterations were observed in the sperm morphology, testicular, epididymis, liver and kindney histology, or testosterone levels. These findings unveiled the hazardous effects of DFB on male reproduction after the subacute exposure and special attention should be addressed to the effects of low doses of this pesticide.

Impacts of Plant Size, Density, Herbivory, and Desease on Native Platte Thistle (Cirsium Canescens)

Abstract. Based on prior field observations, we hypothesized that individual and interacting effects of plant size, density, insect herbivory, and especially fungal disease, influenced seedling and juvenile plant growth in native Platte thistle populations (Cirsium canescens Nutt.). We worked at Arapaho Prairie in the Nebraska Sandhills (May - August 2007), monitoring plant growth, insect damage, and fungal infection within different density thistle patches. In the main experiment, we sprayed half of test plants in different density patches with fungicide (Fungonil© Bonide, containing chlorothalonil) and half with a water control. Fungal infection rates were very low, so we found no difference in fungal attack between these treatments. However, plants that received the fungicide treatment had significantly faster growth over the season than did the control plants. At the same time, plants in the fungicide treatment had significantly reduced insect herbivory. These results strongly suggest that the fungicide had insecticidal effects and that insect herbivory significantly decreases juvenile Platte thistle growth. Further, damage by insect herbivores tended to be higher for larger plants, and herbivory was variable among different patches. However, plant density did not appear to have a large effect on the amount of insect herbivory that individual juvenile Platte thistle plants received. In the second experiment, we examined germination and survival success in relationship to seed density, and found that germination success was higher in areas of lower seed density. In the third experiment, we tested germination for filled seeds categorized primarily by color variation and size, and found no difference in germination related to either color or seed weight. We conclude that seed density, but not seed quality as estimated by color or size, affects germination success. Further, although herbivory was not significantly affected by plant density at any of the scales examined, insect herbivory significantly reduces the growth and success of juveniles of this characteristic native sand prairie plant.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coil, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAD) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut CAD hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C265) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 angstrom, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. (C) 2012 Elsevier Ltd. All rights reserved.

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Integrated landmark and outline-based morphometric methods efficiently distinguish species of Euglossa (Hymenoptera, Apidae, Euglossini)

Morphometric methods permit identification of insect species and are an aid for taxonomy. Quantitative wing traits were used to identify male euglossine bees. Landmark- and outline-based methods have been primarily used independently. Here, we combine the two methods using five Euglossa. Landmark-based methods correctly classified 84% and outline-based 77%, but an integrated analysis correctly classified 91% of samples. Some species presented significantly high reclassification percentages when only wing cell contour was considered, and correct identification of specimens with damaged wings was also obtained using this methodology.