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.

Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an encloglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections. (C) 2009 Elsevier Ltd. All rights reserved.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

SILVA, Fatima C. B. L. et al. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets. Insect Biochemistry and Molecular Biology, v. 36, p. 561-569, 2006.ISSN: 0965-1748.DOI: 10.1016/j.ibmb.2006.04.004.

Prey captured and used in Polistes versicolor (Olivier) (Hymenoptera: Vespidae) Nourishment

As vespas sociais são predadoras de muitas espécies de insetos e o estudo de suas presas pode revelar seu potencial para programas de controle biológico de pragas. Foram realizadas 240h de coleta de presas em 32 colônias de Polistes versicolor (Olivier) no município de Juiz de Fora, MG, de março de 2000 a fevereiro de 2001. As presas capturadas por P. versicolor foram, principalmente, das ordens Lepidoptera (95,4%) e Coleoptera (1,1%) além de 3,4% de indivíduos não identificados. A espécie mais coletada foi Chlosyne lacinia saundersii Doubleday & Hewitson (13,5%) (Lepidoptera: Nymphalidae) e o número total estimado de presas capturadas por colônia de P. versicolor foi de 4.015 indivíduos por ano. Isso mostra que a espécie pode ser utilizada em programas de manejo integrado de pragas de insetos herbívoros, principalmente lagartas desfolhadoras.

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.

Spermatogenesis chronology in Achroia grisella Fabricius (Lepidoptera, Pyralidae) comparing eupyrene and apyrene spermiogenesis

Achroia grisella exhibit dichotomous spermatogenesis producing two types of sperm, one is eupyrene that possesses nucleus, and the other is apyrene that lacks it. Transmission electron microscopy of spermatogenesis morphology is described considering sperm type which will appear at sonic point during insect development, and differences that mark the two types of sperm formation. The differences between them are only really visible during spermiogenesis even though they were determined before meiosis. Both forms were seen in the larval stage, but there is a little difference in the time of their appearance. Eupyrene cysts were seen from the 8(th) larval stage, whereas apyrene were only found after the 10(th) stage. In early insect development stages, eupyrene cysts predominate, but as the insect ages, they are overtaken by apyrene. Although some eupyrene cysts are still present in young adult testis, the majority are apyrene. As eupyrene sperm is formed, bundles migrate to the seminal vesicle therefore in early pupae eupyrene sperm are already present there whereas apyrene cells arrive later. The exact mechanism and determining factors responsible for apyrene sperm origin are still to be clarified. The probable causes of apyrene sperm appearance are discussed as well as its role in the sperm competition.

Reproductive biology of Abolboda pulchella and A. poarchon (Xyridaceae: Poales)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Histochemical and ultrastructural cytochemistry of glycogen on ovarioles of Neoponera villosa ants (Hymenoptera: Ponerinae)

In Neoponera villosa ants, we found ovaries of the polytrophic meroistic type which is characterized by the presence of nurse cells forming together with the oocyte, the so-called follicles. The nurse cells have the primary function of supplying the oocyte with RNA, but they contribute to the supply of other elements such as glycogen. With the objetive of detecting the presence of this substance in the ovarioles of workers and queens of N.villosa ante the ovaries were removed and processed according to electron microscopy technic for glycogen detection. Glycogen is a common element in insect oocytes and is abundantly distributed in the cytoplasm of N.villosa workers and queens. However, in ovarian follicles it can only be detected at stages ET and lit of development. Glycogen synthesis probably occurs predominantly in nurse cells which transfer it into the oocyte through the nourish pore. This process requires high energy expenditure that justify the large numbers of mitochondria associated with glycogen in the nurse cell cytoplasm. The amount of glycogen in the nurse cells of queens is slightly greater than workers.

A preliminary study of insect fauna on pig carcasses located in sugarcane in winter in southeastern Brazil

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

Population dynamics of Leptopharsa heveae (Hemiptera: Tingidae) and Erythmelus tingitiphagus (Hymenoptera: Mymaridae) in rubber tree plants

The tingid Leptopharsa heveae, known as the lace bug, occurs in large populations in rubber tree plantations, limiting the production of latex due to losses of photosynthetic area and weakening of the infested trees. The alternative for the use of insecticides would be the release of biological control agents, but little is known about the natural enemies of L. heveae. The parasitoid Erythmelus tingitiphagus parasitizes eggs of the lace bug in rubber tree plantations. The knowledge of the population dynamics and the peak of occurrence of economically important insect species and their natural enemies are indispensable requirements for the establishment of efficient and rational control strategies. The objective of this work was to study the population dynamics of L. heveae and E. tingitiphagus in rubber tree plantations in the county of Itiquira, MT, Brazil. Mature folioles were collected weekly from the lower stratum of the canopy of the tree clones RRIM 600, PR 255, GT 1, PB 235 and PB 217, from August/2005 to February/2006. The parasitoid was observed during the whole study period. The population peaks of the populations of the L. heveae and E. tingitiphagus in the study area occurred at the end of October and beginning of November. This result demonstrates that measures for the control of L. heveae and population increase of E. tingitiphagus must be adopted before the peak population of this pest in cultivated rubber plantations.

Histochemical and ultrastructural cytochemistry of glycogen in ovarioles of Neoponera villosa ants (hymenoptera : ponerinae)

In Neoponera villosa ants, we found ovaries of the polytrophic meroistic type which is characterized by the presence of nurse cells forming together with the oocyte, the so-called follicles. The nurse cells have the primary function of supplying the oocyte with RNA, but they contribute to the supply of other elements such as glycogen. With the objetive of detecting the presence of this substance in the ovarioles of workers and queens of N. viillosa ants the ovaries were removed and processed according to electron microscopy technic for glycogen detection. Glycogen is a common element in insect oocytes and is abundantly distributed in the cytoplasm of N. villosa workers and queens. However, in ovarian follicles it can only be detected at stages II and III of development. Glycogen synthesis probably occurs predominantly in nurse cells which transfer it into the oocyte through the nourish pore. This process requires high energy expenditure that justify the large numbers of mitochondria associated with glycogen in the nurse cell cytoplasm. The amount of glycogen in the nurse cells of queens is slightly greater than workers.

Changes in amounts of total salivary gland proteins of Lutzomyia longipalpis (Diptera: Psychodidae) according to age and diet

Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission. © 2008 Entomological Society of America.

Anti-inflammatory activity of Lychnophora passerina, Asteraceae (Brazilian arnica)

Ethnopharmacological relevance: Lychnophora passerina (Asteraceae), popularly known as arnica, is used to treat inflammation, pain, rheumatism, contusions, bruises and insect bites in Brazilian traditional medicine. Materials and methods: The anti-inflammatory activity of crude ethanolic extract of aerial parts of L. passerina and its ethyl acetate and methanolic fractions had their abilities to modulate the production of NO, TNF-α and IL-10 inflammatory mediators in LPS/IFN-γ-stimulated J774.A1 macrophages evaluated. Moreover, the crude ethanolic extract and derived fractions were also in vivo assayed by carrageenan-induced paw oedema in mice. Results: In vitro assays showed remarkable anti-inflammatory activity of L. passerina crude ethanolic extract (EE) and its ethyl acetate (A) and methanolic (M) fractions, through the inhibition of production of NO and TNF-α inflammatory mediators and induction of production of IL-10 anti-inflammatory cytokine. In vivo assays showed anti-inflammatory activity for EE 10% ointment, similar to the standard drug diclofenac gel. The A and M fraction ointments 20% presented anti-inflammatory activity. Conclusion: The results obtained showed that possible anti-inflammatory effects of EE and its A and M fractions may be attributed to inhibition pro-inflammatory cytokines production, TNF-α and NO and to increased IL-10 production. EE, A and M ointments showed topical in vivo anti-inflammatory activity. The in vivo anti-inflammatory activity of EE of L. passerina may be related to synergistic effects of different substances in the crude extract. Therefore, traditional use of aerial parts of L. passerina in the inflammatory conditions could be beneficial to treat topical inflammatory conditions, as evidenced by the present study. © 2011 Elsevier B.V. All rights reserved.

Responses of aquatic insect functional diversity to landscape changes in Atlantic forest

The Atlantic Forest domain, one of the 25 world's hotspots for biodiversity, has experienced dramatic changes in its landscape. While the loss of species diversity is well documented, functional diversity has not received the same amount of attention. In this study, we evaluated functional diversity of insects in streams utilizing three indices: functional diversity (FD), functional dispersion (FDis), and functional divergence (FDiv), seeking to understand the roles of three predictor sets in explaining functional patterns: (1) bioclimatic and landscape variables; (2) spatial variables; and (3) local environmental variables. We determined the amount of variation in different measures of functional diversity that was explained by each predictor set and their interplays using variation partitioning. Our study showed that variation in functional diversity is better explained by a set of variables linked to different scales dependent on spatial structures, indicating the importance of landscape and mainly environmental variables in the functional organization of aquatic insect communities, and that the relative importance of predictor sets depends on the indices considered. Variation in FD was better explained by the interplay among the three predictor sets and by local environmental variables, whereas variation in FDis was better explained by spatial variables and by the interplay between environmental and spatial variables. Variation in FDiv was not significantly explained by any predictors. Our study adds more evidence on the harmful effects caused by landscape changes on biodiversity in the Atlantic Forest, suggesting that these effects also influence the functional organization of stream insect communities. © 2013 The Author(s) Journal compilation © 2013 by The Association for Tropical Biology and Conservation.