The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

A preliminary investigation of pre-dispersal seed predation by Acanthoscelides schrankiae horn (Coleoptera: Bruchidae) in Mimosa bimucronata (DC.) Kuntze trees

This is the first record of Acanthoscelides schrankiae Horn, feeding in seeds of Mimosa bimucronata (DC.) Kuntze. We investigated the pattern of oviposition and seed exploitation by A. schrankiae, and the distribution of mature fruits and seed predation in the inflorescences. We also compared the percentage of predated seeds, the total dry weight of fruits and non-predated seeds, the percentage of aborted seeds, and the percentage of non-emergent insects, among different quadrants of the M. bimucronata canopy. To determine the occurring species, the emergence of bruchids and parasitoids was observed in the laboratory, resulting altogether, only in individuals of A. schrankiae and Horismenus sp. (Hymenoptera: Eulophidae) species, respectively. Mean number of fruits produced in the median region of inflorescence was significantly higher than in the inferior and superior regions, and the frequencies (observed and expected) of predated and non-predated seeds differed among the different regions of inflorescence. Females of A. schrankiae laid their eggs on fruits, and larvae, after emergence, perforated the exocarp to reach the seeds. Most fruits presented one to three eggs and only one bruchid larva was observed in each seed. The highest value of the rate number of eggs/fruit and the highest percentage of predated seeds were recorded in April. Dry weight of fruits (total) and seeds (non-predated), proportions of predated seeds, seed abortions, and non-emergent seed predators, were evenly distributed in the canopy.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin. Copyright ©2007 John Wiley & Sons, Ltd.

Mitochondrial genome sequence of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae): Mitochondrial genes utility to investigate the evolutionary history of Coleoptera and its bioluminescence

The Coleoptera order is the richest group among Metazoa, but its phylogenetics remains incompletely understood. Among Coleoptera, bioluminescence is found within the Elateroidea, but the evolution of this character remains a mystery. Mitochondrial DNA has been used extensively to reconstruct phylogenetic relationships, however, the evolution of a single gene does not always correspond to the species evolutionary history and the molecular marker choice is a key step in this type of analysis. To create a solid basis to better understand the evolutionary history of Coleoptera and its bioluminescence, we sequenced and comparatively analyzed the mitochondrial genome of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae). © 2007 Elsevier B.V. All rights reserved.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors. © 2007 The Authors.

The structural origin and biological function of pH-sensitivity in firefly luciferases

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered. © The Royal Society of Chemistry and Owner Societies.

Pyrearinus termitilluminans larval click beetle luciferase: Active site properties, structure and function relationships and comparison with other beetle luciferases

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, KM values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging. © The Royal Society of Chemistry and Owner Societies 2009.

Controle de Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) em instalações para frangos de corte

Searching for a new alternative to A. diaperinus control, it was evaluated the action of Spinosad in two concentrations (250ppm, 400ppm) and two doses (0.05 L/m 2 and 0.1 L/m 2), applied in poultry broiler facilities naturally infested by this coleoptera. Assessments of the infestation were held in weekly intervals, during 49 days after treatment, using traps. The percentage of effectiveness were calculated from the results of the number of adults and/or larval stages in control and treated groups. Spinosad at the concentration of 250ppm, applied at a dose of 0.1L/m2, can be considered ineffective against these beetles, however the application of 400ppm at a dose of 0.1L/m2 showed high efficacy and short residual period. The dose of 0.1L/m 22 of Spinosad at the concentration of 400ppm demonstrated, between treatments, better effectiveness against coleopters, reaching efficacy of 100% against larvae of A. diaperinus, observed after the seventh day post-treatment.

Predatory behavior of Canthon virens (Coleoptera: Scarabaeidae): A predator of leafcutter ants

We present a detailed description of the predatory behavior of the beetle Canthon virens Mannerheim, 1829, on the leafcutter ant Atta sp. We observed 51 acts of predation, which were also recorded on film and subjected to behavioral analysis. Canthon virens exhibited 28 behaviors while predating upon Atta sp. queens. Adult beetles search for queens while flying in a zigzag pattern, 15 to 20cm above the ground. After catching a queen, the predator stands on its back and starts cutting the queen cervix. Once the prey is decapitated, the predator rolls it until an insurmountable obstacle is reached. The distance from the site of predation to the obstacle can vary widely and is unpredictable. The beetle rolling the queen also buries it in a very peculiar way: first, it digs a small hole and pulls the queen inside, while another beetle is attached to the prey. The burial process takes many hours (up to 12) and may depend on the hardness of the soil and the presence of obstacles. In general, one or two beetles are found in a chamber with the queen after it is buried. They make the brood balls, which serve as food for the offspring. This study contributes to the knowledge of the predatory behavior of Canthon virens, a predator poorly studied in Brazil and widespread in the country. Copyright © 2012 Luiz Carlos Forti et al.

Nitidulidae (Coleoptera) Associados A Frutos de Café (Coffea Arabica L.)

This study reports sap beetles from fruits of a coffee crop in Cravinhos, SP, Brazil. Fruits were collected directly from plants and, in laboratory, from the fruits at the cherry state we obtained 20 adults of three species: Carpophilus nepos Murray, 1864, Colopterus niger Murray, 1864 and Nitops sordidus Erichson, 1847. This is the first report of association between these insects and coffee fruits.

Seleção de isolados de beauveria bassiana (Bals.) Vuill. para o controle de sitophilus zeamais (Mots.) (Coleoptera: Curculionidae)

Sitophilus zeamais (Mots.) (Coleoptera: Curculionidae) is considered a major pest of maize, responsible for reducing grain quality and making the corn inappropriate for industrial use and human consumption. S. zeamais has been controlled exclusively with chemical products. The objective of this research was to select isolates of Beauveria bassiana (Bals.) Vuill. to control S. zeamais. Beetles were immersed in conidia suspensions of each isolate for five seconds and placed in a gerbox container with maize grains. In pathogenicity tests, the isolates that caused the highest mortality to the maize weevil were ESALQ-447 (68.0%), CCA-UFES/ Bb-36 (57.3%) and CCA-UFES/Bb-31 (51.3%). ESALQ-447 was the most virulent, with an LC50 of 1.7 × 107 conidia/ml and shows promise for controlling maize weevils. These isolates of B. bassiana can be used as effective substitutes for conventional chemical control, normally carried out with phosphine. Further tests should be performed under field and semi-field conditions to develop an appropriate strategy for the use of this entomopathogen to manage S. zeamais.

Selective defaunation affects dung beetle communities in continuous Atlantic rainforest

Overhunting has caused severe decline or local extinction in many large-bodied mammals with direct consequences on plant regeneration, yet little is known about indirect impacts of selective defaunation on commensal species. Cascading effects of species extinction across dependent species groups are likely to occur in coprophagous beetles, because these invertebrates rely on mammal dung for food and nesting material. Both mammals and dung beetles provide important ecosystem services and cascading effects are likely to lead to rapid functional losses. In this study, we described changes in dung beetle communities across a gradient of selective defaunation in continuous Brazilian Atlantic rain forest. We compared the dung beetle assemblages in seven sites with different mammalian biomass and composition. The reduction in the mammalian biomass had a major effect on dung beetle communities by (1) increasing dung beetle abundance with decreasing overall mammal, primate and large mammal biomasses, (2) decreasing dung beetle species richness with decreasing overall mammal biomass and (3) decreasing dung beetle size with decreasing large mammal biomass. Moreover, our study demonstrated the importance of the composition of mammal communities in structuring dung beetle communities. This study documented how selective changes in mammalian biomass and composition affect dung beetle species communities, which in turn may have cascading consequences for the ecosystem. Since most of tropical ecosystems are facing dramatic changes in mammalian composition, it is urgent to evaluate the functional losses associated with such co-extinctions. © 2013 Elsevier Ltd.

Diversidade e distribuição sazonal de Carabidae (Insecta, Coleoptera) em diferentes culturas

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Análise de fauna, flutuação populacional e preferência pelo hábitat de Carabidae e Staphylinidae (Coleoptera) na região de Guaíra, Estado de São Paulo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)