Sphenophorus Levis Research Articles

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the pectin methylesterase from the sugar cane weevil Sphenophorus levis.

Pectin methylesterase removes the methyl groups from the main chain of pectin, the major component of the middle lamella of the plant cell wall. The enzyme is involved in plant cell-wall development, is part of the enzymatic arsenal used by microorganisms to attack plants and also has a wide range of applications in the industrial sector. Therefore, there is a considerable interest in studies of the structure and function of this enzyme. In this work, the pectin methylesterase from Sphenophorus levis was produced in Pichia pastoris and purified. Crystals belonging to the monoclinic space group C2, with unit-cell parameters a = 122.181, b = 82.213, c = 41.176 Å, β = 97.48°, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.1 Å resolution. Structure refinement and model building are in progress.

Sugarcane straw and the populations of pests and nematodes

The green cane harvesting represented a significant change in sugarcane ecosystem due to the presence of straw left on the soil and to the absence of fire. These two factors may affect the populations of pests and their natural enemies. Among the pests benefit from the green cane harvesting stand out the spittlebug, Mahanarva fimbriolata, the curculionid Sphenophorus levis and sugarcane borer, Diatraea saccharalis. In areas of green cane harvesting, the population of these species grew faster than in areas of burnt cane. On the other hand, there are virtually no records of attacks by lesser cornstalk borers in areas of green cane harvesting. Populations of plant parasitic nematodes and the beetles Migdolus fryanus, very important pests of sugarcane, were apparently not affected by the green cane harvesting. Despite the absence of more consistent information, it appears that populations of ants and the giant borer Telchin licus can increase in green cane areas, due primarily to the difficulty of pest control. The partial or total removal of straw from the field represents an additional change to the ecosystem that could alter the status of pests and nematodes. It is likely that spittlebug, the curculionid S. levis and sugarcane borer populations decrease if a portion of the straw is removed from the field. However, the pest populations in areas where the straw is collected will not return to their original conditions at the time of burnt cane harvesting because the absence of fire will be maintained.

Eficiência de nematoides entomopatogênicos e inseticidas químicos contra Sphenophorus levis e Leucothyreus sp. em cana-de-açúcar

O bicudo-da-cana-de-açúcar, Sphenophorus levis (Coleoptera: Dryophthoridae), é uma importante praga em canaviais do Estado de São Paulo. Suas larvas destroem o rizoma da planta, causando prejuízos de até 30 t ha-1 ano-1 de cana. Três experimentos de campo foram conduzidos para avaliar a eficácia de nematoides entomopatogênicos e de inseticidas químicos no controle do bicudo-da-cana-de-açúcar e do escarabeídeo Leucothyreus sp. O primeiro consistiu de 10 tratamentos: 1) tiametoxam (Actara 250WG) 500 g ha-1 de produto comercial (p.c.); 2) tiamethoxam 1,0 kg ha-1 p.c.; 3) tiametoxam 1,5 kg ha-1 p.c.; 4) Steinernema brazilense 10(8) juvenis infectivos (JI) ha-1 + tiametoxam 250 g ha-1 p.c.; 5) S. brazilense (10(8) JI ha-1) + tiametoxam 500 g ha-1 p.c.; 6) Heterorhabditis indica (10(8) JI ha-1) + tiametoxam 250 g ha-1 p.c.; 7) H. indica (10(8) JI ha-1) + tiametoxam 500 g ha-1 p.c.; 8) S. brazilense (10(8) JI ha-1); 9) H. indica (10(8) JI ha-1); e 10) Testemunha. No segundo, foram considerados cinco tratamentos: 1) tiametoxam (Actara 250WG) 800 g ha-1 p.c.; 2) fipronil (Regente 800WG) 250 g ha-1 p.c.; 3) S. brazilense (10(8) JI ha-1); 4) S. brazilense (10(8) JI ha-1) + tiametoxam (Actara 250WG) 200 g ha-1 p.c.; e 5) Testemunha. O terceiro incluiu todos os tratamentos do segundo experimento, mais a mistura S. brazilense (10(8) JI ha-1) + fipronil (Regente 800WG) 62,5 g ha-1 p.c. Todos os tratamentos com inseticidas e nematoides proporcionaram maior produção de cana, quando comparados às respectivas testemunhas, que foram mais danificadas por S. levis, mas sem diferenças significativas. O nematoide S. brazilense foi mais eficaz no controle deste inseto, quando comparado com H. indica, mas sem diferenças significativas. S. brazilense proporcionou 50% de controle das larvas de Leucothyreus sp., e os melhores tratamentos para controle deste inseto foram as misturas do nematoide com fipronil (78%) e tiametoxam (83%).

Caracterização de novos isolados de Bacillus thuringiensis para o controle de importantes insetos-praga da agricultura

A bactéria Bacillus thuringiensis Berliner produz um corpo de inclusão paraesporal (cristal) de natureza proteica, formado durante a esporulação, que atua de forma eficiente no controle de insetos-praga de culturas economicamente importantes. Esse cristal é constituído de proteínas Cry, que são codificadas pelos genes cry; um isolado pode ser caracterizado pelo conteúdo de genes cry que apresenta. Visando caracterizar novos isolados no combate de insetos-praga pertencentes às ordens Lepidoptera e Coleoptera, 76 isolados bacterianos foram analisados molecularmente e tiveram seu potencial de controle avaliado por meio de bioensaios com larvas de Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie e Tenebrio molitor Linnaeus. As análises moleculares indicaram 11 isolados (14,5% da coleção), contendo genes lepidóptero-específicos e 17 (22,37%) com genes coleóptero-específicos. As análises de patogenicidade revelaram dois isolados com alto potencial de controle para lagartas de S. frugiperda, um para larvas de S. levis e seis prejudiciais ao desenvolvimento das larvas de T. molitor. Esses isolados de B. thuringiensis podem ser promissores no controle biológico das referidas pragas.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase ( Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 °C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and r Sl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC ( k cat /K m = 37.53 mM S −1), which is similar to other insect cathepsin L-like enzymes. r Sl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. r Sl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 ( K i = 0.196 nM), indicating that this protease is a potential target for pest control.

Leviserpin: A Serine Peptidase Inhibitor (Serpin) from the Sugarcane Weevil Sphenophorus levis

Serine peptidase inhibitors (serpins) form a superfamily of proteins covering abroad spectrum of different biological functions. Here we describe the inhibitory characterization of leviserpin, the first serpin from the sugar cane weevil Sphenophorus levis. Leviserpin was able to inhibit bovine trypsin by the formation of the covalent complex serpin-peptidase, demonstrated by SDS-PAGE and mass spectroscopy analysis. We also have determined the cleavage site at the reactive center loop, by the analysis of the polypeptides released from de C-terminus of leviserpin. Moreover we investigated the mRNA expression of leviserpin in different stages of S. levis development. Thus the specificity of leviserpin, in addition with its mRNA coding being transcribed through all lifecycle of the insect, can suggest a possible role in defense mechanism by regulating the action of prophenoloxidase (proPO) activating enzyme.

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with kcat/Km values of 20.0±1.1μM−1s−1 and 30.0±0.5μM−1s−1, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.

Microbial diversity in the larval gut of field and laboratory populations of the sugarcane weevil Sphenophorus levis (Coleoptera, Curculionidae)

The sugarcane weevil, Sphenophorus levis, is a wide-spread sugarcane pest in Brazil. Sphenophorus levis may depend on microorganisms that inhabit its intestinal tract. We examined the diversity of the gut microbiota of S. levis, which was characterized using culture-dependent and culture-independent methods. Analysis of 16S rRNA amplified directly from the gut community revealed the presence of 14 genera, one group from the Candidatus category, one uncultured group assigned to the family Flavobacteriaceae, and one uncultured group assigned to the family Enterobacteriaceae; all of them are members of the Alpha-Proteobacteria, Beta-Proteobacteria, Gamma-Proteobacteria, Firmicutes, and Bacteroidetes phyla. Microorganisms isolated through culture-dependent methods were classified according to morphological parameters and by 16S rRNA gene sequences. In addition to bacteria, four filamentous fungi were isolated. A higher bacterial diversity was observed in field populations of larvae than in laboratory populations, according to the Shannon index (Field H' = 3.36; Laboratory H' = 3.26). Five genera of bacteria and two filamentous fungi were found to have cellulolytic activity. This is the first report of S. levis gut microbiota; it may contribute to development of strategies for controlling this sugarcane pest.

Virulência de nematóides entomopatogênicos (Nematoda: Rhabditida) a Sphenophorus levis (Coleoptera: Curculionidae)

O bicudo da cana-de-açúcar, Sphenophorus levis (Coleoptera: Curculionidae), é uma importante praga de solo dos canaviais no Estado de São Paulo, ocasionando prejuízos de até 30 t de cana/ha/ano. Visando ao controle biológico desta praga, esse estudo teve por objetivo avaliar a virulência de 17 isolados de nematóides entomopatogênicos do gênero Steinernema e Heterorhabditis contra adultos de S. levis, e o efeito dos quatro nematóides mais virulentos em três dosagens cada um. A pesquisa foi realizada em condições de laboratório, sendo realizados quatro ensaios para o estudo de virulência. Os nematóides selecionados como mais virulentos em cada teste foram S. brazilense IBCB n6, Heterorhabditis sp. IBCB n10, Heterorhabditis sp. IBCB n24 e Heterorhabditis sp. IBCB n44, sendo os únicos que diferenciaram significativamente das respectivas testemunhas com 45%, 40%, 20% e 31% de mortalidade do inseto na concentração de 240 juvenis infectivos (JI)/inseto. No teste de dose, os isolados de Heterorhabditis identificados como IBCB n10, IBCB n24 e IBCB n44 destacaram-se quanto à virulência para os adultos do bicudo da cana-de-açúcar, diferindo significativamente da testemunha na maior dose avaliada (1200 JI/inseto) com níveis de mortalidade do inseto de 60%, 65,7% e 74,3% respectivamente.

Atração de adultos de Sphenophorus levis Vaurie (Coleoptera: Curculionidae) a fragmentos vegetais em diferentes estados de conservação

The occurrence of the sugarcane weevil Sphenophorus levis Vaurie is important in sugarcane in some regions in Brazil. Damage is caused by the larvae as they bore into the nodes and can reach 30 ton/ha/year. Many control alternatives have been attempted, but none were satisfactory, except for the use of toxic baits. Therefore, it is necessary to optimize their efficiency or to propose new techniques. The objective of the current study was to evaluate the attractiveness of adults of S. levis to sugarcane nodes and pineapple peelings in an 'Y' tube olfactometer. The sugarcane internodes were treated with 10% molasses, and tested after different periods of fermentation (24, 48 e 72h), at different times of the day (diurnal and nocturnal) and with both sexes. These tests were carried out in order to correlate the response of S. levis to ethyl acetate and ethanol release as a result of the fermentation process. The release of both compounds was monitored by gas chromatography-mass spectrometry (GC-MS). Our data indicated that sugarcane internodes mixed with 10% molasses fermented for 24h and 48h were the most attractive to S. levis (up to 90%). Pineapple peelings attracted 62.5% of the tested insects. The olfactory response was higher during the day, and no differences were found between the sexes. The production of ethanol in all plant substrates was higher than ethyl acetate, but we could not establish a clear correlation with the insect response to baits.

Identificação de genes cry de Bacillus thuringiensis no controle de Sphenophorus levis, o bicudo da cana-de-açúcar

A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis, por meio da caracterização morfológica e molecular, identificando as diferentes subclasses dos genes cry3 e cry35 e determinar a patogenicidade contra Sphenophorus levis, uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1163 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis. O material genético foi purificado pela matriz de troca iônica "Instagene Matrix" e submetido a PCR com iniciadores gerais cry3 e cry35 identificando-se 30 isolados contendo genes com potencial para o controle de coleópteros, os quais juntamente com as linhagens-padrão de B. thuringiensis var. tenebrionis, B. thuringiensis var. morrissone e B. thuringiensis var. tolworthi foram utilizados para a realização do bioensaio. Através de análise discriminante alocaram-se os isolados em quatro grupos quanto à toxicidade de B. thuringiensis. Os grupos ficaram assim definidos: um grupo que promovem até 10% de mortalidade contendo as testemunhas e duas linhagens;um grupo que causou 39% de mortalidade contendo três linhagens padrão e dez isolados; um grupo com 52% de mortalidade contendo treze isolados e um grupo com 70% de mortalidade contendo cinco isolados, os quais devem ser considerados promissores no controle biológico de S. levis.

Production of a His‐tagged canecystatin in transgenic sugarcane and subsequent purification

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.

Synthesis of (S)-(+)-2-methyl-4-octanol: male-specific compound released by sugarcane weevil Sphenophorus levis (coleoptera: curculionidae)

(S)-2-Methyl-4-octanol (1) is a male-specific compound released by the sugarcane weevil Sphenophorus levis, being its biological activity associated with the aggregation behaviour of the species. Continuing our studies on the chemical ecology of this species, this work describes an expeditious synthesis of alcohol (1), starting from the easily available D-mannitol (2), in a route that employ the known (R)-glyceraldehyde acetonide (4) as key intermediate. GC analysis of an acetyl derivative of the final product, using a chiral stationary phase column, revealed an enantiomeric excess of 99.5%. Compound (1) has been identified as component of the male-produced aggregation pheromone in several curculionid species.

Identification of male-specific chiral compound from the sugarcane weevil Sphenophorus levis.

Comparative gas chromatographic analyses of airborne volatiles produced by males and females of the sugarcane weevil Sphenophorus levis, showed one male-specific compound. Gas chromatography-mass spectrometry data indicated an aliphatic alcohol that was identified as 2-methyl-4-octanol. Both optical isomers were synthesized in five steps by employing commercially available (R)- and (S)-2.2-dimethyl-1,3-dioxolane-4-methanol as starting material. Enantiomeric resolution by gas chromatography with a chiral column demonstrated that the natural alcohol possessed the S configuration. Preliminary indoor observations suggested that the alcohol elicited aggregation behavior among adults. The same compound has been previously described as an aggregation pheromone in several other curculionid species.