Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.

Respostas da lagarta‑do‑cartucho a milho geneticamente modificado expressando a toxina Cry 1A(b)

O objetivo deste trabalho foi avaliar parâmetros biológicos da lagarta-do-cartucho do milho (Spodoptera frugiperda), alimentada com híbridos de milho Bt, que expressam a toxina Cry 1A(b), e com seus respectivos isogênicos não Bt. Os experimentos foram realizados no laboratório da Embrapa Milho e Sorgo, em Sete Lagoas, MG. Os parâmetros avaliados foram: sobrevivência de larvas após 48 horas, sobrevivência da fase larval e pré-imaginal, biomassa de larvas aos 14 dias de idade, biomassa de pupas, período de desenvolvimento larval, e não preferência alimentar de larvas do primeiro ínstar. Larvas de S. frugiperda apresentam menor sobrevivência nas primeiras 48 horas de alimentação e durante toda a fase larval, na maioria dos híbridos de milho Bt, em comparação ao milho não Bt. A biomassa de larvas e pupas foi sempre menor no milho Bt, e o período larval e o pré-imaginal, maior. Houve interação entre a toxina Cry 1A(b) e a base genética dos híbridos transgênicos, quanto à sobrevivência e à biomassa larval. Larvas recém-eclodidas de S. frugiperda apresentam preferência pela alimentação em milho não Bt.

Avaliação de tecnologias de refúgio no cultivo de milho transgênico

O objetivo deste trabalho foi comparar o uso de refúgio tradicional em áreas separadas com a tecnologia alternativa de refúgio pela mistura de sementes não transgênicas no saco ("refuge in the bag" - RIB) em diferentes proporções. Foram utilizados os híbridos comerciais transgênicos AG 7000YG e DKB 390YG com refúgios plantados com as respectivas cultivares convencionais. Avaliaram-se sete tratamentos: RIB com quatro proporções de mistura de sementes não transgênicas (2,5, 5,0, 7,5 e 10%) no saco; refúgio tradicional, com 10% da área plantada exclusivamente com cultivar não transgênica; e área totalmente cultivada com plantas transgênicas ou totalmente com plantas não transgênicas. O híbrido DKB 390YG foi o mais produtivo. Para este híbrido, não foram observadas diferenças de produtividade entre os tratamentos, com exceção do controle inteiramente convencional, que produziu menos. Com o híbrido AG 7000YG, a tecnologia RIB com proporções de 5,0 e 7,5% de sementes não transgênicas apresentou as maiores produtividades, significativamente superiores às do refúgio tradicional, que não diferiu do controle convencional. Nos tratamentos RIB, as diferenças nas intensidades de dano por Spodoptera frugiperda não se refletiram em diferenças na produtividade. O refúgio no saco é alternativa viável para substituir o método de refúgio utilizado atualmente.

Caracterização do gene vip3A e toxicidade da proteína Vip3Aa50 à lagarta-do-cartucho e à lagarta-da-soja

O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por "Western blot". Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL50 de 20,3 e 79,6 ng cm-2, respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A.

Manejo de lepidópteros-praga na cultura do milho com o evento Bt piramidado Cry1A.105 e Cry2Ab2

O objetivo deste trabalho foi avaliar a eficácia do evento piramidado (MON 89034), que expressa as proteínas Cry1A.105 e Cry2Ab2, no controle dos principais lepidópteros-praga da cultura do milho no Brasil, Spodoptera frugiperda, Helicoverpa spp. e Diatraea saccharalis. Os ensaios foram conduzidos em quatro regiões do país, com o híbrido DKB 390, submetido a seis tratamentos: híbrido com o evento piramidado, híbrido com o evento que expressa apenas a proteína Cry1A(b) (MON 810) e híbrido convencional (não Bt), todos com e sem manejo integrado de S. frugiperda. Para o evento piramidado, não foi necessário o controle químico em nenhum dos locais avaliados. Diferenças significativas foram observadas entre os tratamentos quanto aos danos e à presença de lagartas. Em geral, essas variáveis foram mais baixas no híbrido com o evento piramidado e mais altas no híbrido convencional, sem controle químico. Sob alta infestação, o controle químico reduziu os danos causados por S. frugiperda e D. saccharalis, tanto no evento que expressa apenas uma proteína, como no híbrido convencional. Com base nos danos causados pelos insetos, o evento piramidado Cry1A.105 e Cry2Ab2 é eficiente no controle dos principais lepidópteros-pragas do milho no Brasil.

Identification of parasitoid species using PCR amplification and restriction enzyme digestion

Spodoptera frugiperda is a pest of great economic importance in the Americas. It is attacked by several species of parasitoids, which act as biological control agents. Parasitoids are morphologically identifiable as adults, but not as larvae. Laboratory rearing conditions are not always optimal to rear out parasitic wasps from S. frugiperda larvae collected from wild populations, and it frequently happens that parasitoids do not complete their life cycle and stop developing at the larval stage. Therefore, we explored ways to identify parasitoid larvae using molecular techniques. Sequencing is one possible technique, yet it is expensive. Here we present an alternate, cheaper way of identifying seven species of parasitoids (Cotesia marginiventris, Campoletis sonorensis, Pristomerus spinator, Chelonus insularis, Chelonus cautus, Eiphosoma vitticolle and Meteorus laphygmae) using PCR amplification of COI gene followed by a digestion with a combination of four restriction endonucleases. Each species was found to exhibit a specific pattern when the amplification product was run on an agarose gel. Identifying larvae revealed that conclusions on species composition of a population of parasitic wasps can be biased if only the emerging adults are taken into account.

Relação entre toxicidade de proteínas Vip3Aa e sua capacidade de ligação a receptores intestinais de lepidópteros-praga

Resumo:O objetivo deste trabalho foi avaliar a toxicidade de novas proteínas Vip3Aa e sua capacidade de ligação a vesículas de membrana da microvilosidade apical (VMMA) do intestino de lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalise Heliothis virescens. Proteínas expressas pelos genes vip3Aa42 e vip3Aa43 mostraram-se tóxicas a S. frugiperda (CL50 de 78,2 e 113 ng cm-2, respectivamente) e A. gemmatalis(CL50 de 239,2 e 57,5 ng cm-2, respectivamente), e pouco tóxicas a H. virescens (CL50>5.000 ng cm-2). Os ensaios de ligação às VMMA mostraram que as proteínas unem-se de forma efetiva aos receptores nas vesículas das espécies avaliadas, mas essa capacidade de ligação somente é efetiva na ativação da toxicidade para as populações avaliadas de S. frugiperdae A. gemmatalis.

Determinação de nitrato e nitrito em chás de peles de pescados empregados para tratamento de asma brônquica na região sul do Rio Grande do Sul

The objective of this paper was to determine nitrate and nitrite anions in toasted fish skin tea, which the population of southern Rio Grande do Sul believes to have pharmacological properties for trating asthma. Two methods were compared, an official and one described by Guozhen et al., that has the advantage of not needing reduction of nitrate to nitrite on a Cd column. The samples were the skin of the fish species Balistes capriscus (peixe porco) and Menticirrhus littoralis (papa-terra), both crude and toasted, as traditionally used. The method of Guozhen et al. was used due to its performance (detection limit 0.005 µmol/L NO3- and 0.02 µmol/L - NO2- and mean recuperation of 100 and 97% respectively for each anion) and results similar to those of the official methods under conditions used by the population, the estimated daily intake, of the teas is equivalent to 0.03 to 0.05 mg of nitrate and 0.007 to 0.014 mg of nitrite. These values are in accord with safety recommendations and don't represent a health risk.

Constituintes químicos e atividade inseticida dos extratos de frutos de Trichilia elegans E T. catigua (Meliaceae)

Phytochemical investigation of the fruits extracts of Trichilia elegans and Trichilia catigua (Meliaceae) has led to the identification of the limonoids 11β-acetoxyobacunone, cedrelone, methylangolensate and epimeric mixture of photogedunin besides known coumarins (scoparone, scopoletin, umbeliferone) and the steroids stigmasterol, β-sitosterol, sitostenone and campesterol. The structures of the compounds were proposed by spectroscopic analysis and comparison with literature data. An evaluation of the insecticidal activity of the fruits extracts of Trichilia ssp. was carried out and the extracts of T. elegans revealed to have strong insecticidal activity and the extracts of T. catigua showed moderate larval mortality on Spodoptera frugiperda.

Corn yield response to weed and fall armyworm controls

The interference imposed the by weeds on corn decreases practically all vegetative characteristics. As consequence, the green ear and grain yield are also reduced. Losses due to the fall armyworm (Spodoptera frugiperda) attack can reduce corn grain yield up to 34%. In general, weed and insect control issues are addressed separately in research papers. Nevertheless, interaction between weeds and insects may exist. This study aimed to evaluate green ear and corn grain yield response to weed and fall armyworm control. A completely randomized block design with split-plots and five replicates was adopted. Corn cultivar AG 1051 was grown under weedy conditions or with control by hand hoeings performed at 20 and 40 days after planting. Fall armyworm control (applied to subplots) was performed with sprays of water (control), deltamethrin (5g active ingredient ha-1); neem oil, at 0.5% (diluted in water), and neem leaf extract at 5%. Each product was sprayed three times, at seven-day intervals, starting at the 7th day after planting, using 150 L ha-1 of the tank solution. Dry mass of the above-ground part, internode diameter, leaf length, leaf width, leaf area, green ear yield and grain yield of corn were reduced due to the lack of weed control. Fall armyworm control in the weeded plots did not influence green ear yield and grain yield, except green mass of marketable, husked ears, which was reduced when the caterpillar was not controlled. Without weed control, neem extracts and deltamethrin sprays provided highest yields of number and total weight of green ears with husks, number and weight of marketable ears with husks and number of marketable ears without husks. The best results for husked ear mass and for grain yield were obtained with neem extract and deltamethrin, respectively.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Diagnosi de l’estat ecològic de les poblacions de nàiades als recs i rieres de l’Estany de Banyoles: propostes de gestió per la seva conservació

Estudi sobre la situació actual de les nàiades dels recs de l’estany de Banyoles, concretament de les espècies unio elongatulus penchinatianus, potomida littoralis subreniformis i anodonta cygnea. Amb les dades recollides es fa una proposta de pla de gestió per a la conservació, protecció i repoblació d’aquestes espècies a la zona

Susceptibility of different insect pupae to the bacterial symbiont, Xenorhabdus nematophila, isolated from the entomopathogenic nematode Steinernema carpocapsae

Cells of the bacterial symbiont Xenorhabdus nematophila from the entomopathogenic nematode, Steinernema carpocapsae entered the pupae of Plutella xylostella after 15 minutes treatment with suspensions containing the bacterial cells. Secretions of Xenorhabdus nematophila, in either broth or water, were found lethal to the pupae of P. xylostella when applied in moist sand. The bacterial symbiont Xenorhabdus nematophila was found lethal to the pupae of greater wax moth (Galleria mellonella), beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella) and black vine weevil (Otiorhynchus sulcatus) in the absence of the nematode vector and the cells of X. nematophila entered the haemocoele of the pupae.

Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.

First contribution of mites (Acari) to the forensic analysis of hanged corpses: a case study from Spain

This case study from North Spain, highlights the importance of the collection of mites in addition to insects, from crime scenes or corpses subjected to environmental constraints that reduce or minimise insect activity, such as hanged corpses. In addition, this analysis highlights the relevance of arthropods’ collection in the field, even after the corpse has been moved away for autopsy. Four species of mites, phoretic on carrion (Silphidae) and rove (Staphylinidae) beetles, complemented and reinforced the autopsy analysis as well as the scarce information provided by insect activity. Poecilochirus carabi Canestrini & Canestrini, 1882 and Poecilochirus (Physoparasitus) davydovae Hyatt, 1980 (Mesostigmata: Parasitidae) were found in association with two Silphidae, Nicrophorus Fabricius, 1775 and Necrodes Leach, 1815, only when sampled in the autopsy room; this is suggestive of host-switching of mites and was likely due to the lack of availability of specific carriers in the field. The interpretation of the activity of Parasitidae mites both in the field and the autopsy room allows a better understanding of the timing and circumstances of decomposition. Phoretic deutonymphs of Pelzneria Scheucher 1957 (Astigmata: Histiostomatidae) were highly abundant, mostly P. crenulata Oudemans, 1909 and are reported for the first time on a Staphylinidae rove beetle, Creophilus maxillosus (L., 1758). Surprisingly, in this case study no Pelzneria were associated with the Silphidae found, which are their most common hosts, such as Necrodes littoralis (L., 1758) and Nicrophorus interruptus (Stephens, 1830). All histiostomatids were removed from the staphylinid (rove beetle) collected from the soil, at the scene of death, suggesting a recent arrival of the beetle. The occurrence of Staphylinidae beetles and their associated mites, such as Parasitidae and Pelzneria, and the information they provided would have been easily overlooked or lost if only the autopsy sampling would have been considered in the analysis of the case. The four mite species are reported for the first time for the Iberian Peninsula.