Establishment of Sinoxylon anale Lesne (Coleoptera, Bostrichidae) in Brazil: Identification, Host Plants, Distribution, and Damage.

Damage from Sinoxylon anale Lesne, a woodboring beetle not previously known to be established in Brazil, was observed in young jabuticaba trees (Plinia cauliflora, Myrtaceae) in a nursery in the municipality of Laranjal Paulista, state of São Paulo. We immediately advised MAPA ("Ministério da Agricultura, Pecuária e Abastecimento") and collected samples from the nursery and from different hosts in nearby areas, to identify the specimens and investigate the dynamics of the infestation in the jabuticaba trees. Sinoxylon anale was also collected in ethanol-baited and ultraviolet-light traps and in dry branches of the native species pau-jacaré (Piptadenia gonoacantha, Fabaceae) and inga (Inga vera, Fabaceae), and the exotic pau-d'água (Dracaena fragrans, Asparagaceae) in the municipality of Piracicaba, state of São Paulo. These collections established that S. anale larvae and adults develop in dead branches of four new host plants. Taxonomic studies using morphological parameters and DNA barcoding confirmed the identification of S. anale. An illustrated key to the three Sinoxylon species now recorded in Brazil is provided, and the COI gene sequences have been made available in a public database. Sinoxylon anale probably attacked the young jabuticaba trees after they were killed by larvae of long-horned beetles (Cerambycidae). So far, S. anale has been found established only in two locations in the same area of the state of São Paulo.

Nitrogen fixation in the stag beetle, Ceruchus piceus (Coleoptera: Lucanidae): could insects contribute more to ecosystem nitrogen budgets than previously thought?

Nitrogen (N) is a key nutrient required by all living organisms for growth and development, but is a limiting resource for many organisms. Organisms that feed on material with low N content, such as wood, might be particularly prone to N limitation. In this study, we investigated the degree to which the xylophagous larvae of the stag beetle Ceruchus piceus (Weber) use associations with N-fixing bacteria to acquire N. We paired acetylene reduction assays by cavity ring-down absorption spectroscopy (ARACAS) with 15N2 incubations to characterize rates of N fixation within C. piceus. Not only did we detect significant N fixation activity within C. piceus larvae, but we calculated a rate that was substantially higher than most previous reports for N fixation in insects. While taking these measurements, we discovered that N fixation within C. piceus can decline rapidly in a lab setting. Consequently, our results demonstrate that previous studies, which commonly keep insects in the lab for long periods of time prior to and during measurement, may have systematically under-reported rates of N fixation in insects. This suggests that within-insect N fixation may contribute more to insect nutrition and ecosystem-scale N budgets than previously thought.

DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues.

DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae.

The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. Cry7Ab and Cry1Ba were the most effective in controlling X. arvicola larvae due to the significant reduction in larvae survival (32.9 and 25.9 days, respectively), and by causing serious alterations in the larvae during the remaining months of their development. The developmental stage of the prepupal and pupal stages was not affected by the previous ingestion of Cry proteins. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, applying them only at the moment when the larvae hatch from the egg outside the grapevine wood (this would only be useful and justified if the economic threshold is exceeded) in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.

Influence of the amount of food resources in the foraging behavior of Nasutitermes corniger (Motschulsky) / Influência da quantidade de madeira no comportamento de forrageamento de Nasutitermes corniger (Motschulsky)

Food scarcity or abundance are factors regulating termites' foraging behavior in general. The aim of the present study is to evaluate the influence of four amounts of Eucalyptus grandis on foraging behavior events shown by worker and soldier of Nasutitermes corniger during laboratory tests. The tests were carried out with adult and active N. corniger colonies found in nests collected in the field, which were stored in glass cubes connected to the test arenas. Four different amounts of wood blocks were used in the tests and each amount concerned a treatment and defined a different experimental group: 1, 2, 3 and 4 blocks/arenas, with 5 repetitions. Each test lasted 60 minutes and consisted in observing, or not, the occurrence of behavioral events shown by foragers when they had contact with the treatment. The duration of each event was recorded, whenever it was observed. The number of recruited foragers and the number of workers consuming the blocks were recorded at the end of each test applied to each treatment. Nasutitermes corniger presented the three behavioral events in all treatments; however, there was not significant difference between treatments in the occurrence of the two first events, in the time taken from test start to the occurrence of a new event, in the number of recruited termites and in the number of gnawing workers. Only workers' mass recruiting was influenced by the amount of wood available. The occurrence of this event was significantly higher in treatments with greater amounts of wood. Thus, N. corniger adjusts its mass recruitment behavior in response to available food amount, which should be considered when developing baiting system for its control. (AU)

A escassez ou abundância de alimento são fatores que regulam o forrageamento do térmitas em geral. No presente estudo, avaliou-se a influência de quatro quantidades de madeira de Eucalyptus grandis em eventos comportamentais do forrageamento exibidos por operárias e soldados de Nasutitermes corniger em testes de laboratório. Os testes foram realizados com colônias maduras e ativas de N. cornigerpresentes em ninhos coletados a campo, os quais foram acondicionados em cubas de vidro conectadas a arenas testes. Blocos de madeira de mesma dimensão foram ofertados aos térmitas em quatro quantidades: 1, 2, 3 e 4 blocos/arena, com 5 repetições. Cada teste durou 60 minutos e consistiu na observação ou não da ocorrência de eventos comportamentais exibidos pelos forrageadores quando foi dado acesso a cada tratamento. Quando cada evento foi observado, o tempo de sua duração foi registrado. Ao final de cada teste registrou-se, em cada tratamento, o número de térmitas forrageadores recrutados e o número de operários consumindo os blocos. Observou-se que N. corniger exibiu os três eventos comportamentais em todos os tratamentos. Todavia, não houve diferença significativa entre os tratamentos com relação à ocorrência dos dois primeiros eventos, aos tempos transcorridos desde o início do teste até a ocorrência de cada evento, aos números de térmitas recrutados e ao número de operários em roedura. Apenas a ocorrência de recrutamento em massa dos operários foi influenciada pela quantidade de madeira ofertada, sendo que foi significativamente superior quando se ofertou os blocos de madeira na maior quantidade. Dessa forma, N. corniger ajusta seu comportamento de recrutamento em massa em resposta a quantidade de alimento disponível, o que deve ser considerado ao se desenvolver um sistema de isca para seu controle. (AU)

Molecular Identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) From Frass by Loop-Mediated Isothermal Amplification.

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.

Durabilidad natural de la madera de Ochroma pyramidale Urb. en el municipio de Atrato, Colombia / Natural durability of the wood of Ochroma pyramidale Urb. in the municipality of Atrato, Colombia / Durabilidade natural da madeira de Ochroma pyramidale Urb. no município de Atrato, Colômbia

RESUMEN Se evaluó la durabilidad natural de la madera de O, pyramidale en el municipio de Atrato, Chocó. En cada uno de los ambientes: bajo dosel de árboles BDA, libre exposición LE y bajo sombra de plástico negro BS, se empleó un diseño de bloques completamente al azar conformado por tres tratamientos y tres repeticiones de 30 probetas cada uno. Los resultados del análisis de varianza sugieren que no existe diferencia significativa para el contenido de humedad y la pérdida de masa. Se experimentó un aumento en el contenido de humedad de 119,32% y 90,63% de las probetas ubicadas a LE y BS, respectivamente. Basados en estas características, la madera de O. pyramidale se ubica en la categoría poco resistente, por lo que se puede inferir que, no es apta para construcciones que queden en contacto directo con el suelo, donde es afectada por diversos agentes de biodeterioro.

ABSTRACT The natural durability of O, pyramidalewood was evaluated in the municipality of Atrato, Chocó. In each of the environmental conditions: under canopy of UCT trees, free exposure FE and under black plastic shade UBP a completely random block design was used, consisting of three treatments and three repetitions of 30 wood samples each. The results of the analysis of variance suggest that there is no significant difference for moisture content and mass loss. An increase in moisture content of 119.32% and 90.63% of the wood specimens located at LE and BS, respectively, was observed. Based on these characteristics, O. pyramidalewood is located in the low resistance category, so it can be inferred that it is not suitable for constructions in direct contact with the ground, where it is affected by various biodeteriorating agents.

RESUMO A durabilidade natural da madeira O. pyramidalefoi avaliada no munic'pio de Atrato, Chocó. Em cada um dos ambientes: sob uma copa de árvores BDA, exposição livre LE e sob uma sombra de plástico preto BS, foi utilizado um desenho de bloco completamente aleatório, consistindo em três tratamentos e três repetições de 30 espécimes cada um. Os resultados da análise de variância sugerem que não há diferença significativa para o conteúdo de umidade e perda de massa. Um aumento no teor de umidade de 119,32% e 90,63% foi experimentado por peças de teste localizadas no LE e na BS, respectivamente. Com base nestas caracter'sticas, a madeira O. pyramidaleestá na categoria de baixa resistência, pelo que se pode inferir que não é adequada para construções que estejam em contacto directo com o solo, onde é afectada por vários agentes biodeteriorantes.

Occurrence of Scolytinae in mangrove with impact trap and in wood of five forest species / Ocorrência de Scolytinae em manguezal com armadilha de impacto e na madeira de cinco espécies florestais

Scolytinae is a beetle group belonging to Order Coleoptera; these insects play an essential role in wood degradation in forest ecosystems, since they build galleries that enable substrate colonization by other saprophytic organisms, as well as nutrient cycling. Thus, the aim of the current study was to evaluate the occurrence of Scolytinae in the wood of 5 tree species exposed to mangrove environment, as well as to simultaneously survey Scolytinae specimens captured in ethanol-baited impact traps placed in the same environment. The study was carried out in a mangrove area located in Santa Cruz neighborhood - RJ. Five freshly-harvested Clitoria fairchildiana, Rhyzophora mangle, Corymbia citriodora, Melia azedarach and Eucalyptus pellita wood logs (1 m long and 5-10 cm diameter) were arranged perpendicular to the ground (1 m above it) and spaced 30 cm away from each other. Five impact traps were set up 50 m away from each other, 1.3 m above the ground. Insects were collected for 5 months. One hundred and thirty (130) Scolytinae individuals (14 species in 2 genera) were recorded in the wood logs; the relative frequency (Fr) of the species comprised Xyleborus affinis (33.9%) and Hypothenemus sp.4 (17.7%), which represented 51.6% of the total number of captured individuals. Hypothenemus sp.6 specimens were not collected in ethanol-baited traps, but the wood of C. fairchildiana. E. pellita did not show insect infestation. The traps captured 798 individuals (24 species belonging to 8 genera); the frequency of X. affinis (25.3%) and Hypothenemus eruditus (14.5%) represented 39.8% of the total number of captured insects.

Scolytinae é um grupo da Ordem Coleoptera que tem função importante na degradação da madeira em ecossistemas florestais, através da construção de galerias, facilitam a colonização por outros organismos saprófitas neste substrato, auxiliando a ciclagem de nutrientes. Nesse contexto, este trabalho teve como objetivo avaliar à ocorrência de Scolytinae na madeira de cinco espécies arbóreas expostas em ambiente de mangue, bem como realizar simultaneamente, o levantamento dos escolitíneos capturados por armadilha de impacto iscada com etanol no mesmo ambiente. O estudo foi realizado numa área de manguezal, no Bairro de Santa Cruz-RJ. Cinco toras de madeira recém abatidas de Clitoria fairchildiana, Rhyzophora mangle, Corymbia citriodora, Melia azedarach e Eucalyptus pellita, de 1 m e diâmetro variando 5-10 cm, foram dispostas perpendicularmente ao solo a uma altura de 1 m e espaçadas de 30 cm. Cinco armadilhas de impacto foram instaladas a uma distância de 50 m entre si a uma altura de 1,3 m em relação ao solo. Os insetos foram coletados durante o período de 5 meses. Nas madeiras, registrou-se 130 indivíduos de Scolytinae (14 espécies em 2 gêneros) cuja frequência relativa (Fr) das espécies foi: Xyleborus affinis Eichhoff (33,9%) e Hypothenemus sp.4 (17,7%) representando 51,6% do total de indivíduos capturados. Hypothenemus sp.6 não foi coletada em armadilha etanólica, sendo capturada na madeira de C. fairchildiana. Em E. pellita não houve infestação. Nas armadilhas foram capturados 798 indivíduos (24 espécies em 8 gêneros), cuja a Fr de X. affinis(25,3%) e Hypothenemus eruditus Westwood(14,5%) representou 39,8%.

Identification and control of wood-deteriorating fungi / Identificação e controle de fungos deterioradores de madeira

Wood is rich in organic compounds; thus, it is susceptible to attacks by several deteriorating agents because they acknowledge such attacks as a necessary energy supply. Fungi stand out among these agents since they can attack the wood in trees (before felling) and in several post-felling stages. Fungi are biological agents that use organic compounds as food sources, and that is the reason why they degrade most chemical components found in wood. Wood attacked by fungi suffers significant value loss, since these degrading agents affect their mechanical and aesthetic properties. Fungi ­ which are classified as staining, molding and rotting ­ are one of the main responsible for the biggest losses in the timber industry. Wood can be virtually used in a whole range of environments, a fact that makes it susceptible to attacks by different fungal species, since the ideal conditions for fungal development change from species to species. In other words, the fact that one or more fungal species are capable of deteriorating wood is directly related to conditions wood is subjected to. Thus, it is essential knowing how to evaluate fungal attacks, symptoms to be taken into consideration at the time to identify the type of attacking organism and the attack stage, as well as the control and prevention measures to be applied to these organisms.(AU)

Rica em compostos orgânicos, a madeira é suscetível ao ataque de diversos agentes deterioradores por ser reconhecida como suprimento necessário de energia. Entre esses agentes, destacam-se os fungos, cujo início de ataque pode se dar ainda na árvore, antes do abate e nas diversas fases posteriores. Os fungos são agentes biológicos que necessitam de compostos orgânicos como fontes de alimento e, por essa razão, degradam grande parte dos componentes químicos da madeira. As madeiras, quando atacadas por fungos, sofrem grandes perdas de valor, afetando suas propriedades mecânicas e estéticas. Os fungos são considerados um dos principais responsáveis pelos maiores prejuízos na indústria madeireira e são classificados como manchadores, emboloradores e apodrecedores. Praticamente, a madeira pode estar em uso em uma variedade de ambientes; sabendo-se que as condições ideais de desenvolvimento de um fungo são diferentes para cada espécie, a madeira ficará suscetível ao ataque por diferentes espécies de fungos. Ou seja, o fato de uma ou mais espécies de fungos serem encontradas deteriorando a madeira está diretamente relacionado às condições em que ela se encontra. Dessa forma, torna-se importante saber como avaliar os ataques feitos por fungos, os sintomas a serem considerados para identificar o tipo de organismo e o estágio do ataque, bem como as medidas de controle e prevenção desses organismos.(AU)

First record of the genus Oodera Westwood, 1874 (Hymenoptera, Pteromalidae, Cleonyminae, Ooderini) from the Arabian Peninsula, with the description of four new species.

The genus Oodera Westwood, 1874 (Hymenoptera, Pteromalidae, Cleonyminae) is recorded for the first time for the Arabian Peninsula, from the Kingdom of Saudi Arabia and the Sultanate of Oman. The present study is based on specimens reared from xylophagous beetle larvae of the family Buprestidae (Coleoptera) infesting dead Acacia trees from Al-Dakhiliyah and Dhofar governorates in Oman and Al-Baha, Asir and Riyadh regions in Saudi Arabia. Four new species, Oodera arabica sp. nov., O. omanensis sp. nov., O. rapuzzii sp. nov., and O. similis sp. nov. are described, illustrated and compared with closely related Oodera species. An illustrated key and the xylophagous host records of the species are also provided.

Occurrence of termites (Isoptera) on living and standing dead trees in a tropical dry forest in Mexico.

Termites play a key role as ecosystem engineers in numerous ecological processes though their role in the dynamics of wood degradation in tropical dry forests, particularly at the level of the crown canopy, has been little studied. In this study, we analysed the occurrence of termites in the forest canopy by evaluating the density and proportion of living and standing dead trees associated with termites in deciduous and riparian habitats of the tropical dry forest in Chamela, Mexico. The results indicated that 60-98% of standing dead trees and 23-59% of living trees in Chamela were associated with termites. In particular, we found that the density of standing dead trees was higher in deciduous forests (0.057-0.066 trees/m2) than in riparian forests (0.022 and 0.027 trees/m2), even though the proportion of trees was not significantly different among habitats. Additionally, we found a higher density of trees associated with termites in trees of smaller size classes (0.01-0.09 trees/m2) than in larger class sizes (0-0.02 trees/m2). Interestingly, 72% of variation in the density of trees associated with termites is explained by the density of standing dead trees. Overall, these results indicate that standing dead tree availability might be the main factor regulating termite populations in Chamela forest and suggest that termites could play a key role in the decomposition of above-ground dead wood, mediating the incorporation of suspended and standing dead wood into the soil.

Heterologous expression in Pichia pastoris and characterization of a ß-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose.

A ß-glucosidase (BG), PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed in the methylotrophic yeast Pichia pastoris, purified, and biochemically characterized. Post-translational modification and N-terminal sequencing analysis demonstrated that the expression product was comprised of two polypeptides with different N-terminal sequences, presumably due to the presence of lysine-arginine (KR) sequence in the putative mature region. Substrate specificity analysis showed that PaBG1b hydrolyzed a broad range of substrates including cellohexaose, with the preference for aryl ß-d-fucosyl linkage and laminaribiose. Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM-1s-1, respectively. In addition, PaBG1b was not inhibited by cellobiose up to the highest concentration tested (100mM). Collectively, our work demonstrates that PaBG1b is a potentially valuable BG for commercial bioethanol production from cellulose.

Cellulose degrading bacteria isolated from industrial samples and the gut of native insects from Northwest of Argentina.

The raw materials used to produce bioethanol mostly are food crops, which has led to conflicts on food security. It is, therefore, recommended the gradual replacement for second generation substrates such as lignocellulosic materials. Herein, cellulolytic bacteria were isolated from the gut content of native larvae from Lepidoptera, Coleoptera, and adults of Isoptera. Few environmental samples from the pulp and paper feedstock were also assessed. A total of 233 isolates were obtained using enrichment cultures and classic criteria. Interestingly, several halo-forming colonies were found to be bacterial consortia that presented difficulties to take apart the microbial members. Those pure isolates which hydrolyzed cellulose in larger extend (45 strains) were selected and identified by means of 16S rRNA sequence analysis. Firmicutes was the prevalent phylum (62.2%) being Bacillus spp. the most frequent genus, while Paenibacillus, Brevibacillus, Cohnella, and Staphylococcus species were less frequent. The phylum Actinobacteria (6.7%) was represented by isolates related to Agromyces spp. and Microbacterium spp. Regarding Gram-negative bacteria (31.1%), the more depicted genus was Pseudomonas spp., and members of Achromobacter spp., Enterobacter spp., and Bacteroidetes phylum were also selected. These native bacterial strains are expected to enlarge the cellulolytic toolbox for efficient biomass deconstruction.

A simple and rapid method for DNA isolation from xylophagous insects.

Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts. A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described. This method was successfully applied to PCR and restriction analysis, indicating removal of common inhibitors. DNA isolated by the CTAB-PVP method could be used in most molecular analyses.