Functional analysis of nuclear receptor genes in molting and metamorphosis of the cigarette beetle, Lasioderma serricorne.

Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva-pupa-adult transitions and caused 64.44-100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67-95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.

Coleoborers in Bambusa sp. in the Southern Amazon / Coleobrocas em Bambusa sp. na Amazonia Meridional

Bamboo (Bambusa sp.) is a grass species with high potential for exploitation, however, this raw material is easily attacked by xylophagous agents such as Coleoptera. The objective of this study is thus to analyse the presence of wood-boring beetles associated with Bambusa sp. in Alta Floresta, Mato Grosso, Brazil. The study was carried out in a bamboo plantation, from August 2016 to July 2017. Two models of ethanolic traps were used, PET Santa Maria and ESALQ-84. Three families of wood-boring beetles were observed in this study: Bostrichidae, Cerambycidae and Curculionidae. 2,144 individuals were identified, belonging to 19 species, in four subfamilies and 11 tribes, and the species Xyleborus affinis was the most frequently collected. Thus, this study constitutes an important contribution to the knowledge of coleoborers associated with bamboo plantations, with the registration of the occurrence of species, as well as contributing to the knowledge of the entomofauna associated with species cultivated in the Southern Amazon.

O bambu (Bambusa sp.) é uma espécie de gramínea com alto potencial de exploração de fibras, no entanto, essa matéria-prima é facilmente atacada por agentes xilófagos, como os coleópteros. O objetivo deste estudo foi analisar a presença de besouros associados a Bambusa sp. em Alta Floresta, Mato Grosso, Brasil. O estudo foi realizado em um plantio de bambu, no período de agosto de 2016 a julho de 2017. Foram utilizados dois modelos de armadilhas etanólicas, o PET Santa Maria e ESALQ-84. Três famílias de coleobrocas foram observadas neste estudo: Bostrichidae, Cerambycidae e Curculionidae. Foram identificados 2,144 indivíduos, pertencentes a 19 espécies, em quatro subfamílias e 11 tribos, e Xyleborus affinis foi a espécie com maior número de indivíduos coletados. Assim, este estudo constitui-se em uma importante contribuição para o conhecimento de coleobrocas associadas a plantios de bamboo, com o registro de ocorrência de espécies, bem como contribui para o conhecimento da entomofauna associada a espécies cultivadas na Amazônia Meridional.

Chewing insects, pollinators, and predators on Acacia auriculiformis A. Cunn. ex Beth (Fabales: Fabaceae) plants fertilized with dehydrated sewage sludge / Insetos mastigadores, polinizadores e predadores em plantas de Acacia auriculiformis A. Cunn. ex Beth (Fabales: Fabaceae) fertilizada com lodo de esgoto desidratado

Fertilization with dehydrated sewage sludge can speed up the recovery process of degraded areas due to nutrients concentration, favoring the development of pioneer plants such as Acacia auriculiformis A. Cunn. ex Beth (Fabales: Fabaceae) and the emergence of insects. This study aimed the evaluation of chewing, pollinating insects, predators, their ecological indices and relationships on A. auriculiformis plants fertilized with dehydrated sewage sludge. The experimental design was completely randomized with two treatments (with and without dehydrated sewage sludge) and 24 repetitions. The prevalence of chewing insects Parasyphraea sp. (Coleoptera: Chrysomelidae), Nasutitermes sp. (Blattodea: Termitidae), and Tropidacris collaris (Stoll, 1813) (Orthoptera: Romaleidae), defoliation, and ecological indices of abundance of Coleoptera and Orthoptera were observed on fertilized A. auriculiformis. Acacia auriculiformis plants, with a superior number of branches/tree, revealed greater abundance of Coleoptera and Orthoptera, species richness of pollinating insects, defoliation, numbers of Parasyphraea sp. and T. collaris. The ones with larger leaves/branches displayed greater abundance of species richness of Coleoptera and Diabrotica speciosa (Germar, 1824) (Coleoptera: Chrysomelidae). Therefore, the use of A. auriculiformis plants, fertilized with dehydrated sewage sludge, is promising in the recovery of degraded areas due to the ecological indices increase of chewing and pollinators insects and spiders in the analyzed area.

A fertilização com lodo de esgoto desidratado pode acelerar o processo de recuperação de áreas degradadas devido à concentração de nutrientes, favorecendo o desenvolvimento de plantas pioneiras tais como Acacia auriculiformis A. Cunn. ex Beth (Fabales: Fabaceae) e de seus insetos. O objetivo deste trabalho foi avaliar os insetos mastigadores, polinizadores e predadores e seus índices e relações ecológicas em plantas de A. auriculiformis fertilizadas com lodo de esgoto desidratado, em área degradada, durante 24 meses. O delineamento foi inteiramente casualizados com dois tratamentos (com e sem adubação com lodo de esgoto desidratado) e 24 repetições (uma repetição = uma planta). O maior número de insetos mastigadores Parasyphraea sp. (Coleoptera: Chrysomelidae), Nasutitermes sp. (Blattodea: Termitidae) e Tropidacris collaris (Stoll, 1813) (Orthoptera: Romaleidae), de desfolha, e do índice ecológico abundância de Coleoptera e de Orthoptera foram maiores em plantas de A. auriculiformis fertilizadas do que nas não fertilizadas com lodo de esgoto desidratado. Plantas de A. auriculiformis, com maior quantidade de galhos/árvore, apresentaram maiores abundâncias de Coleoptera e Orthoptera, riqueza de espécies de insetos polinizadores, desfolha e números de Parasyphraea sp. e T. collaris, e as com maior folhas/galho os de riqueza de espécies de Coleoptera e Diabrotica speciosa (Germar, 1824) (Coleoptera: Chrysomelidae). Por tanto, a utilização de A. auriculiformis, adubada com lodo de esgoto desidratado, é promissora na recuperação de áreas degradadas devido ao aumento dos índices ecológicos de insetos mastigadores, polinizadores e aranhas na área.

Depredación, reproducción y desarrollo de la catarina Hippodamia convergens (Coleoptera: Coccinellidae) / Predation, reproduction, and development of the lady beetle Hippodamia convergens (Coleoptera: Coccinellidae)

Introducción: Mantener poblaciones de insectos depredadores en el agroecosistema ofreciéndoles presas y recursos alimenticios provenientes de las plantas es importante para mejorar el control biológico y contribuir con la conservación. Objetivo: Determinar si el consumo de presas y recursos alimenticios florales potencia los atributos biológicos del depredado Hippodamia convergens. Métodos: Las larvas de Hippodamia convergens fueron alimentadas con dos especies de áfidos, mientras que los adultos fueron alimentados con áfidos, espigas de maíz y dos especies de plantas no cultivadas. La presencia de azúcar en el intestino del depredador se comprobó mediante la prueba de antronas frías, y la presencia de polen mediante el método de acetólisis. Los experimentos demográficos se realizaron durante los primeros 40 días después de la emergencia de la hembra bajo condiciones controladas (25 °C ± 0.5; 75 % ± 1.75 HR; L12:D12). Resultados: H. convergens no consumió fructosa de Sorghum halepense, Parthenium hysterophorus o Zea mays, sino únicamente polen. En condiciones controladas (25 °C ± 0.5, 75 % ± 1.75 HR), el tiempo de desarrollo (huevo-adulto) del depredador fue más corto (21.36 días) cuando consumió Rhopalosiphum maidis en lugar de Uroleucon nigrotibium (24.6 días), mientras que la supervivencia (L1- adulto) y la proporción sexual no cambió. La fecundidad promedio fue mayor (55.5 huevos / 40 días) al consumir U. nigrotibium con polen de P. hysterophorus, que solo U. nigrotibium (22.5 huevos / 40 días), o R. maidis con polen de Zea mays (11 huevos / 40 días). La tasa intrínseca de crecimiento natural fue mayor al consumir U. nigrotibium con polen de P. hysterophorus (0.055), que U. nigrotibium (0.034) o R. maidis con polen de Z. mays (0.019). La tasa de depredación (L1-L5) fue mayor al consumir R. maidis (0.65) que U. nigrotibium (0.51). Conclusiones: Las especies de áfidos y su combinación con polen de plantas no cultivadas afectan de manera diferente el tiempo de desarrollo, reproducción y tasa de depredación de H. convergens.

Introduction: Keeping populations of predatory insects in the agroecosystem by offering them prey, as well as food resources from plants, is important for enhancing conservation biological control. Objective: To determine if the consumption of prey and floral food resources enhances the biological attributes of the predator Hippodamia convergens. Methods: We fed the beetle larvae two species of aphids; and the adults were fed aphids, maize tassels, and two non-cultivated plant species. We checked gut sugar in the predator by the cold anthrone test and pollen presence by the acetolysis method. Demographic experiments were done in the first 40 days after female emergence, under controlled conditions (25 °C ± 0.5; 75 % ± 1.75 Relative Humidity; Light 12 h: Darkness 12 h). Results: H. convergens did not consume fructose, but only pollen from Sorghum halepense, Parthenium hysterophorus or Zea mays. Developmental time (egg-adult) of the predator was shorter (21.4 days) when it consumed Rhopalosiphum maidis than Uroleucon nigrotibium (24.6 days); survival (L1-adult) and sex ratio did not change. Average fecundity was higher (55.5 eggs / 40 days) when consuming U. nigrotibium with pollen from P. hysterophorus, than U. nigrotibium (22.5 eggs / 40 days) or R. maidis with pollen from Zea mays (11 eggs / 40 days). The intrinsic rate of natural growth was higher when consuming U. nigrotibium with pollen from P. hysterophorus (0.055), than U. nigrotibium (0.034) or R. maidis with pollen from Z. mays (0.019). Predation rate (L1-L5) was higher when consuming R. maidis (0.65) than U. nigrotibium (0.51). Conclusions: Aphid prey species and its combination with pollen from maize and non-cultivated plants affected development time, reproduction, and predation rate of H. convergens.

Oncideres saga Dalman: first record damaging Stryphnodendron adstringens Mart. Coville (Fabaceae) in Brazil / Oncideres saga Dalman: primeiro registro em Stryphnodendron adstringens Mart. Coville (Fabaceae) no Brasil

Oncideres females girdle tree branches of the Fabaceae family, interrupting the sap flow and turning the wood conditions ideal for their larvae development. The bark of Stryphnodendron adstringens (Mart.) Coville, a species native to the Brazilian Cerrado, is widely used in the traditional medicine. The objectives were to report, for the first time, Oncideres saga (Dalman), using S. adstringens as a host and to describe the pattern of branch girdling and oviposition distribution by this insect on these branches. The diameter at the base and the length of the girdled branches were measured and the number of incisions made by the O. saga females to oviposit, per branch section (basal, median and apical), counted. The emerged specimens were counted and the diameter of the exit holes measured. The average diameter at the base of the girdled branches was 2.5 ± 0.16 cm and the length was 90.6 ± 4.6 cm. The average number of incisions per branch was 37.7 ± 2.7. Damage by O. saga can reduce the growth and cause losses on S. adstringens, a tree with great extractivism potential.

As fêmeas de Oncideres anelam galhos de árvores da família Fabaceae, interrompendo o fluxo de seiva, tornando a madeira ideal para o desenvolvimento de suas larvas. A casca de Stryphnodendron adstringens (Mart.) Coville uma espécie nativa do cerrado brasileiro, é amplamente utilizada na medicina tradicional. Os objetivos foram relatar, pela primeira vez, Oncideres saga (Dalman), usando S. adstringens como hospedeiro e descrever o padrão dos galhos anelados e a distribuição das posturas desse inseto. O diâmetro na base e o comprimento dos galhos anelados foram medidos e o número de incisões feitas pelas fêmeas por seção do galho (basal, mediana e apical) quantificados. Os espécimes emergidos foram contados e o diâmetro dos orifícios de emergência medidos. O diâmetro médio, dos galhos anelados, na base foi de 2,58 ± 0,16 cm e o comprimento de 90,6 ± 4,6 cm. O número médio de posturas por galho foi 37,7 ± 2,7. Danos por O. saga podem reduzir o crescimento e causar perdas em S. adstringens, árvore com grande potencial extrativista.

Anadenanthera colubrina (Fabaceae) logs in the Atlantic Forest biome: first host plant for Thoracibidion lineatocolle (Col: Cerambycidae) and a new host for Temnopis megacephala (Col: Cerambycidae) / Troncos de Anadenanthera colubrina (Fabaceae) no bioma da Mata Atlântica: primeira planta hospedeira de Thoracibidion lineatocolle (Col: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Col: Cerambycidae)

Wood-boring beetles develop in live trees and dead wood, performing ecological services such as decomposition and regulation of forest resources. Species of the Cerambycidae family, widely distributed in the world, bore into the trunks of trees and dead wood in native and cultivated areas. The objective is to report the first host plant for Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) and a new host plant for Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) in the Brazilian Atlantic Forest biome. Three logs, with one-meter-long by 20 cm in diameter, were cut from the trunk of a healthy Anadenanthera colubrina (Fabaceae) tree in October 2013 and tied in the understory at 1.5m high in the Rio Doce State Park, Minas Gerais State, Brazil. The logs, exposed in the forest, were each removed after 40, 80 and 120 days and stored individually in a cardboard box in the "Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)" in the Rio Doce State Park. A total of 94 individuals of T. lineatocolle and 228 of T. megacephala emerged from the A. colubrina logs. This is the first report of a host plant for T. lineatocolle and a new host plant for T. megacephala.

Besouros broqueadores se desenvolvem em árvores vivas e madeira morta, realizando serviços ecológicos como decomposição e regulação de recursos da floresta. Espécies da família Cerambycidae, amplamente distribuídas no mundo, perfuram o caule de árvores e madeira morta em áreas nativas e cultivadas. O objetivo é relatar a primeira planta hospedeira de Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) no bioma da Mata Atlântica brasileira. Três toras, com um metro de comprimento por 20 cm de diâmetro, foram cortadas de uma árvore sadia de Anadenanthera colubrina (Fabaceae) em outubro de 2013 e amarradas no sub-bosque a 1,5m de altura no Parque Estadual do Rio Doce, estado de Minas Gerais, Brasil. As toras, expostas na floresta, foram removidas, cada uma, após 40, 80 e 120 dias e armazenadas, individualmente, em caixas de papelão no “Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)” no Parque Estadual do Rio Doce. Um total de 94 indivíduos de T. lineatocolle e 228 de T. megacephala emergiu das toras de A. colubrina. Esse é o primeiro registro de uma planta hospedeira para T. lineatocolle e o de uma nova planta hospedeira para T. megacephala.

Notes on the biological development of the darkling beetle Blaps nefrauensis nefrauensis Seidlitz, 1893 (Coleoptera: Tenebrionidae) / Notas sobre o desenvolvimento biológico do besouro escuro Blaps nefrauensis nefrauensis Seidlitz, 1893 (Coleoptera: Tenebrionidae)

Several endemic species of Blaps occur in Tunisia, and the species Blaps nefrauensis nefrauensis has been reported in Moulares (urban zone in west-central Tunisia), where it lives and reproduces in home gardens and old buildings. The aim of this work is to study the life cycle of the darkling beetle, considering both field and laboratory rearing conditions. As a result, the beetle species has different developmental stages (egg, larva, prepupa, pupa, and adult) that last about 15 months. Each year during the same period, adults emerge (early summer) and expire (late autumn), larvae hatch (late summer) and pupate (early summer). There is only one generation per year. Females began laying eggs in late July. The eggs were ovoid, white, and about 2.7 mm in length and 1.5 mm in width. Embryogenesis took an average of nine days. The first instar larvae were at initially only 4.5 mm long and ivory white in color. A brief description of the newly egg hatched larva was provided; thus, the nerve fibers innervating the apical setae in the antennae and ligula were detected. Further light microscopic examination of the embryo before hatching from the egg pointed out that the antennal sensilla are protected during the embryogenesis stage.

Várias espécies endêmicas de Blaps ocorrem na Tunísia, e a espécie Blaps nefrauensis nefrauensis foi relatada em Moulares (zona urbana no centro-oeste da Tunísia), onde vive e se reproduz em jardins domésticos e prédios antigos. O objetivo deste trabalho é estudar o ciclo de vida do besouro escuro, considerando as condições de criação em campo e em laboratório. Como resultado, a espécie de besouro tem diferentes estágios de desenvolvimento (ovo, larva, prepupa, pupa e adulto) que duram cerca de 15 meses. Todos os anos, durante o mesmo período, os adultos emergem (início do verão) e expiram (final do outono), as larvas eclodem (final do verão) e se tornam pupas (início do verão). Existe apenas uma geração por ano. As fêmeas começaram a botar ovos no final de julho. Os ovos eram ovóides, brancos, com cerca de 2,7 mm de comprimento e 1,5 mm de largura. A embriogênese demorou em média nove dias. As larvas de primeiro instar tinham inicialmente apenas 4,5 mm de comprimento e uma cor branca marfim. Foi fornecida uma breve descrição da larva recém-eclodida; assim, as fibras nervosas que inervam as cerdas apicais nas antenas e ligulas foram detectadas. Um exame microscópico de luz posterior do embrião antes da eclosão do ovo mostrou que as sensilas antenais são protegidas durante o estágio de embriogênese.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis.

An insect's capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period - results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Aegerolysins from the fungal genus Pleurotus - Bioinsecticidal proteins with multiple potential applications.

The aegerolysin proteins ostreolysin A6, pleurotolysin A2 and erylysin A are produced by mushrooms of the genus Pleurotus. These aegerolysins can interact specifically with sphingolipid-enriched membranes. In particular, they strongly bind insect cells and to artificial lipid membranes that contain physiologically relevant concentrations of the main invertebrate-specific sphingolipid, ceramide phosphoethanolamine. Moreover, the aegerolysins permeabilise these membranes when combined with their protein partner pleurotolysin B, which contains a membrane-attack-complex/perforin domain. These aegerolysin/ pleurotolysin B complexes show strong and selective toxicity towards western corn rootworm larvae and adults and Colorado potato beetle larvae. Their insecticidal activities arise through aegerolysin binding to ceramide phosphoethanolamine in the insect midgut. This mode of membrane binding is different from those described for similar aegerolysin-based complexes of bacterial origin (e.g., Cry34Ab1/Cry35Ab1), or other Bacillus thuringiensis proteinaceous crystal toxins, which associate with protein receptors. The ability of Pleurotus aegerolysins to specifically interact with sphingolipid-enriched domains in mammalian cells can be further exploited to visualize lipid rafts in living cells, and to treat certain types of tumours and metabolic disorders. Finally, these proteins can strongly enhance fruiting initiation of P. ostreatus even when applied externally. In this review, we summarise the current knowledge of the potential biotechnological and biomedical applications of the Pleurotus aegerolysins, either alone or when complexed with pleurotolysin B, with special emphasis on their bioinsecticidal effects.

Serotonin signaling suppresses the nutrition-responsive induction of an alternate male morph in horn polyphenic beetles.

Environment-responsive development contributes significantly to the phenotypic variation visible to selection and as such possesses the potential to shape evolutionary trajectories. However, evaluation of the contributions of developmental plasticity to evolutionary diversification necessitates an understanding of the developmental mechanisms underpinning plastic trait expression. We investigated the role of serotonin signaling in the regulation and evolution of horn polyphenism in the beetle genus Onthophagus. Specifically, we assessed the role of serotonin in development by determining whether manipulating serotonin biosynthesis during the larval stage alters body size, developmental rate, and the formation of relative adult trait size in traits characterized by minimal (genitalia), moderate (elytra), and pronounced (horns) nutrition-responsive development in O. taurus. Second, we assessed serotonin's role in evolution by replicating a subset of our approaches across four species reflecting ancestral as well as derived conditions. Lastly, we employed immunohistochemical approaches to begin assessing whether serotonin may be acting via the endocrine or nervous system. Our results show that pharmacological manipulation of serotonin signaling affects overall size, developmental rate, and the body size threshold separating alternate male morphs. Threshold body sizes were affected across species, regardless of the severity of horn polyphenism, and independent of the precise morphological location of horns. However, histological assessments suggest it is unlikely serotonin functions as a neurotransmitter and instead may rely on other mechanisms that remain to be identified. We discuss the most important implications of our results for our understanding of the evolution of and through plasticity in horned beetles and beyond.

First field estimation of greenhouse gas release from European soil-dwelling Scarabaeidae larvae targeting the genus Melolontha.

Arthropods are a major soil fauna group, and have the potential to substantially influence the spatial and temporal variability of soil greenhouse gas (GHG) sinks and sources. The overall effect of soil-inhabiting arthropods on soil GHG fluxes still remains poorly quantified since the majority of the available data comes from laboratory experiments, is often controversial, and has been limited to a few species. The main objective of this study was to provide first insights into field-level carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) release of soil-inhabiting larvae of the Scarabaeidae family. Larvae of the genus Melolontha were excavated at various sites in west-central and southern Germany, covering a wide range of different larval developmental stages, larval activity levels, and vegetation types. Excavated larvae were immediately incubated in the field to measure their GHG production. Gaseous carbon release of individual larvae showed a large inter- and intra-site variability which was strongly correlated to larval biomass. This correlation persisted when upscaling individual CO2 and CH4 production to the plot scale. Field release estimates for Melolontha spp. were subsequently upscaled to the European level to derive the first regional GHG release estimates for members of the Scarabaeidae family. Estimates ranged between 10.42 and 409.53 kt CO2 yr-1, and 0.01 and 1.36 kt CH4 yr-1. Larval N2O release was only sporadically observed and not upscaled. For one site, a comparison of field- and laboratory-based GHG production measurements was conducted to assess potential biases introduced by transferring Scarabaeidae larvae to artificial environments. Release strength and variability of captive larvae decreased significantly within two weeks and the correlation between larval biomass and gaseous carbon production disappeared, highlighting the importance of field measurements. Overall, our data show that Scarabaeidae larvae can be significant soil GHG sources and should not be neglected in soil GHG flux research.

Optimal Extraction of Ocimum basilicum Essential Oil by Association of Ultrasound and Hydrodistillation and Its Potential as a Biopesticide against a Major Stored Grains Pest.

The essential oil of basil (Ocimum basilicum) has significant biological activity against insect pests and can be extracted through various techniques. This work aimed to optimize and validate the extraction process of the essential oil of O. basilicum submitted to different drying temperatures of the leaves and extracted by the combination of a Clevenger method and ultrasound. The biological activity of the extracted oil under different conditions was evaluated for potential control of Sitophilus zeamais. The extraction method was optimized according to the sonication time by ultrasound (0, 8, 19, 31 and 38 min) and hydrodistillation (20, 30, 45, 60 and 70 min) and drying temperature (20, 30, 45, 60 and 70 °C). The bioactivity of the essential oil was assessed against adults of S. zeamais and the effects of each variable and its interactions on the mortality of the insects were evaluated. The best yield of essential oil was obtained with the longest sonication and hydrodistillation times and the lowest drying temperature of leaves. Higher toxicity of the essential oil against S. zeamais was obtained by the use of ultrasound for its extraction. The identification and the relative percentage of the compounds of the essential oil were performed with a gas chromatograph coupled to a mass selective detector. The performance of the method was assessed by studying selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. The LOD and LOQ values for linalool were 2.19 and 6.62 µg mL-1 and for estragole 2.001 and 6.063 µg mL-1, respectively. The coefficients of determination (R2) were >0.99. The average recoveries ranged between 71 and 106%, with coefficient of variation ≤6.4%.

RNA interference-mediated control of cigarette beetle, Lasioderma serricorne.

The cigarette beetle (CB; Lasioderma serricorne) is a pest on many stored products including tobacco. Fumigation is the common control method currently used. However, the options for controlling this pest are limited, due to resistance issues and phasing out of currently used chemical insecticides. Here, we evaluated RNA interference (RNAi) as a potential method for controlling the CB. RNA isolated from different stages was sequenced and assembled into a transcriptome. The CB RNA sequences showed the highest homology with those in the red flour beetle, Tribolium castaneum. Orthologs of proteins known to function in RNAi pathway were identified in the CB transcriptome, suggesting that RNAi may work well in this insect. Also, 32 P-labeled double-stranded RNA (dsRNA) injected into CB larvae and adults was processed to small interference RNAs. We selected 12 genes that were shown to be the effective RNAi targets in T. castaneum and other insects and identified orthologs of them in the CB by searching its transcriptome. Injection of dsRNA targeting genes coding for GAWKY, Kinesin, Sec23, SNF7, and 26S proteasome subunit 6B into the CB larvae caused 100% mortality. Feeding dsRNA targeting SNF7 and 26S proteasome subunit 6B by sucrose droplet assay induced more than 90% mortality, which is 1.8 times higher than the mortality induced by dsGFP control (53%). These data demonstrate an efficient RNAi response in CB, suggesting that RNAi could be developed as an efficient method to control this pest.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne.

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Mitonuclear mismatch alters performance and reproductive success in naturally introgressed populations of a montane leaf beetle.

Coordination between nuclear and mitochondrial genomes is critical to metabolic processes underlying animals' ability to adapt to local environments, yet consequences of mitonuclear interactions have rarely been investigated in populations where individuals with divergent mitochondrial and nuclear genomes naturally interbreed. Genetic variation in the leaf beetle Chrysomela aeneicollis was assessed along a latitudinal thermal gradient in California's Sierra Nevada. Variation at mitochondrial cytochrome oxidase II (COII) and the nuclear gene phosphoglucose isomerase (PGI) shows concordance and was significantly greater along a 65 km transect than 10 other loci. STRUCTURE analyses using neutral loci identified a southern and northern subpopulation, which interbreed in the central drainage Bishop Creek. COII and PGI were used as indicators of mitochondrial and nuclear genetic variation in field and laboratory experiments conducted on beetles from this admixed population. Fecundity, larval development rate, running speed and male mating frequency were higher for beetles with geographically "matched" than "mismatched" mitonuclear genotypes. Effects of mitonuclear mismatch were largest for individuals with northern nuclear genotypes possessing southern mitochondria and were most pronounced after heat treatment or at high elevation. These findings suggest that mitonuclear incompatibility diminishes performance and reproductive success in nature, effects that could intensify at environmental extremes.

Graphene quantum dots as cysteine protease nanocarriers against stored grain insect pests.

Storing grains remain vulnerable to insect pest attack. The present study developed a biopesticide using biomolecules and their encapsulation in nanoparticles. A 25 kDa cysteine protease extracted from seeds of Albizia procera (ApCP) was encapsulated in graphene quantum dots (GQDs). The insecticidal activity of ApCP, with or without GQDs, against two stored grain insect pests, Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored. Insects were exposed to three concentrations 7.0, 3.5 and 1.7 mg of ApCP per a gram of wheat flour and grains. The insecticidal activity of ApCP encapsulated with GQDs was improved compared to that of ApCP without GQDs for both insect pests. The number of eggs and larvae of T. castaneum was reduced by 49% and 86%, respectively. Larval mortality was increased to 72%, and adult eclosion of T. castaneum was reduced by 98% at a 7.0 mg/g concentration of ApCP with GQDs compared to that of ApCP without GQDs. Exposure to 7.0 mg/g ApCP with GQDs, the number of R. dominica eggs and larvae was reduced by 72% and 92% respectively, larval mortality was increased by 90%, and eclosion was reduced by 97%. The extraction, purification, characterization, quantification and encapsulation of ApCP with GQDs were also studied. Cysteine protease nanocarriers have the potential to control stored grain insect pests.

Notch signaling patterns head horn shape in the bull-headed dung beetle Onthophagus taurus.

Size and shape constitute fundamental aspects in the description of morphology. Yet while the developmental-genetic underpinnings of trait size, in particular with regard to scaling relationships, are increasingly well understood, those of shape remain largely elusive. Here we investigate the potential function of the Notch signaling pathway in instructing the shape of beetle horns, a highly diversified and evolutionarily novel morphological structure. We focused on the bull-headed dung beetle Onthophagus taurus due to the wide range of horn sizes and shapes present among males in this species, in order to assess the potential function of Notch signaling in the specification of horn shape alongside the regulation of shape changes with allometry. Using RNA interference-mediated transcript depletion of Notch and its ligands, we document a highly conserved role of Notch signaling in general appendage formation. By integrating our functional genetic approach with a geometric morphometric analysis, we find that Notch signaling moderately but consistently affects horn shape, and does so differently for the horns of minor, intermediate-sized, and major males. Our results suggest that the function of Notch signaling during head horn formation may vary in a complex manner across male morphs, and highlights the power of integrating functional genetic and geometric morphometric approaches in analyzing subtle but nevertheless biologically important phenotypes in the face of significant allometric variation.

A specific type of insulin-like peptide regulates the conditional growth of a beetle weapon.

Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) has been identified as a major physiological mechanism underlying the nutrient-dependent regulation of sexually selected weapon growth in animals. However, the molecular mechanisms that couple nutritional state with weapon growth remain largely unknown. Here, we show that one specific subtype of insulin-like peptide (ILP) responds to nutrient status and thereby regulates weapon size in the broad-horned flour beetle Gnatocerus cornutus. By using transcriptome information, we identified five G. cornutus ILP (GcorILP1-5) and two G. cornutus insulin-like receptor (GcorInR1, -2) genes in the G. cornutus genome. RNA interference (RNAi)-mediated gene silencing revealed that a certain subtype of ILP, GcorILP2, specifically regulated weapon size. Importantly, GcorILP2 was highly and specifically expressed in the fat body in a condition-dependent manner. We further found that GcorInR1 and GcorInR2 are functionally redundant but that the latter is partially specialized for regulating weapon growth. These results strongly suggest that GcorILP2 is an important component of the developmental mechanism that couples nutritional state to weapon growth in G. cornutus. We propose that the duplication and subsequent diversification of IIS genes played a pivotal role in the evolution of the complex growth regulation of secondary sexual traits.

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.