Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests.

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen.

Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrA(O)) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrA(O) strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution.

Repellent and fumigant toxicity of essential oil from Thymus persicus against Tribolium castaneum and Callosobruchus maculatus.

Repellent and insecticidal activity of the essential oil extracted from Thymus persicus (Roniger ex Reach. F.) Jalas was evaluated against two stored-product beetles Tribolium castaneum (Herbst) and Callosobruchus maculatus (F.). Dry flowering aerial parts of the plant were subjected to hydro distillation using a modified Clevenger-type apparatus. The repellent and fumigant toxicity were tested against 1-7 days old adult beetles at 27 +/- 1 degrees C and 65 +/- 5% RH in dark condition. The repellency on C. maculatus and T. castaneum at highest concentration (2 microL/mL acetone) was 82.40% and 70.40% respectively. Fumigation bioassays showed that C. maculatus adults were significantly more susceptible (LC50 = 2.39 microL/L air) to the essential oil than T. castaneum adults (LC50 = 234.42 microL/L air). It could be concluded that T. persicus may have potential for applications in management of stored-product pests because of its safety, strong repellency and fumigant toxicity.

Alergia ocupacional por monosensibilización a Tribolium Confusum

Introducción: Como agente causante de asma en el panadero se ha señalado, en general, las proteínas presentes en dicha materia prima, pertenecientes tanto al mismo cereal como a algunos de los insecto que a menudo infestan estas materias vegetales. Caso Clínico: Presentamos un caso de alergia ocupacional causada por Tribotium confusum en una trabajadora de panadería, en donde despachaba pan, que resultó ser monosensible a dicho insecto, y que presentaba síntomas de asma bronquial, rinitis, conjuntivitis y angioedema al contacto con harina contaminada. Diagnosticamos a la paciente de asma mediante la medición del pico de flujo respiratorio y la prueba de broncodilatación. Descartaremos la alergia a otros alérgenos habituales y de la panadería como de alergenos alimentarios. Confirmamos la sensibilización a Tribolium confusum mediante prueba cutánea y de provocación nasal positiva. Los extractos para pruebas in vivo se obtuvieron de la harina que utilizaban en la panadería tras cultivo y estudio entomológico preciso. Diagnóstico definitivo: Rinoconjuntivitis, Asma bronquial, Angioedema de origen ocupacional por sensibilización a Tribolium confusum. Los síntomas son producidos por harina de panificadora infestada con T confusum (Insecta, Coleóptera, Tenebrionidae, Ulominae) que contenía abundantes ejemplares preimaginales y adultos de dicho insecto. Discusión: Ante el estudio de un paciente con asma en la panadería, nos debemos de plantear la existencia de alergia a diferentes alergenos ocultos o, incluso, aún por describir, pese a existir sensibilidad a otros alergenos comunes. como las proteínas de los cereales o las enzimas utilizadas en los procesos de panificación (AU)

The cauative agent in baker's asthma is generally accepted to be the proteins present in tlour, which can be derived from the cereal itself or from some of the insect that frequently infest these vegetable products. We present a case of occupational allergy due to Tribolium confusum sensitisation in a female bakery worker; the patient was monosensitied to that insectand evidenced symptoms of bronchial asthma, rhinitis, conjuntivitis and angiedema upon contact witb contaminated flour. The diagnosis of bronchial asthma was confirmed by ex piratory peak flow measurement and bronchodilator testing, and sensitiation to other comrnon allergen and bakery allergens was ruled out. Sensitisation to Tribolium confusum was confirmed by the positive result of the kin and nasal challenge tests. The extracrs used for the in vivo tests were prepared from the flour the patient used in the bakery. after culture and exhaustive entomological study. The difinitive diagnosis was rhinoconjunctivitis, bronchial asthma and angioedema of occupational origin due to Tribolium confusum sensitisation. The symptoms were induced by bakery flour infested with T confusum (lnsecta. Choleoptera,Tenebrionidae. Ulominae). which contained abundant specimens. both of pre-imagotages and of the adult insect. In the study of a patient with bakers asthma one mu t always consider the possibility of allergic sensitisation to various occult or even as yet undicovered allergens, even when there in confirmed sensitisation to other more common allergen such a cereal proteins or the enzymes used in the baking proces (AU)