Jeffrey C. Lord

Desiccant Dusts Synergize the Effect of Beauveria bassiana (Hyphomycetes: Moniliales) on Stored-Grain Beetles

Abstract Diatomaceous earth (DE) is a desiccant insecticide and most efficacious in low humidity. It acts on insect cuticle by absorbing lipids, and perhaps by cuticular abrasion. Beauveria bassiana (Balsamo) Vuillemin, an entomopathogenic fungus, is most efficacious in high humidity and has a complex interaction with cuticular lipids. Interaction between these materials may enhance insect control performance. Assays with stored-grain beetles were conducted with B. bassiana at rates of 11, 33, 100, and 300 mg of conidia per kilogram of grain with and without single rates of DE that killed 10% or less of the target beetles. The assays revealed synergism in effects on adult Rhyzopertha dominica (F.) and Oryzaephilus surinamensis (L.) at all doses. There was statistically significant synergism for adult Cryptolestes ferrugineus (Stephens) and larval R. dominica but at only one B. bassiana rate for each target. Both amorphous silicon dioxide, a sorptive dust, and diamond dust, an abrasive, showed synergistic interaction with B. bassiana on adult R. dominica. These results may provide a basis for a least-toxic approach to control of stored-product beetles and for efficacy-enhancing formulation of entomopathogenic fungi.

Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge

To investigate gene expression in Tribolium castaneum exposed to Beauveria bassiana, reference genes for qPCR were evaluated. Of these, beta-actin, alpha-tubulin, and RPS6 were not stable. The most stable were ribosomal protein genes, RPS3, RPS18, and RPL13a. Syntaxin1, syntaxin6, and E-cadherin may be appropriate for some experimental systems.

A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophilas cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophilas tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.

Efficacy of Beauveria bassiana for Red Flour Beetle When Applied with Plant Essential Oils or in Mineral Oil and Organosilicone Carriers

Abstract The carriers mineral oil and Silwet L-77 and the botanical insecticides Neemix 4.5 and Hexacide were evaluated for their impacts on the efficacy of Beauveria bassiana (Balsamo) Vuillemin conidia against red flour beetle, Tribolium castaneum (Herbst), larvae. The dosages of liquid treatments were quantified by both conidia concentration in the spray volume and conidia deposition on the target surface. The latter approach allowed comparison with dry, unformulated conidia. The median lethal concentrations of B. bassiana in 0.05% Silwet L-77 solution or without a carrier were approximately double that for conidia in mineral oil. Carriers had highly significant effects on the efficacy of B. bassiana. The lower efficacy of conidia in aqueous Silwet L-77 may have been the result of conidia loss from the larval surface because of the siloxane’s spreading properties. Neemix 4.5 (4.5% azadirachtin) delayed pupation and did not reduce the germination rate of B. bassiana conidia, but it significantly reduced T. castaneum mortality at two of four tested fungus doses. Hexacide (5% rosemary oil) caused significant mortality when applied without B. bassiana, but it did not affect pupation, the germination rate of conidia, or T. castaneum mortality when used in combination with the fungus.

Analysis of transcriptome data in the red flour beetle, Tribolium castaneum

The whole genome sequence of Tribolium castaneum, a worldwide coleopteran pest of stored products, has recently been determined. In order to facilitate accurate annotation and detailed functional analysis of this genome, we have compiled and analyzed all available expressed sequence tag (EST) data. The raw data consist of 61,228 ESTs, including 10,704 obtained from NCBI and an additional 50,524 derived from 32,544 clones generated in our laboratories. These sequences were amassed from cDNA libraries representing six different tissues or stages, namely: whole embryos, whole larvae, larval hindguts and Malpighian tubules, larval fat bodies and carcasses, adult ovaries, and adult heads. Assembly of the 61,228 sequences collapsed into 12,269 clusters (groups of overlapping ESTs representing single genes), of which 10,134 mapped onto 6,463 (39%) of the 16,422 GLEAN gene models (i.e. official Tribolium gene list). Approximately 1,600 clusters (13% of the total) lack corresponding GLEAN models, despite high matches to the genome, suggesting that a considerable number of transcribed sequences were missed by the gene prediction programs or were removed by GLEAN. We conservatively estimate that the current EST set represents more than 7,500 transcription units.

Cuticular Lipids of the Booklouse, Liposcelis bostrychophila: Hydrocarbons, Aldehydes, Fatty Acids, and Fatty Acid Amides

The booklouse, Liposcelis bostrychophila, is an increasingly common pest of stored food products worldwide. We report here the cuticular lipid composition of this pest (the first report of the hydrocarbons of any member of the Order Psocoptera and the first report of fatty acid amides as cuticular components for any insect). No unsaturated hydrocarbons were present. A homologous series of n-alkanes (C21–C34), monomethyl alkanes (3-, 4-, 5-, 7-, 9-, 11-, 12-, 13- and 15-methyl-) with a carbon chain range of C28–C42, and dimethyl alkanes (3, 7-; 9, 13-; 11, 15-; 13, 17-; 9, 21-; 11, 19-; and 13, 21-); with a carbon number range of C31–C43 were identified. The relative abundances of these hydrocarbons were low, comprising approximately 0.0125% of total biomass. The amides were a homologous series (C16–C22 in chain length), with the major amide being stearoyl amide. In addition to the amides, free fatty acids (C16:1, C16:0, C18:2, C18:1, and C18:0 in chain length) and three straight chain aldehydes (C15, C16, and C17:1 in chain length) also occurred as cuticular components. These findings are discussed in terms of the chemical and physiological ecology of this species.

Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum.

The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability as normalizers in comparisons across tissues and/or developmental stages. In less diverse samples, such as comparisons within developmental stages or tissue only, normalizers for mRNA were more stable and consistent. Overall, the genes for ribosomal proteins rps6, rpl13a, rps3, and rps18 were the most stable normalizers for broad scale gene expression analysis in T. castaneum. However, their stability ranking was dependent upon the instrument as well as the analysis program. These data emphasize the need to optimize normalizers used in all real-time polymerase chain reaction experiments specifically for the experimental conditions and thermocycler and to carefully evaluate data generated by computational algorithms.

Bacillus thuringiensis Cry3Aa toxin increases the susceptibility of Crioceris quatuordecimpunctata to Beauveria bassiana infection

The spotted asparagus beetle, Crioceris quatuordecimpunctata (Coleoptera: Chrysomelidae), is one of the most devastating pests of asparagus in China. Sprayed synthetic pesticides have been used to control C. quatuordecimpunctata damage, but they pose problems because of residues and harm to natural enemies. Neither the microbial coleopteran-specific toxin from Bacillus thuringiensistenebrionis, Cry3Aa, nor the fungal pathogen Beauveria bassiana have sufficient activity to effectively control C. quatuordecimpunctata damage to asparagus. However, second instar C. quatuordecimpunctata larvae exposed to a sublethal dose of Cry3Aa toxin demonstrated significantly higher larval mortality when exposed to B. bassiana. Our results suggest that a combination of Cry3Aa and B. bassiana may be effective in reducing damage by C. quatuordecimpunctata larvae to asparagus.

Control of Sawtoothed Grain Beetles (Coleoptera: Silvanidae) in Stored Oats by Using an Entomopathogenic Fungus in Conjunction with Seed Resistance

Abstract We tested the hypothesis that the entomopathogenic fungus Beauveria bassiana would be more efficacious on oat cultivars that prolonged the immature developmental period of sawtoothed grain beetle, Oryzaephilus surinamensis (L.), a storage pest. However, percentage of reduction in progeny production was similar on whole ‘Don’ and ‘Paul’ oats treated with fungus, even though immature developmental time was longer on whole ‘Don’ than on ‘Paul’ oats. In our initial test at 10 mg of conidia per kilogram of oats, the number of beetle progeny produced was reduced by 38–67% in whole oats, and there was no effect of the fungus on insects developing on cracked oats. Therefore, we conducted two dose–response studies that showed that adding 150 mg of conidia per kilogram to cracked or whole ‘Paul’ oats resulted in a 70 and 98% reduction, respectively, in number of progeny produced. No further reduction was obtained by adding 200 mg of conidia per kilogram of cracked or whole ‘Paul’ oats. Presence of the fungus did not affect developmental time in any of our tests. A previous study showed that cleaned oats should limit insect population growth to allow long-term storage of oats without insect damage. However, the current study shows that if the oats are not cleaned, and not cleaning oats is the normal storage practice, then B. bassiana could be used to help control sawtoothed grain beetles.

Enhanced Efficacy of Beauveria bassiana for Red Flour Beetle with Reduced Moisture

Abstract Red flour beetle, Tribolium castaneum (Herbst), is a major pest of stored and processed grains that is tolerant of Beauveria bassiana (Balsamo) Vuillemin under most conditions. Laboratory assays demonstrated that desiccating conditions improved the efficacy of the fungus. For T. castaneum larvae exposed to B. bassiana at different vapor pressure deficits (VPDs), the LC50 potency ratios were 2.3 for 2.42 kPa and 3.9 for 3.31 kPa compared with 1.06 kPa. There were significant effects of moisture on B. bassiana-associated mortality even with brief exposure to low VPD before or at the beginning of exposure to fungus. When T. castaneum larvae were held at 3.31 kPa 1 d before exposure to B. bassiana and/or in the first 1 or 2 d of exposure to the fungus, there was significantly greater mortality for all of the low moisture treatments than occurred with continuous incubation at 1.06 kPa. B. bassiana efficacy for adult T. castaneum was significantly better for VPDs of 3.56 or 3.78 than 1.06 kPa. Weight gain over 4 d of incubation with moisture held constant was significantly less at a VDP of 3.31 kPa than at 1.87 or 1.06 kPa. Treatment with fungus did not affect weight gain significantly. There was no significant difference in larval water content among treatments.