RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella

Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951. Abbreviation / RNAi: RNA interference PCR: polymerase chain reaction RT-PCR: reverse transcription-PC

Effects of Pink Bollworm Resistance to Bacillus thuringiensis on Phenoloxidase Activity and Susceptibility to Entomopathogenic Nematodes

Widespread planting of crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on many key agricultural pests to evolve resistance to Bt. Fitness costs can slow the evolution of Bt resistance. We examined effects of entomopathogenic nematodes on fitness costs of Bt resistance in the pink bollworm,Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), a major pest of cotton,Gossypium hirsutum L., in the southwestern United States that is currently controlled by transgenic cotton that produces Bt toxin Cry1Ac. We tested whether the entomopathogenic nematodes Steinernema riobrave Cabanillas, Poinar, and Raulston (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) affected fitness costs of resistance to Cry1Ac in two laboratory-selected hybrid strains of pink bollworm reared on non-Bt cotton bolls. The nematode S. riobrave imposed a recessive fitness cost for one strain, and H. bacteriophora imposed a fitness cost affecting heterozygous resistant individuals for the other strain. Activity of phenoloxidase, an important component of insects\u27 immune response, did not differ between Bt-resistant and Bt-susceptible families. This suggests phenoloxidase does not affect susceptibility to entomopathogenic nematodes in Bt-resistant pink bollworm. Additionally, phenoloxidase activity does not contribute to Bt resistance, as has been found in some species. We conclude that other mechanisms cause higher nematode-imposed mortality for pink bollworm with Bt resistance genes. Incorporation of nematode-imposed fitness costs into a spatially explicit simulation model suggests that entomopathogenic nematodes in non-Bt refuges could delay resistance by pink bollworm to Bt cotton

Chronological Age-Grading of House Flies by Using Near-Infrared Spectroscopy

The sensitivity and accuracy of near-infrared spectroscopy (NIRS) was compared with that of the pteridine fluorescence technique for estimating the chronological age of house flies, Musca domestica (L.). Although results with both techniques were significantly correlated with fly age, confidence limits on predicted ages generally were smaller with NIRS. Young flies could be readily differentiated from old flies by using NIRS. Age predictions using the pteridine method are dependent upon size, sex, and temperature at which adult flies are exposed. In contrast, those factors do not need to be determined for age-grading using NIRS. Classification accuracy using the NIRS method was similar for whole flies, fresh heads, dried heads, and ethanol-preserved heads. The NIRS method was also suitable for predicting age of stable flies, Stomoxys calcitrans (L.), and face flies, Musca autumnalis De Geer. NIRS has several advantages over the measurement of pteridine levels for age-grading field-collected flies, including speed and portability of instrumentation, and not needing to determine sex, size, and temperatures to which adult flies were exposed

Transposon insertion causes cadherin mis-splicing and confers resistance to Bt cotton in pink bollworm from China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wildtype or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory-and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.China's Key Project for Breeding Genetically Modified Organisms [2016ZX08012-004]; Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture [2018-67013-27821]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An initial event in insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the NMR solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~ 6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation of the protein:carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~ 102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to sub-micromolar concentrations. The structural model thus derived from the present studies for N-βGRP:laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple helical form of laminarin on the basis of an X-ray crystallographic structure of N-βGRP:laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements carried out with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of βGRP:β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway

Similar Genetic Basis of Resistance to Bt Toxin Cry1Ac in Boll-Selected and Diet-Selected Strains of Pink Bollworm

Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F1 progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research

Molecular Genetic Basis of Lab- and Field-Selected Bt Resistance in Pink Bollworm

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world’s most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world’s top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries

Survival (mean and SE) in greenhouse bioassays with resistant (Bt4R) and susceptible (SOM-07) strains of pink bollworm on Bt cotton and non-Bt cotton plants.

<p>Survival (mean and SE) in greenhouse bioassays with resistant (Bt4R) and susceptible (SOM-07) strains of pink bollworm on Bt cotton and non-Bt cotton plants.</p