Characterization of Iflavirus in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae).

Iflavirus is a group of viruses distributed mainly in arthropod species. We surveyed Tribolium castaneum iflavirus (TcIV) in different laboratory strains and in Sequence Read Archives (SRA) in GenBank. TcIV is highly specific to only T. castaneum and is not found in seven other Tenebrionid species, including the closely related species T. freemani. The same strains from different laboratories and different strains displayed largely different degrees of infections in the examination of 50 different lines by using Taqman-based quantitative PCR. We found that ~63% (27 out of 43 strains) of T. castaneum strains in different laboratories are positive for TcIV PCR with large degrees of variation, in the range of seven orders of magnitude, indicating that the TcIV is highly fluctuating depending on the rearing conditions. The TcIV was prevalent in the nervous system with low levels found in the gonad and gut. The transovarial transmission was supported in the experiment with surface-sterilized eggs. Interestingly, TcIV infection did not show observable pathogenicity. TcIV offers an opportunity to study the interaction between the virus and the immune system of this model beetle species.

Effects of Diet and Temperature on the Life History of the Redlegged Ham Beetle (Coleoptera: Cleridae).

We investigated the effects of various foods and different rearing temperatures on the survival and development of the redlegged ham beetle Necrobia rufipes (De Geer) (Coleoptera: Cleridae), a serious pest of dry-cured hams. The diets tested were dried pet food, finely shredded copra, shredded cheese, dry-cured ham, ground fish meal, and mature larvae of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). As indicated by the growth index and k-values, N. rufipes populations grew fastest on the pet food and slowest on the copra. On the other hand, N. rufipes fed both ham and T. castaneum larvae produced significantly heavier larvae. The adult beetles lived significantly longer on cheese compared to other foods. Studies conducted to determine the developmental rates of N. rufipes fed pet food at temperatures of 22, 25, 28, and 31°C showed that the total developmental time and longevity of N. rufipes significantly varied at different temperatures tested. The shortest developmental time (93.32 d) was observed at 28°C. Oviposition rate was highest for females reared at 25°C while lowest at 22°C. Based on the development, reproduction, and thermal requirements, the most suitable temperature for N. rufipes was between 28 and 31°C. The results also revealed that larval cannibalism prevailed among adult beetles.

Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel ß-sheets organized into ß-sandwich layers. The amino-terminal domains 1-3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long ß-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4-6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

Differential localization of Hessian fly candidate effectors in resistant and susceptible wheat plants.

Hessian fly Mayetiola destructor is a notorious pest of wheat. Previous studies suggest that Hessian fly uses effector-based mechanisms to attack wheat plants during parasitism, but no direct evidence has been reported to support this postulation. Here, we produced recombinant proteins for five Family-1 candidate effectors and antibodies. Indirect immunostaining and western blots were carried out to examine the localization of Hessian fly Family-1 proteins in plant and insect tissues. Confocal images revealed that Family-1 putative effectors were exclusively produced in the basal region of larval salivary glands, which are directly linked to the mandibles' ducts for effector injection. The five Family-1 proteins were detected in infested host plants on western blots. Indirect immunostaining of sectioned host tissues around the feeding site revealed strikingly different localization patterns between resistant and susceptible plants. In susceptible plants, the Family-1 proteins penetrated from the feeding cell into deep tissues, indicative of movement between cells during nutritive cell formation. In contrast, the Hessian fly proteins were primarily limited to the initially attacked cells in resistant plants. The limitation of effectors' spread in resistant plants was likely due to wall strengthening and rapid hypersensitive cell death. Cell death was found in Nicotiana benthamiana in association with hypersensitive reaction triggered by the Family-1 effector SSGP-1A2. Our finding represents a significant progress in visualizing insect effectors in host tissues and mechanisms of plant resistance and susceptibility to gall midge pests.

Efficacy of Sulfuryl Fluoride Against Fourth-Instar Pecan Weevil (Coleoptera: Curculionidae) in Pecans for Quarantine Security.

The efficacy of sulfuryl fluoride was evaluated for control of fourth-instar pecan weevil, Curculio caryae (Horn), at 25°C for a 24-h exposure. Larvae, collected as they naturally emerged from pecans, were used to artificially infest pecan nuts. Infested nuts were fumigated with six concentration by time (CT) treatment dosages of sulfuryl fluoride (0-750 g-h/m3) within air-tight, glass containers. The sulfuryl fluoride concentration in each fumigation container was analyzed 30 min after sulfuryl fluoride introduction and just prior to termination of the experiment. Mean sulfuryl fluoride CT dosages were calculated from sulfuryl fluoride measurements and were used for probit analysis. The lethal accumulated dosage (LAD99) of sulfuryl fluoride for pecan weevil was 1052.0 g-h/m3 with a 95% C.I. of 683.21-2,573.0 g-h/m3. For the confirmatory trial, we used two sulfuryl fluoride CT dosage treatments, 1,100 and 1,300 g-h/m3, and a nonfumigated control. All larvae were dead in both fumigation treatments by 14-d postfumigation. Due to higher mortality in the nonfumigated control in the confirmatory trial compared to that of the dose-response trial, 1300 g-h/m3 was selected as the sulfuryl fluoride CT dosage for a proposed quarantine treatment schedule. Fumigating pecans with sulfuryl fluoride can control larval pecan weevil infestations in commercially traded nuts and maintain compliance with quarantine regulations both within and outside the United States.

Efficacy of Controlled Atmosphere Treatments to Manage Arthropod Pests of Dry-Cured Hams.

Research here explored the use of controlled atmospheres (CA) for managing arthropod pests that infest dry-cured hams. Experiments were conducted with low oxygen (O2) achieved with low pressure under a vacuum, high carbon dioxide (CO2), and ozone (O3). Results showed that both low O2 and high CO2 levels required exposures up to 144 h to kill 100% of all stages of red-legged ham beetle, Necrobia rufipes (De Geer) (Coleoptera: Cleridae) and ham mite Tyrophagus putrescentiae (Schrank) (Sarcoptiformes: Acaridae) at 23 °C. In addition, both low O2 and high CO2 had no significant mortality against the ham beetle and ham mites at short exposures ranging from 12 to 48 h. Ham beetles were more tolerant than ham mites to an atmosphere of 75.1% CO2 and low pressure of 25 mm Hg, which imposed an atmosphere estimated at 0.9% O2. Both low O2 and high CO2 trials indicated that the egg stages of both species were more tolerant than other stages tested, but N. rufipes eggs and pupae were more susceptible than larvae and adults to high concentration ozone treatments. The results indicate that O3 has potential to control ham beetles and ham mites, particularly at ≈166 ppm in just a 24 h exposure period, but O3 is known from other work to have poor penetration ability, thus it may be more difficult to apply effectively than low O2 or high CO2. would be. CA treatment for arthropod pests of dry-cured hams show promise as components of integrated pest management programs after methyl bromide is no longer available for use.

Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum.

BACKGROUND: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide. RESULTS: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression. CONCLUSIONS: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.

Fumigation of bed bugs (Hemiptera: Cimicidae): effective application rates for sulfuryl fluoride.

The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), has resurged recently as a domestic pest in North America with very limited options for decisive control. We report efficacy studies with sulfuryl fluoride (SF) toward use as a structural fumigant to control bed bugs. Laboratory studies were conducted in which eggs, adults, and nymphs from a pesticide susceptible laboratory population were fumigated for 24 h using SF at 99.8% purity in airtight, 3.8-liter glass containers under two temperatures, 25 degrees C and 15 degrees C. Bed bugs were placed in separate ventilated glass vials and wrapped in mattress padding before fumigation. The gas concentration within each jar was determined using quantitative gas chromatography-mass spectrometry. Dose-response trials using eggs of known age (48-96 h) were conducted at five or six target concentrations measured as concentration x time accumulated dosages (g-h/m3) and one untreated control at each temperature. Each target dose was replicated in four different fumigation containers (replicates), with at least 32 eggs per replicate. The number of hatched and unhatched eggs postfumigation, and number of live and dead nymphs that resulted from hatched eggs, were evaluated daily for at least 1 wk after egg hatch. The lethal accumulated dosage (LAD99) for bed bug eggs was 69.1 (95% fiducial limits [FLs] of 62.9-79.5) g-h/m3 at 25 degrees C and 149.3 (95% FLs of 134.4-177.9) g-h/m3 at 15 degrees C. Confirmatory trials with dosages of 1.5x the LAD99 were conducted at 25 degrees C and 1.5x the threshold mortality dose at 15 degrees C with at least 15 adults, 13 late-instar nymphs and 79 eggs of known age per replicate. At 25 degrees C, a target dosage of 103.7 g-h/m3 resulted in 100% mortality of adults and late-instar nymphs. Nymphs emerged and survived from two of 439 eggs treated with SF dosages that were 6-7 g-h/m3 less than the target dosage. No nymphs emerged from eggs fumigated with dosages > 97.9 g-h/m3 in the validation study. Therefore, the threshold dosage for complete egg mortality (97.9 g-h/m3) was used, rather than the LAD99, to calculate the monitored field dosage rate of 148.2 g-h/m3 (= 1.5 x 97.9 g-h/m3) for control of all life stages of bed bugs at 25 degrees C. Based on these results, at 15 degrees C, 1.5x the threshold dosage for complete egg control (189.7 g-h/m3) was used to calculate a target dosage of 285 g-h/m3 for the confirmatory trial, which resulted in 100% mortality of adults, late-instar nymphs, and eggs.

Anatomical localization and stereoisomeric composition of Tribolium castaneum aggregation pheromones.

We report that the abdominal epidermis and associated tissues are the predominant sources of male-produced pheromones in the red flour beetle, Tribolium castaneum and, for the first time, describe the stereoisomeric composition of the natural blend of isomers of the aggregation pheromone 4,8-dimethyldecanal (DMD) in this important pest species. Quantitative analyses via gas chromatography-mass spectrometry showed that the average amount of DMD released daily by single feeding males of T. castaneum was 878 ± 72 ng (SE). Analysis of different body parts identified the abdominal epidermis as the major source of aggregation pheromone; the thorax was a minor source, while no DMD was detectable in the head. No internal organs or obvious male-specific glands were associated with pheromone deposition. Complete separation of all four stereoisomers of DMD was achieved following oxidation to the corresponding acid, derivatization with (1R, 2R)- and (1S, 2S)-2-(anthracene-2,3-dicarboximido)cyclohexanol to diastereomeric esters, and their separation on reversed-phase high-performance liquid chromatography at -54°C. Analysis of the hexane eluate from Porapak-Q-collected volatiles from feeding males revealed the presence of all four isomers (4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) at a ratio of approximately 4:4:1:1. A walking orientation bioassay in a wind tunnel with various blends of the four synthetic isomers further indicated that the attractive potency of the reconstituted natural blend of 4:4:1:1 was equivalent to that of the natural pheromone and greater than that of the 1:1 blend of (4R,8R)/(4R,8S) used in commercial lures.

Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle.

The insect arginine vasopressin-like (AVPL) peptide is of special interest because of its potential function in the regulation of diuresis. Genome sequences of the red flour beetle Tribolium castaneum yielded the genes encoding AVPL and AVPL receptor, whereas the homologous sequences are absent in the genomes of the fruitfly, malaria mosquito, silkworm, and honeybee, although a recent genome sequence of the jewel wasp revealed an AVPL sequence. The Tribolium receptor for the AVPL, the first such receptor identified in any insect, was expressed in a reporter system, and showed a strong response (EC(50)=1.5 nM) to AVPL F1, the monomeric form having an intramolecular disulfide bond. In addition to identifying the AVPL receptor, we have demonstrated that it has in vivo diuretic activity, but that it has no direct effect on Malpighian tubules. However, when the central nervous system plus corpora cardiaca and corpora allata are incubated along with the peptide and Malpighian tubules, the latter are stimulated by the AVPL peptide, suggesting it acts indirectly. Summing up all the results from this study, we conclude that AVPL functions as a monomer in Tribolium, indirectly stimulating the Malpighian tubules through the central nervous system including the endocrine organs corpora cardiaca and corpora allata. RNA interference in the late larval stages successfully suppressed mRNA levels of avpl and avpl receptor, but with no mortality or abnormal phenotype, implying that the AVPL signaling pathway may have been near-dispensable in the early lineage of holometabolous insects.