Coleopteran Insect Pests Research Articles

Bio-efficacy and Persistence of Inert Dust Formulations as Stored-grain Protectants against Sitophilus oryzae (L.)


 
 
 Inert dust formulations of natural origin act as promising alternatives in controlling storage infestations caused by deleterious coleopteran insect pests. In that context, laboratory studies were conducted in order to evaluate the insecticidal activity, progeny inhibition and persistence of three commercially available dust formulations, namely, diatomaceous earth, zeolite and cloisite 20A against Sitophilus oryzae adults, a major coleopteran pest of stored grains, throughout 90 days of storage period. Adulticidal and persistence bioassays were conducted to evaluate the toxicity of binary combinations of inert dusts using their sub-lethal doses towards the test insect for 60 days of storage period. Further, ultrastructural architecture of the test insect pest species was examined via Field Emission Scanning Electron Microscopy (FE-SEM), and Energy Dispersive X-Ray Analysis (EDX) to study the uptake/penetration pattern of inert dust particles through the cuticular layers of target insect pest. All inert dust formulations exhibited very efficacious toxic and progeny inhibition activities and extraordinary mortality percentages irrespective of the inert dust used at the end of initial 30 day-long storage period. Thereafter, mortality percentages gradually declined with the progress of the storage time period, declining in the order of, diatomaceous earth>zeolite>cloisite 20A. All the dust formulations successfully inhibited the progeny production and the lowest average progeny production was recorded at the end of initial storage period. Progeny inhibition decreased with the progress of storage time. Binary combinations of inert dust formulations exhibited additive and antagonistic effects against S. oryzae and similarly, the mortality percentages gradually decreased with the prolongation of the storage period. The FE-SEM and EDX micrographs clearly indicated the presence of dust particles and the distribution of their constituting elements on the cuticular layer of the exposed insect pests with the appearances of abrasions and scratches that may have led to dehydration and eventual death of S. oryzae. Thus, findings of the present study suggest that, naturally-derived diatomaceous earth, zeolite and cloisite 20A can be used as eco-friendly means as stored grain protectants against S. oryzae populations successfully in the storage grain systems.
 Keywords: Sitophilus oryzae, Diatomaceous earth, Cloisite 20A, Zeolite
 
 

Virulence of Native Isolates of Entomopathogenic Nematodes for the Management of White Grubs

An alternative to chemical control, entomopathogenic nematodes are very effective against insect pests. Native isolates of Steinernema siamkayai, S. carpocapsae, Heterorhabditis indica, H. bacteriophora, Heterorhabditis sp. and Oscheius sp. have been evaluated for their virulence against white grubs. Under laboratory conditions, S. glaseri was more susceptible to A. communis in potato. S. glaseri@ 2.5 x10 9 IJ/ ha showed the highest grub mortality of 83.33% and 19.04% tuber damage. The same dosage of Heterorhabditis sp. in pot culture caused 18.66% tuber damage and 74.05% larval mortality. Steirnernema glaseriand Heterorhabditis sp. effectiveness against the coleopteran insect pests of Anomala communis<?i> in potato was conducted under field conditions. The maximum grub mortality with S. glaseri at 2.5 x10 9 IJ/ ha was 71.33%. With Heterorhabditis sp. at 2.5 x10 9 IJ/ ha, the yield increased by 14.14 t/ ha while tuber damage was 24.99%. However, the nematode S. glaseri and Heterorhabditis sp. has its best ability to live at the lowest temperature in the field trial, Anomala communiswas successfully managed by S. glaseri followed Heterorhabditis sp. in potato. The natural distribution of the native isolates of EPNs may be used for local insect pest management.

Ozone Efficiency on Two Coleopteran Insect Pests and Its Effect on Quality and Germination of Barley.

Simple SummaryRhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) are notorious global pests, destroying various stored grains, including barley, wheat, oats, maize, and rice. Ozone (O3) is a promising fumigant to control pests in stored grain since it can safely and rapidly auto-decompose without leaving residues, however, relatively few studies have focused on the toxicity of O3 on stored grain pests in stored barley. In this study we not only explored the susceptibility of all life stages of R. dominica and T. castaneum in barley seeds to different durations of gaseous O3, but also investigated the effect of O3 on germination ability, seedling growth, and quality of barley. O3 was effective against all life stages of two species in barley under sufficient exposure times without negative impacts affecting the commercial quality of barley. However, the germination ability and seedling growth were adversely impacted at longer O3 exposure times. Thus, it is imperative to select an optimal O3 exposure time to achieve the desired functional outcome, such as malting, animal feeding, and human consumption.Ozone (O3) is a potential fumigant to control pests in stored grain since it can safely and rapidly auto-decompose without leaving residues. In this study, the efficacy of O3 on all life stages of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) in barley and the physiological effects on barley and its quality were investigated. Complete control of all life stages of pests was obtained at 700 ppm for 1440 min of ozone exposure without negatively impacting the contents of soluble protein, moisture content, seed colour, hardness, and the weight of thousand barley seeds. The eggs and pupae of these two insects were the more tolerant stages than their larvae and adults. Prolonged exposure times (40 to 1440 min) and mortality assessment intervals (1, 2, and 7 days) increased O3 efficacy due to the reaction characteristics and delayed toxicity. Aging barley seeds appeared to be more sensitive to prolonged ozone duration than new seeds. A total of 20 and 40 min could promote germination rate, and longer O3 exposure (1440 min) was unfavourable for germination and seedling growth. Thus, it is imperative to select an optimal O3 exposure time to transfer ozone into quality contributors of final products and achieve the desired functional outcomes.

Laboratory and field studies on the combined application of Beauveria bassiana and fipronil against four major stored-product coleopteran insect pests.

In the current study we have tested the application of Beauveria bassiana (Hypocreales: Cordycipitaceae) alone and in combination with fipronil at two doses against Tribolium castaneum (Coleoptera: Tenebrionidae), Rhyzopertha dominica (Coleoptera: Bostrychidae), Sitophilus granarius (Coleoptera: Curculionidae), and Trogoderma granarium (Coleoptera: Dermestidae) under laboratory and field conditions. At laboratory conditions, the combination of B. bassiana with the highest dose of fipronil produced the highest mortality. At different temperatures, mortality was increased with the increase in temperature. Maximum mortality was observed at 30°C, followed by 25°C and 20°C for all tested species. Different treatments significantly reduced the progeny number in comparison to control groups for all tested species at all temperatures. In the persistence trial, all treatments that included the combinations of B. bassiana with fipronil produced significantly higher mortalities than the single treatments for all tested species over a period of 6months. Furthermore, all treatments significantly reduced the number of progenies of all insect species in comparison with the control groups over the same storage period. In field trials, mortalities of all tested insect species were significantly higher on wheat treated with B. bassiana, fipronil, or their combinations than on controls for an entire storage period of 180days. Overall, R. dominica was found the most susceptible species followed by S. granarius, T. castaneum, and T. granarium. The findings of the current study suggest that the use of B. bassiana and fipronil as grain protectants may provide elevated control against major stored-grain insect species during a prolonged period of storage.

Multiple and non-recessive resistance to Bt proteins in a Cry2Ab2-resistant population of Helicoverpa zea

Transgenic corn and cotton expressing Cry and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) have been planted for managing lepidopteran and coleopteran insect pests for more than two decades in the U.S. Evolution of insect resistance to these Bt proteins is a major threat for the continued efficacy of Bt technology. Understanding patterns of cross-resistance and dominance of resistance in target insects is essential for resistance management and pest control. The bollworm, also called corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt crops in the U.S. In this study, we evaluated the responses of Cry2Ab2-resistant (RR), susceptible (SS), and heterozygous (RS) strains of H. zea to the Cry1Ac, Cry1A.105, Cry2Ab2 and Vip3Aa39 proteins using diet-overlay bioassays. The Cry2Ab2-resistant H. zea also possessed high levels of resistance to Cry1Ac and Cry1A.105, but were very susceptible to Vip3Aa. The resistance to Cry1 and Cry2 proteins in RR H. zea varied from incompletely recessive to completely dominant depending on the protein concentration. These data suggest that the RR H. zea possess multiple non-recessive resistance to Cry1/Cry2 proteins. These results have significant implications for understanding field-evolved resistance of H. zea to Cry1 and Cry2 proteins in U.S. Bt corn and cotton fields and should contribute to developing effective management strategies for control of H. zea.

Two Splice Isoforms of Leptinotarsa Ecdysis Triggering Hormone Receptor Have Distinct Roles in Larva-Pupa Transition.

Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. In Drosophila melanogaster, the ethr gene produces two functionally distinct splicing isoforms, ethra and ethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of both ethra and ethrb in a Coleopteran insect pest, the Colorado potato beetle Leptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions of ethr. RNAi of dsethr, dsethra, or dsethrb by the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All the ethra depleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of the ethrb RNAi larvae developmentally halted at the prepupae stage. The remaining Ldethrb hypomorphs became pupae, with blackened wings and highly-expressed burs, pburs and four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition in L. decemlineata.

Soil Application of Metarhizium anisopliae JEF-314 Granules to Control, Flower Chafer Beetle, Protaetia brevitarsis seulensis

Root-feeding Scarabaeidae, particularly white grubs are considered among the most harmful coleopteran insect pests in turfgrass. In this work, sixteen entomopathogenic fungal species were assayed against flower chafer beetle, Protaetia brevitarsis (Coleoptera: Scarabaeidae) and Metarhizium anisopliae JEF-314 showed high virulence. The control ability of the isolate JEF-314 has been in detail tested for a model insect flower chafer beetle. Further analyses showed insect stage-dependent virulence where the fungal virulence was the highest against smaller instar larvae. Additionally, we confirmed that millet-based solid cultured granule was effective against the soil-dwelling larval stage. The isolate also showed a similar ability for a representative pest (Popillia spp.) in laboratory conditions. Our results clearly suggest a high potential of M. anisopliae JEF-314 to control the flower chafer beetle, possibly resulting in controlling of root-feeding white grubs in turfgrass. Based on the insect life cycle and susceptibility to the fungus, late spring and summer time would be the optimum time to apply JEF-314 granules for an effective control. Further characterization of the efficacy of the fungus under field conditions against the Scarabaeidae beetles might provide an efficient tool to control this beetle in an environment-friendly way.

Proteomic insights into the immune response of the Colorado potato beetle larvae challenged with Bacillus thuringiensis

Bacillus thuringiensis (Bt) toxins constitute effective, environmentally safe biopesticides. Nevertheless, insects' tolerance to Bt is influenced by environmental factors affecting immunity. To understand larval immune response in the devastating coleopteran insect pest Colorado potato beetle (CPB), we undertook a proteomic analysis of hemolymph of non-treated control larvae and larvae consuming non-lethal doses of spore-crystal mixtures containing the coleopteran-active Cry3Aa toxin. Results revealed lower amount of proteins involved in insect growth and higher amount of immune response-related proteins in challenged insects, sustaining the larval weight loss observed. Additionally, we found a potential regulatory role of the evolutionary conserved miR-8 in the insect's immune response relying on antimicrobial peptides (AMPs) production. Upon toxin challenge, different patterns of hemolymph AMPs expression and phenoloxidase activity were observed in CPB larvae reared on different Solanaceae plants. This suggests that diet and diet-associated insect midgut microbiota might modulate this insects' tolerance to non-lethal doses of Bt.

In the search for new secondary metabolites with biopesticidal properties

Secondary metabolites are involved in diverse functions in plants, including defense and protective processes. Information concerning the biosynthesis of secondary metabolites in plants points at a constitutive or induced chemical defense, generated for protection against a variety of phytopathogenic attacks. Our phytochemical studies are aimed at finding biopesticides of botanical origin. Some plant taxa of American distribution are toxic to selected insects, fungi and bacterial strains, and their effect has been associated with the presence of phenolics, phenylpropanoids and terpenes. We have isolated some diterpenes, triterpenes, sesquiterpene lactones, flavonoids, and phenylpropanoids from members of the plant families Araucariaceae, Asteraceae, Calceolariaceae, Celastraceae, and Rhamnaceae. In addition, we have identified a number of chemical derivatives of these compound classes from the plants. A major finding indicates that compounds or their derivatives that possess antioxidant, antifungal, insect growth regulator or insecticidal activity and enzymatic inhibitors are natural compounds. Insecticidal activities were assayed against strains of lepidopteran, dipteran, and coleopteran insect pests that affect many crops. Antifungal and antibacterial activities were assayed against phytopathogenic species of filamentous fungi and bacterial strains that are pests on many crops. Our results indicate that the plant-derived compounds obtained from the abovementioned plants have excellent insect growth regulatory activity and a good potency as antifungal agents. However, little is known about the effects of these natural compounds and their derivatives on insect pests. The natural compounds that we have isolated represent a valuable resource for future studies of plant chemical defense and the role of these substances in chemical ecology.

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.

Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis

Three-dimensional (3D) models for the 79.2kDa activated Cry1Ib9 and 77.4kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally β0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), β6(T390-Q393), β7(V398-W404), β8 (V418-W425), β9 (E453-N454), β10 (S470-I479) where as in domain III the changes were observed at positions β19 (R601-F607), β20 (609-L613), β21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), β1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at β2 (E292-L295), β3(V299-L308), β4(I340-F347), β5(D356-P368), β6(I375-T377), β7(V389-F394), β8(K398-N405), β9(Y416-Y427), β10 (T436-Y439), β12(G476-H495), β12A (M503-I504) where as in domain III main variations observed at positions of β18 (P583-I593), β19(F604-S610), β20(P611-L615), β21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.

Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests

The occurrence of Bacillus entomopathogenic bacteria on a Tunisian biodynamic farm was determined by examining 75 samples from olive tree ( Olea europaea L.) habitats. A total of 40 Bacillus isolates were characterized according to their phenotypic, physiological and biochemical parameters. Isolates of the species Bacillus subtilis, Bacillus mycoides, Brevibacillus brevis, Paenibacillus polymyxa, Bacillus licheniformis, Bacillus sp. (1), Bacillus sp. (2) and a standard strain Btk HD-1 were used separately in feeding bioassays on fresh artificial diet against larvae of lepidopterans Prays oleae (Bernard) and Palpita unionalis (Hübner) and coleopterans Hylesinus oleiperda (F.) and Phloeotribus scarabaeoides (Bernard), which are olive tree pests. Larvae were successfully reared on an artificial diet with 25 g powdered olive tree leaves. Compared to the control data, only Btk and the isolates of B. licheniformis, P. polymyxa and B. brevis were entomopathogenic. Larval mortality assessed 7 days post-treatment showed high mortality rates with Btk to lepidopteran larvae (86.6% for P. oleae and 80.9% for P. unionalis) and low mortality against coleopteran pests. B. brevis isolates showed high mortality rates against P. oleae (up to 67.9%). B. licheniformis isolates caused up to 59.2% larval mortality for P. oleae and 43.6% for P. unionalis. Highest coleopteran mortality was achieved by P. polymyxa isolates (up to 55%). According to the 16S rDNA results, isolates of each of the three entomopathogenic strains were similar. Proteins in the strain supernatants were toxic to P. oleae larvae with LC50 values of 10.0 ( B. brevis), 12.5 ( B. licheniformis) and 37.6 μg/ml ( P. polymyxa). Also, P. polymyxa showed an LC50 of 12.4 mg/l against P. scarabaeoides. Our results suggest that entomopathogenic Bacillus present locally in the biodynamic farm could be used in biological control programmes of olive tree pests.

Control of coleopteran insect pests through RNA interference

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

Field Studies Assessing Arthropod Nontarget Effects in <I>Bt</I> Transgenic Crops: Introduction

TEN YEARS AGO THE Þrst crops modiÞed through genetic engineering to produce novel in-plant protectants became commercially available in the United States. Since that time, the adoption and use of crops with built-in resistance to lepidopteran and coleopteran insect pests and tolerance to certain herbicides has grown rapidly in both industrial and developing countries worldwide. By 2004, 17 countries had adopted transgenic crops ranging from corn and cotton producing insecticidal proteins of Bacillus thuringiensis Berliner (Bt) to corn, cotton, soybean, and canolacontaining genetic constructs conferring tolerance to the herbicides glyphosate or glufosinate. It is estimated that in 2004, 81 million hectares of transgenic crops were cultivated in these 17 countries, the United States leading the way with 47.6 million hectares (James 2004). In 2004, 32% of the Þeld corn and 46% of the upland cotton acreage in the United States was planted with genetically modiÞed cultivars producing one or more Bt toxins (USDA 2004). The rapid and widespread adoption of transgenic crops in the United States and elsewhere has prompted extensive debate over multiple issues related to human safety and environmental risk. The putative environmental risks that have been articulated include outcrossing of nontransgenic plants through pollen drift, horizontal transfer of transgenes to unrelated organisms, loss of susceptibility to Bt toxins in target pests, disruption of ecosystem processes, and direct or indirect effects on nontarget organisms and biodiversity. There has been equal discourse expounding the potential beneÞts associated with the use of transgenic crops in agricultural production systems, including signiÞcant reductions in use of conventional, broad-spectrum insecticides, improved suppression of target pests, improved yields, reductions in production costs leading to increased proÞtability, and increased opportunities for biological control. Governmental agencies such as the U.S. Environmental Protection Agency (EPA) in the United States require toxicological testing of a limited number of representative nontarget organisms as one component of the commercial registration process for transgenic crops producing insecticidal proteins. However, there have been calls by research advisory groups (e.g., National Research Council, EPA-FIFRA ScientiÞc Advisory Panels) and private advocacy groups for

Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say)

Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis δ-endotoxins.In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.

Transgenes for Pest and Disease Resistance

Using a leaf disc transformation procedure andAgrobacterium- mediated gene transfer, transgenic apple and strawberry plants have been produced that carry a gene encoding a cowpea trypsin inhibitor (CpTl). This gene is known to confer resistance to several lepidopteran and coleopteran insect pests. Scientific and regulatory aspects of this type of work are discussed.