Toxicity and repellency of origanum essential oil and its components against Tribolium castaneum (Coleoptera: Tenebrionidae) adults

The components of Origanum vulgare L. essential oil showing insecticidal activity and repellency against red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), adults were analysed by GC-MS. All constituents were identified, and the main components were carvacrol (67.2%), p-cymene (16.2%), γ-terpinene (5.5%), thymol (4.9%), and linalool (2.1%). In a vapor phase fumigant assay, the origanum oil was more effective in closed conditions (LD₅₀ = 0.055 mg/cm³) than in open conditions (LD₅₀ > 0.353 mg/cm³). This suggests that toxicity is exerted largely in the vapor phase. Based on 24-h LD₅₀ values, the toxicity of caryophyllene oxide (0.00018 mg/cm³) was comparable with that of dichlorvos (0.00007 mg/cm³). In addition, thymol, camphene, α-pinene, p-cymene, and γ-terpinene showed good insecticidal activity (LD₅₀ = 0.012–0.195 mg/cm³). In repellency tests using 9 constituents of origanum oil, caryophyllene oxide showed complete repellency at 0.03 mg/cm². Hydrogenated monoterpenoids, such as thymol, α-pinene, carvacrol, and myrcene, elicited strong repellency at 0.03 and 0.006 mg/cm². Repellency depended on both time and concentration. These results indicate that origanum oil and its components could be potential candidates as a fumigant and repellent for managing T. castaneum adults.     

A family of chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera)

Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR). To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs) and 26 olfactory receptors (Ors) was identified. The receptors have seven transmembrane domains (7TMs) and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b), which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO(2) receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG). These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species.     

Hydrocarbon Repellents Isolated from Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae)

Seven unsaturated hydrocarbons were isolated from the volatiles of two species of flour beetles, Tribolium castaneum and T. confusum, and identified as 1-pentadecene, 1-heptadecene, 1,8-heptadecadiene, 1-tetradecene, 1-hexadecene, 1,6-pentadecadiene and heptadecatriene by GLC, GLC–MS, NMR, IR and micro-ozonolysis. This is the first report of the presence of these compounds in red flour beetles, and the last four compounds are also reported for the first time in confused flour beetles. These compounds are strong repellents for the same species or each other. The roles of these compounds are discussed.     

Pheromone production by male Tribolium castaneum (Coleoptera: Tenebrionidae) is influenced by diet quality

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), the red flour beetle, is a common cosmopolitan pest exploiting a variety of stored products. We experimentally manipulated diet nutritional quality by using non-nutritive filler to examine how this influenced pheromone production and olfactory attractiveness of T. castaneum adult males. Volatiles released by individual males reared on high versus low nutrition diets were collected using solid phase microextraction, and gas chromatography coupled to mass spectrometry was used to identify and quantify the Tribolium aggregation pheromone 4, 8-dimethyldecanal (DMD). Males kept on high nutrition diet showed a three-fold increase in daily DMD production, which suggests the possibility that this pheromone could act as a condition-dependent mating signal. In pitfall trap assays, there was no significant difference in the mean response of virgin females to discs kept with low versus high nutrition males, although discs carrying male cues were significantly more attractive than blank discs. These results suggest that DMD production rates by T. castaneum males will depend on the nutritional quality of various stored products, but such differences may not alter males' ability to attract females.     

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.     

Genetic structure of Tribolium castaneum (Coleoptera: Tenebrionidae) populations in mills

The red flour beetle, Tribolium castaneum (Herbst), is primarily found associated with human structures such as wheat and rice mills. Such structures are predicted to be spatially isolated resource patches with frequent population bottlenecks that should influence their genetic structure. Genetic diversity and differentiation among nine populations of T. castaneum collected from wheat and rice mills (ranging from <1-5,700 km apart) were investigated using eight polymorphic loci (microsatellites and other insertion-deletion polymorphisms, each with 3-14 alleles). Seventy-two locus-by-population combinations were evaluated, of which 31 deviated significantly from Hardy-Weinberg equilibrium, all because of a deficiency of heterozygotes. AMOVA analysis indicated significant differences among populations, with 8.3% of the variation in allele frequency resulting from comparisons among populations, and commodity type and geographic region not significant factors. Although there were significant differences in genetic differentiation among populations (F(ST) values = 0.018-0.149), genetic distance was not significantly correlated with geographic distance. Correct assignment to the source population was successful for only 56% of individuals collected. Further analyses confirmed the occurrence of recent genetic bottlenecks in five out of nine populations. These results provide evidence that populations of T. castaneum collected from mills show spatial genetic structure, but the poor ability to assign individuals to source populations and lack of isolation by distance suggest greater levels of gene flow than predicted originally.     

Selection for high and low amylase activity in adult flour beetles (Tribolium confusum) (Coleoptera,Tenebrionidae)

As a result of selection for high amylase activity in two strains of the flour beetle Tribolium confusum, mean activity considerably increased. Selection for low activity was ineffective in one strain, and mean activity increased, contrary to expectation, in the downward-selection line of the other.Heritabilities of amylase activity were 0.52 and 0.18 for the two strains (estimated by offspring-midparent regression in generation 1). Environmental factors have considerable effects on activity.The outcome of selection and possible models for the genetic control of amylase activity in Tribolium are discussed.     

Anti-infectious bronchitis virus (IBV) activity of 1,8-cineole: effect on nucleocapsid (N) protein

In the present study, anti-IBV (infectious bronchitis virus) activity of 1,8-cineole was studied by MTT assay, as well as docking and molecular dynamic (MD) simulations. The CC50 of 1,8-cineole was above 10 mM. And the maximum noncytotoxic concentration (TD0) of 1,8-cineole was determined to be 3.90 ± 0.22 mM, which was much higher than that of ribavirin (0.78 ± 0.15 mM). 1,8-cineole could inhibit IBV with an IC(50) of 0.61 mM. MTT assay showed that the inhibition of IBV by 1, 8-cineole appears to occur moderately before entering the cell but much strongly after penetration of the virus into the cell. In silico simulations indicated that the binding site of 1,8-cineole was located at the N terminus of phosphorylated nucleocapsid (N) protein, with interaction energy equaling -40.33 kcal mol(-1). The residues TyrA92, ProA134, PheA137, AspA138 and TyrA140 had important roles during the binding process and are fully or partially conserved in various IBV strains. Based on spatial and energetic criteria, 1,8-cineole interfered with the binding between RNA and IBV N-protein. Results presented here may suggest that 1,8-cineole possesses anti-IBV properties, and therefore is a potential source of anti-IBV ingredients for the pharmaceutical industry.     

Insecticidal effects of extracts of seven plant species on larval development, alpha-amylase activity and offspring production of Tribolium castaneum (Herbst) (Insecta: Coleoptera: Tenebrionidae)

Bioinsecticidal effects of methanol extracts from seven plant species on Tribolium castaneum were investigated. Centaurium erythraea, Peganum harmala, Ajuga iva, Aristolochia baetica, Pteridium aquilinum and Raphanus raphanistrum extracts inhibit growth of larvae. C. erythraea was the most toxic with 63% mortality 10 days after treatment, followed by P. harmala with 58%. C. erythraea and P. aquilinum reduce the emergence rate respectively of 66% and 19%. The duration of larval period was shortened by Launaea arborescens, P. aquilinum and A. iva extracts, whereas R. raphanistrum and P. harmala extracts extend the larval period when compared to the control. Extracts of C. erythraea, P. harmala, A. iva and A. baetica inhibited F1 progeny production. Larvae possess three alpha-amylase isoforms as determined by SDS-PAGE. Larvae fed on treated diet had lower alpha-amylase activity than larvae feed on untreated diet. C. erythraea and P. harmala are the most potent extracts. These plant extracts could be useful to reduce seed damage caused by this pest species.     

Bio-insecticidal activities of halophytic plant extracts against Tribolium confusum (Coleoptera, Tenebrionidae)

Salt marsh plants were tested for their insecticidal activities against larvae of the confused flour beetle Tribolium confusum (Tenebrionidae). 16 aerial part extracts were obtained using organic solvents of increasing polarity and tested for their anti-feedant and toxicity effects. Responses varied with plant material and extract type. Ethyl acetate extracts of F. laevis, S. echioides and. T. boveana and petroleum ether extract of F. laevis presented, anti-feedant property. However, S. fructicosa seemed to be attractive to the tested flour beetle. Mortalities of 97, 87, 97 and 80% were observed by using respectively ethyl acetate extracts of F. laevis, S. echioides and T. boveana and petroleum ether extract of F. laevis, when applied at a dose of 1%, mixed with the insect diet. This preliminary study showed that F. laevis, S. echioides and T. boveana presented potential bio-insecticidal activity with ethyl acetate extracts, similar result was found with petroleum ether extract of F. laevis. More complementary studies are needed for the use of these extracts to control T. confusum.     

Progeny production by Tribolium castaneum (Coleoptera: Tenebrionidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae) on maize previously infested by Sitotroga cerealella (Lepidoptera: Gelechiidae)

A laboratory experiment was conducted to quantify the effects of infestation of maize by Sitotroga cerealella (Olivier) on progeny production by two common secondary colonizers of grain, Tribolium castaneum (Herbst) and Oryzaephilus surinamensis (L.). Adults of both secondary pest species were allowed to oviposit for 3 wk on intact kernels of 'DeKalb 689', mechanically split kernels, kernels that had been infested for 3 mo by S. cerealella, and kernels that had been infested for 6 mo. Progeny of both species reached highest numbers on 6-mo infested maize. Prior infestation for 6 mo by S. cerealella makes maize a more suitable medium for reproduction by T. castaneum and O. surinamensis, much more than can be accounted for by mere disruption of kernel integrity resulting from larval feeding. The results highlight the importance of limiting establishment by S. cerealella on maize in storage.     

Directional and correlated effects of selection on amylase activity,weight and developmental time in Tribolium confusum (Coleoptera,Tenebrionidae)

The interrelationships of amylase activity, weight and developmental time in a strain of Tribolium confusum were studied by selecting, in turn, for one of these traits and monitoring other, non-target characters, in all lines.Experimental lines were selected for high and low amylase activity (AH, AL), for heavy and light adult weight (WH, WL) and for fast and slow development (DF, DS). The variables monitored every generation in all lines were mean amylase activity (measured colorimetrically), mean individual adult weight, median egg-to-pupa developmental time, mean productivity (offspring per fertile pair) and % sterile pairs.The six lines may be grouped by their response to selection in a meaningful way. AH, WH, and DS all had higher amylase activity, heavier adult weight, slower development and lower fitness (lower productivity, higher percent sterility) than the corresponding AL, WL, and DF, although a different variable was under selection in each pair of lines. These results demonstrate that amylase activity, developmental time and individual weight are intercorrelated.     

Comparative insecticidal activity of different plant materials from six common plant species against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the major insect pests of stored grains. Due to export legislation and zero-tolerance for live insect in trade commodities, extensive use of synthetic insecticides is in practice in order to eliminate pest infestations from the lots. Currently, the one and only acceptable chemical to be used in stored grain is phosphine but due to its excessive usage the stored grain pests are becoming resistant against it. Hence discovery of alternative compounds is much needed. In this study we have compared insecticidal efficacy of different plant materials from six commonly grown plants of Pakistan, viz. Allium sativum (Alliaceae), Zingiber officinale (Zingiberaceae), Cymbopogon citratus (Poaceae), Eucalyptus globulus (Myrtaceae), Nicotiana tabacum (Solanaceae), and Azadirachta indica (Meliaceae) against T. castaneum infesting stored wheat, rice, corn and gram pulse. Various plant parts were dried, powdered, and used as admixtures to the stored commodities in the experiments. The results have suggested that A. sativum (garlic) and Z. officinale (ginger) were more effective resulting into 15 times higher adult mortality and 4 to 5 times reduction in grain weight losses when mixed with rice grains. Similarly, A. indica when admixture with wheat checked the population growth in the resources resulting into 3.5 times less adult production compared to controls. A subsequent experiment was conducted to study the dose response of neem seed powder against the beetle pest infesting milled products. Surprisingly, better control was observed either at lowest (1% w/w) or the highest doses (5% w/w). This finding is of great interest to understand the underlying phenomenon which we assume is the ability of T. castaneum to feed selectively in flour mediums, however, further research on this aspect is required to be investigated. The results of this study support the use of botanicals for stored product pest management.     

Variation between field and laboratory populations of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Rates of development and pupal weights were compared between seven field populations and two laboratory strains of Tribolium castaneum. Rates of development did not differ significantly between field populations, but pupal weights did vary. Laboratory strains were both slower to develop and heavier than the field populations. The data support the hypothesis that field populations undergo selection for fast development in transient habitats. The findings are discussed in the context of r and K selection in field populations and laboratory cultures.     

Multiple Origins of Cyclodiene Insecticide Resistance in Tribolium castaneum (Coleoptera: Tenebrionidae)

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a γ-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5–6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution. Received: 14 October 1998 / Accepted: 4 January 1999