Variation in the production and distribution of substituted benzoquinone compounds among genetic strains of the confused flour beetle, Tribolium confusum

Insects often produce chemicals, such as defensive compounds, whose quantity and distribution can affect their fitness. For evolution to produce adaptations, chemical production must be genetically variable. Here we report the results of a study using high-performance liquid chromatography to quantify two important chemical secretions of the flour beetle Tribolium confusum, methyl-1, 4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ). Our results show a distinct difference in the production of the compounds among four genetically distinct strains of T. confusum (b-+, b-I, b-IV, b-Pakistan) with an unusually high amount measured for the b-Pakistan strain. By measuring internal and external benzoquinone levels separately, we were also able to detect differences in production and distribution of the compounds between the strains. Some strains secrete more of the chemicals, whereas other strains appear to sequester the compounds within their bodies. The sexes also differ in total quinone production as well as in their internal to external benzoquinone ratios, suggesting the trait is sex influenced. Finally, a consistent correlation in the amounts of MBQ to EBQ in individual beetles suggests that the substituted benzoquinones share a common precursor or pathway.     

Benzoquinone levels as a function of age and gender of the red flour beetle, Tribolium castaneum

Liquid chromatography with both photodiode array and electrochemical detection was used to analyze as a function of age and gender the levels of two ρ-quinones, methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ), which are found in defensive secretions of the red flour beetle, Tribolium castaneum. We developed a method to simultaneously analyze quinones and hydroquinones excreted from or in homogenates of individual beetles. The major components present in beetle extracts were the benzoquinones and not free or conjugated forms of the hydroquinones. Greater than 95% of the quinone/hydroquinone mixture in extracts was present in the oxidized form. Because of their lability, however, the quinones were quantified indirectly as their hydroquinone derivatives after extraction in dilute acid supplemented with ascorbic acid as a reducing agent. Comparisons of whole body rinses and homogenates revealed that rinses recovered only up to 60% of the total quinones that were extracted after homogenization. The levels recovered also depended on the age and sex of the individual beetles sampled. ρ-Benzoquinones in both male and female beetles increased after adult eclosion and cuticle sclerotization for 40–50 days and then remained at their highest levels (15–21 μg MBQ and 22–32 μg EBQ per beetle) through 80 days posteclosion. Virgin females that were collected 40–80 days after eclosion contained approximately 40% more of these compounds than males of the same age. The build-up of ρ-benzoquinones subsequent to cuticle sclerotization apparently reflects the need for an adequate cuticular barrier for self-protection from these defensive compounds.     

Volatile secretions and epicuticular hydrocarbons of the beetle Ulomoides dermestoides

Many species of tenebrionids produce and secrete a defensive volatile blend containing mainly benzoquinones and alkenes. In this study we characterized the volatile organic compounds (VOC) of the beetle Ulomoides dermestoides (Coleoptera: Tenebrionidae). Solid phase microextraction (SPME) coupled to capillary gas chromatography–mass spectrometry (CGC–MS) analysis was used to identify methyl-1,4-benzoquinone (MBQ), ethyl-1,4-benzoquinone (EBQ), 1-tridecene (C_13:1), and 1-pentadecene (C_15:1), representing more than 90% of the volatile blend. We also used CGC–MS to analyze the epicuticular hydrocarbons of U. dermestoides. Saturated, unsaturated, and branched structures with chain lengths ranging from 13 to 43 carbons were detected. n-pentacosane (C_25:0) and 9,11-pentacosadiene (9,11-C_25:2) were the most abundant components, representing more than 40% of the cuticular hydrocarbons.     

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.     

Plant essential oils: a substitute for conventional insecticides against Tribolium species (Coleoptera: Tenebrionidae)-achievements and challenges

Abstract

The literature on efficacy of plant essential oils and their constituents for the management of Tribolium spp. (T. castaneum and T. confusum), instigating infestation of a variety of stored grains and their products is reviewed here. Both species caused significant losses in the diverse food grains and their products through infestation. Essential oils of higher plants showed potential activity in the management of these infestation driven losses. Researchers have found that essential oils and their chemical constituents have significant toxicity against both the species of Tribolium via different modes of action like repellent, insecticidal, ovicidal, larvicidal, pupicidal, ovipositional and feeding deterrent actions. Concerning repellency and in vivo bioassays, few studies have been done with the essential oils against T. confusum, thus, more investigations are required to find the repellent agents against this insect. There are some essential oil-based botanical insecticides which have been proved effective for the protection of food grains from both beetles during storage. These botanical insecticides also exhibited behaviour altering properties against both beetles, thus, reducing the problem of pest resistance which is a problem with conventional insecticides. Therefore, essential oils-based botanical insecticides may be preferred option than conventional insecticides for protecting stored grains and their products against Tribolium infestation.     

Analysis of orientation behaviour of Tribolium castaneum and T. confusum to synthetic aggregation pheromone

Adult male T. castaneum (Herbst) and T. confusum (du Val) secretes an aggregation pheromone that is attractive to both sexes. Orientation behaviour of the two Tribolium species responding to different concentrations of synthetic aggregation pheromone in still and moving air was studied in an 2.5 m×0.4 m olfactometer. Analysis of Tribolium tracks indicated that the aggregation pheromone stimulated the beetles to walk faster at higher concentrations to increase the frequency and magnitude of turning and to decrease track reversal distances and distances between turns. The mean walking speed of both species was lowest at the highest air speed. The behavioural responses of the beetles to the pheromone in still and moving air were similar, indicating chemotaxis as the major orientation mechanism used by both species to locate an odour source. The beetles showed greater orientation efficiency within a discrete pheromone plume than a diffuse plume.     

Behavioural responses of Oryzaephilus surinamensis, Tribolium castaneum and Tribolium confusum to naturally and artificially damaged durum wheat kernels

Observations on behavioural activity involved in the host selection by secondary pests of stored grains, Oryzaephilus surinamensis (Linnaeus), Tribolium castaneum (Herbst) and Tribolium confusum J. du Val, with respect to intact and mechanically or naturally damaged kernels are reported. Our results indicate that the attraction of secondary pests is facilitated by broken grain kernels, which resulted from either mechanical damage during harvesting and/or binning procedures, or the feeding activity of primary insect pests. Insect damaged kernels were more attractive to O. surinamensis, T. castaneum and T. confusum than whole kernels; in addition insect damaged kernels elicit more attractiveness than mechanically split kernels. The damage caused by primary pests, such as Rhyzopertha dominica (Fabricius) and Sitophilus oryzae (Linnaeus), on whole kernels may facilitate colonization by secondary pests, which continue damaging the cereals. O. surinamensis, T. castaneum and T. confusum utilize the grain volatile odours to distinguish whether the grain kernels of the stored cereals are damaged mechanically or by insects.     

The dietary relationships of Tribolium castaneum (Herbst) with microfungi

The ecology of Tribolium castaneum (Herbst) outside of grain storage facilities is poorly known. However, high densities of T. castaneum adults are known to infest stored cotton seeds (Gossypium hirsutum (L.)) in Queensland, Australia, despite the absence of stored food products in the immediate vicinity. Previous studies suggest that the beetles are attracted to the fungal colonies growing on the residual fibres that remain on the cotton seeds after ginning, but the specifics of this remain unclear, even as to the species of fungi involved. In the current study, 14 fungal species were isolated from stored cotton seeds collected from four sites in Queensland. The feeding preferences of the adult beetles and the developmental success of the larvae were recorded for each fungal species. Gut analyses of T. castaneum adults, after exposure in no-choice feeding tests, showed that the beetles will feed on any one of the 14 fungal species when exposed (over 14 days) to cotton seeds that had been inoculated with one particular fungal species. The developmental success of the larvae varied depending on the fungal species, with most fungal species supporting only low levels of successful development (<30%). Tests with each fungal species and the volatiles they release revealed that neither adult nor larval survival was significantly affected by the presence of any particular fungal isolate. These results indicate that T. castaneum will feed on a variety of fungal species but that none of the tested fungal species on its own provides a good larval diet for T. castaneum.     

Flight directionality of Tribolium castaneum soon after take‐off under glasshouse and field conditions

Flight directionality of the rust‐red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), was investigated under glasshouse and field conditions using sticky traps placed around dense experimental infestations of T. castaneum derived from field‐collected samples. Although beetles of this species are known to fly quite readily, information on flight of beetles away from grain resources is limited. Under still glasshouse conditions, T. castaneum does not demonstrate strong horizontal or vertical trajectories in their initial flight behaviour. Flight was significantly directional in half of the replicates, but trapped beetles were only weakly concentrated around the mean direction of flight. In the field, by contrast, emigration of T. castaneum was strongly directional soon after flight initiation. The mean vector lengths were generally >0.5 which indicates that trapped beetles were strongly concentrated around the calculated mean flight direction. A circular‐circular regression of mean flight vs. mean downwind direction suggested that flight direction was generally correlated with downwind direction. The mean height at which T. castaneum individuals initially flew was 115.4 ± 7.0 cm, with 58.3% of beetles caught no more than 1 m above the ground. The height at which beetles were trapped did not correlate with wind speed at the time of sampling, but the data do indicate that wind speed significantly affected T. castaneum flight initiation, because no beetles (or very few; no more than three) were trapped in the field when the mean wind speed was above 3 m s^?1. This study thus demonstrates that wind speed and direction are both important aspects of flight behaviour of T. castaneum, and therefore of the spatio‐temporal dynamics of this species.     

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6–6.0 in the anterior to 7.0–7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p‐nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin‐, chymotrypsin‐, and elastase‐like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases. © 2009 Wiley Periodicals, Inc.     

Undirectional interspecific mating in Tribolium castaneum and T. confusum: evolutionary and ecological implications

Interspecific mating between two species of Tribolium occurs only between males of T. confusum and females of T. castaneum. The reciprocal mating combination was not observed. This pattern of unidirectional mating resulted in a frequency-independent reduction in the number of inseminated females in mixed populations in comparison with single-species mating groups. The ecological significance in terms of interspecific competition, and the possibility of identifying evolutionarily ancestral and derived species from such data are discussed.

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Accouplements non réciproques entre Tribolium castaneum et T. confusum: conséquences évolutives et écologiques

Résumé Les accouplements interspécifiques entre deux espèces de Tribolium n'ont lieu qu'entre mâles de T. confusum et femelles de T. castaneum. Les combinaisons réciproques n'ont pas été observées. Ce type d'accouplement asymétrique a provoqué une réduction indépendante de la fréquence dans le nombre de femelles inséminées dans les populations par rapport aux groupes d'accouplements monospécifiques. La discussion porte sur la signification écologique dans la compétition interspécifique et les possibilités d'identification des espèces ancestrales et dérivées.

     

Identification of microsatellite markers in the red flour beetle,Tribolium castaneum

We isolated and characterized microsatellite markers for the red flour beetle Tribolium castaneum, an important model species for studies in various areas of evolutionary biology and ecology. A microsatellite‐enriched genomic library was constructed and screened with single stranded oligonucleotide probes [(CCT)₁₇, (AAT)₁₇ and (CAG)₁₇]. Forty‐five primer pairs were designed of which 19 pairs produced successful amplification. Polymorphism screening involved beetles from five beetle strains and revealed 15 polymorphic and four monomorphic markers. The development of polymorphic microsatellite markers will facilitate future ecological and genetic studies involving T. castaneum beetles.     

Response of Tribolium castaneum and Rhyzopertha dominica to various resources,near and far from grain storage

Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) are common cosmopolitan pests of stored grain and grain products. We evaluated the relative attraction of T. castaneum and R. dominica to wheat, sorghum and cotton seeds in the field, near grain storage facilities and well away from storages in southern and central Queensland using multiple trapping techniques. The results show that T. castaneum is more strongly attracted to linted cotton seed relative to wheat, whereas R. dominica did not respond to cotton seed at all and was attracted only to wheat. Significantly more adults of T. castaneum (10–15 times) were attracted to traps placed on the ground, near grain storage, than to equivalent traps that were suspended (1.5 m above the ground) nearby. These results suggest that Tribolium beetles detect and respond to resources towards the end of their dispersal flight, after which they localize resources while walking. By contrast R. dominica was captured only in suspended traps, which suggests they fly directly onto resources as they localize them. The ability of both species to colonize and reproduce in isolated resource patches within the relatively short time of 1 month is illustrated by the returns from the traps deployed in the field (at least 1 km from the nearest stored grain) even though they caught only a few beetles. The results presented here provide novel insights about the resource location behaviours of both T. castaneum and R. dominica. In particular, the relationship of T. castaneum with non‐cereal resources that are not conventionally associated with this species suggests an emphasis on these other resources in investigating the resource location behaviour of these beetles. This new perspective on the ecology of T. castaneum highlights the potential role of non‐cereal resources (such as the lint on cotton seed) in the spread of grain pest infestations.     

Effects of terpinen-4-ol fumigation on protein levels of detoxification enzymes in Tribolium confusum

Terpinen-4-ol has high fumigating activity to stored-grain pests including Tribolium confusum. To understand the detoxification of terpinen-4-ol in insects, proteomic analysis was performed to identify related proteins and pathways in response to terpinen-4-ol fumigation in T. confusum. By using isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy, 4,618 proteins were obtained from T. confusum adults in the present study. Comparative proteomic analysis showed that 148 proteins were upregulated and 137 proteins were downregulated in beetles under the LC₅₀ of terpinen-4-ol treatment for 24 hr. According to functional classifications, differentially expressed proteins (DEPs) were enriched in xenobiotic metabolism pathways. In the detoxification pathway, the levels of 25 cytochrome P450s, 5 glutathione S-transferases, and 2 uridine diphosphate (UDP)-glucuronosyltransferases were changed, most of which were upregulated in T. confusum exposed to terpinen-4-ol. The results indicated that terpinen-4-ol was potentially metabolized and detoxified by enzymes like P450s in T. confusum.     

Population structural changes in single species of flour beetles with ozone as an environmental factor

1. Although a 5 hour monthly exposure for 16 months to 45.0 ppm O₃ at 0.3l/min. reduced the numbers of individuals in single-species populations of T. confusum and T. castaneum, it did not result in elimination or a significant decrease in numbers which would be useful in a control program for these flour beetle species.
2. During the first 5 months of culture, ozone had a stimulating effect on productivity of T. confusum; a similar effect was not noted for T. castaneum.
3. Control of flour beetles by ozone in stored-products seems unlikely; however, ozone in an integrated control program with ionizing radiation might prove beneficial.
4. Biological interpretations are offered to explain the responses of these species to ozone exposures.
5. Further research is warranted to define ozone modifications in pest control programs and on stored-products per se.

     

Quinone production of some species of Tribolium

The quinone secretions of 6 Tribolium species were analyzed qualitatively and quantitatively by gas chromatography. Procedures were developed for collecting and weighing the secretion which prevented its contamination and oxidation of the quinones. Secretions of all species contained 2-methyl- and 2-ethyl-1,4-benzoquinones in amounts ranging from 50.5% in T. brevicornis to 73% in T. confusum. Ratios of 2-methyl- to 2-ethyl-1,4-benzoquinone were 1:1.7 in 2 species and 1:2.6 to 1:2.7 in 4 species. The most prevalent non-quinone in secretions of all species was 1-pentadecene, varying from 15.6% in T. confusum to 19.9% in T. brevicornis. Three unidentified compounds, probably long-chain hydrocarbons, were found and designated biosolvents. BS-2 was present in secretions of all species, BS-1a in 4 species and traces of BS-1b in 2 species. The primary function of 1-pentadecene, and probably the unidentified biosolvents, is quinone solvency. This is discussed in relation to other previously proposed functions and to the recently discovered activity as a sex pheromone.     

Responses of Tribolium castaneum to olfactory cues from cotton seeds,the fungi associated with cotton seeds,and cereals

We tested, in an olfactometer, whether or not Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) responds preferentially to the volatiles that emanate from the fungi associated with cotton [Gossypium hirsutum L. (Malvaceae)] seed over those that emanate from cereals, because cereals are usually portrayed as the primary resources of these beetles. Pairwise comparisons were conducted between cotton seed, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench] (both Poaceae); volatiles were tested from intact seeds and from both water and ethanol extracts. The results demonstrate that T. castaneum is attracted more strongly to cotton seeds with its lint contaminated with fungi, than to the conventional resources of this species (i.e., wheat and sorghum). Further tests prove that it is the fungus on the lint that produces the active volatiles, because the beetles did not respond to sterilized cotton lint (i.e., without the fungi typically associated with it when cotton seed is stored). Tests with five fungal cultures (each representing an unidentified species that was isolated from the field‐collected cotton lint) were variable across the cultures, with only one of them being significantly attractive to the beetles. The others were not attractive and one may even have repulsed the beetles. The results are consistent with the beetles having a strong ecological association with fungi and suggest it would be worth investigating the ecology of T. castaneum from this perspective.     

EXPERIMENTAL STUDIES OF COMMUNITY EVOLUTION II: THE ECOLOGICAL BASIS OF THE RESPONSE TO COMMUNITY SELECTION

Community selection, defined as the differential proliferation and/or extinction of communities, can bring about a response that may be qualitatively different from the response to selection acting at lower levels. This is because community selection can result in genetic changes in all of the species within the community by acting on the interaction among species. In the experiment presented here, a series of one generation assays were performed on the coevolved communities of two species of flour beetles, Tribolium castaneum and T. confusum, discussed by Goodnight (1990). Two community assays and one single-species assay were performed. Taken together, these provide insights into the genetic basis of the response to community selection. The first community assay involved measuring the selected traits on the original coevolved communities that had been subjected to community selection. This assay indicated that all of the selection treatments resulted in a significant response to selection in the original coevolved communities. The single-species assay involved separating the coevolved communities into their constituent single-species populations and again measuring the selected traits on these populations. None of the single-species populations exhibited a significant response to selection; thus the responses to community selection observed in the first community assay are expressed only in a community context. The second community assay again involved separating the coevolved communities into their constituent single-species populations; however, in this assay a competitor of the opposite species that had never been exposed to community selection was added to each population to form a “reconstructed” community. The results of this assay were that for two traits, emigration rate in T. castaneum and emigration rate in T. confusum, the genetic identity of the competing species did not affect the response to selection. This indicates that the competing species was acting like a nonevolving part of the environment. For the other two traits measured, population size in T. castaneum and population size in T. confusum, the results were very different. For these traits there was no detectable response to selection in the reconstructed communities. This indicates that for these traits the response to selection cannot be attributed to a genetic change in either species independently of the other species in the community. Rather it resides in the interaction between the two species.     

Effect of five diatomaceous earth formulations against Tribofium castaneum （Coleoptera： Tenebrionidae）, Oryzaephflus surinamensis （Coleoptera： Silvanidae） and Rhyzopertha dominica （Coleoptera： Bostrychidae）

Laboratory bioassays were conducted to determine the effect of food source on the survival of Tribolium castaneum Herbst, Oryzaephilus surinamensis L. and Rhyzopertha dominica F., after exposure to five diatomaceous earth （DE） formulations： Protect-It, Insecto, Perma-GuardTM, Dryacide and SilicoSec. Adults of these species were exposed to DEs at the rate of 0.5 mg/cm^2 for 1 day on filter paper inside plastic Petri dishes. After exposure, the initial mortality was counted and live individuals of the three species were held for a week in glass vials containing 50 mg wheat flour, rice and whole wheat, respectively. In the second experiment, after 1 day exposure to DEs, beetles were transferred to Petri dishes without food and held for a week to determine if the presence of food source would decrease the mortality of beetles. Experiments were carried out at 27℃ and 55% RH in the dark. The initial mortality in both of the experiments reached 100% for the three species exposed to Protect-It and in the case ofR. dominica and O. surinamensis exposed to Dryacide. In contrast, low level of mortality （〈 10%） was observed for T. castaneum exposed to Perma-GuardTM and Insecto. The mortality after the post-treatment period on food was decreased for the three species exposed to Perma-GuardTM and in the case of T. castaneum and R. dominica exposed to Insecto and SilicoSec. Adults of O. surinamensis were the most susceptible followed by R. dominica and 100% adult mortality was obtained, whereas T. castaneum were the least susceptible beetles to DEs. Protect-It and Dryacide were the most efficient DE formulations and can be used effectively in a stored grain integrated pest management program.     

Critical examination of postulated cladistic relationships among species of flour beetles (genus Tribolium,Tenebrionidae, Coleoptera)

Cytological considerations have led to the hypothesis that Tribolium confusum (CF) evolved from an ancestor similar to T. castaneum (CS) by translocation of an autosome to the X chromsome, and that T. destructor (DEST) was derived from CF. T. brevicornis (BREV) is regarded as the most primitive on morphological grounds. Electrophoretic analyses of 19 strains of CS, 7 of CF, and 1 each of DEST and BREV do not support this postulated evolutionary pathway. CF and CS are much more similar to BREV than they are to each other. (Comparisons of morphological mutations in CS and CF also indicate that the two species are not similar genetically.) DEST and CF are very dissimilar electrophoretically. It is likely that the species evolved independently from an ancestral stock which may be represented by BREV. Recognition of gene homology in different species is the keystone for all attempts at constructing genetically meaningful evolutionary pathways. The difficulties involved in doing so are pointed out.