Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum

As invertebrates lack the molecular machinery employed by the vertebrate adaptive immune system, it was thought that they consequently lack the ability to produce lasting and specific immunity. However, in recent years, it has been demonstrated that the immune defence of invertebrates is by far more complicated and specific than previously envisioned. Lasting immunity following an initial exposure that proves protection on a secondary exposure has been shown in several species of invertebrates. This phenomenon has become known as immune priming. In the cases where it is explicitly tested, this priming can also be highly specific. In this study, we used survival assays to test for specific priming of resistance in the red flour beetle, Tribolium castaneum, using bacteria of different degrees of relatedness. Our results suggest an unexpected degree of specificity that even allows for differentiation between different strains of the same bacterium. However, our findings also demonstrate that specific priming of resistance in insects may not be ubiquitous across all bacteria.     

Identification and characterization of novel ER-based hsp90 gene in the red flour beetle,Tribolium castaneum

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0487-y) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of tandem repeats in Tribolium castaneum genome reveals abundant and highly dynamic tandem repeat families with satellite DNA features in euchromatic chromosomal arms

Although satellite DNAs are well-explored components of heterochromatin and centromeres, little is known about emergence, dispersal and possible impact of comparably structured tandem repeats (TRs) on the genome-wide scale. Our bioinformatics analysis of assembled Tribolium castaneum genome disclosed significant contribution of TRs in euchromatic chromosomal arms and clear predominance of satellite DNA-typical 170 bp monomers in arrays of ≥5 repeats. By applying different experimental approaches, we revealed that the nine most prominent TR families Cast1–Cast9 extracted from the assembly comprise ∼4.3% of the entire genome and reside almost exclusively in euchromatic regions. Among them, seven families that build ∼3.9% of the genome are based on ∼170 and ∼340 bp long monomers. Results of phylogenetic analyses of 2500 monomers originating from these families show high-sequence dynamics, evident by extensive exchanges between arrays on non-homologous chromosomes. In addition, our analysis shows that concerted evolution acts more efficiently on longer than on shorter arrays. Efficient genome-wide distribution of nine TR families implies the role of transposition only in expansion of the most dispersed family, and involvement of other mechanisms is anticipated. Despite similarities in sequence features, FISH experiments indicate high-level compartmentalization of centromeric and euchromatic tandem repeats.     

Translocation of bacteria from the gut to the eggs triggers maternal transgenerational immune priming in Tribolium castaneum

Invertebrates can be primed to enhance their protection against pathogens they have encountered before. This enhanced immunity can be passed maternally or paternally to the offspring and is known as transgenerational immune priming. We challenged larvae of the red flour beetle Tribolium castaneum by feeding them on diets supplemented with Escherichia coli, Micrococcus luteus or Pseudomonas entomophila, thus mimicking natural exposure to pathogens. The oral uptake of bacteria induced immunity-related genes in the offspring, but did not affect the methylation status of the egg DNA. However, we observed the translocation of bacteria or bacterial fragments from the gut to the developing eggs via the female reproductive system. Such translocating microbial elicitors are postulated to trigger bacterial strain-specific immune responses in the offspring and provide an alternative mechanistic explanation for maternal transgenerational immune priming in coleopteran insects.     

Expression of esterases during ontogenesis of the flour beetle Tribolium castaneum (tenebrionidae; coleoptera)

Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze - and -naphthylacetate and -naphthylpropionate with almost equal capacity. -Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate diisopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

Chemical Composition,Toxicity and Repellency of Inula graveolens Essential Oils from Roots and Aerial Parts against Stored-Product Beetle Tribolium castaneum (Herbst)

In this work, essential oils extracted from roots and aerial parts of Inula graveolens by hydrodistillation and their fractions obtained by chromatographic simplification were first investigated for their chemical composition by GC/MS and then evaluated for the first time for their repellency and contact toxicity properties against Tribolium castaneumadults. Twenty-eight compounds were identified in roots essential oil (REO), which accounted for 97.9 % of the total oil composition, with modhephen-8-β-ol (24.7 %), cis-arteannuic alcohol (14.8 %), neryl isovalerate (10.6 %) and thymol isobutyrate (8.5 %) as major constituents. Twenty-two compounds were found in the essential oil from aerial parts (APEO), which accounted for 93.9 % of the total oil, with borneol (28.8 %), caryophylla-4(14),8(15)-dien-6-ol (11.5 %), caryophyllene oxide (10.9 %), τ-cadinol (10.5 %) and bornyl acetate (9.4 %) as main compounds.REO and APEO displayed stronger repellency after 2 h of exposure (80.0 and 90.0 %, respectively) against T. castaneum at the concentration of 0.12 μL/cm². After fractionation, fractions R₄ and R₅ exhibited greater effects (83.3 % and 93.3 %, respectively) than the roots essential oil. Furthermore, the fractions AP₂ and AP₃ showed higher repellency (93.3 and 96.6 %, respectively) than the aerial parts oil. The LD₅₀ values of oils from roots and aerial parts topically applied were 7.44 % and 4.88 %, respectively. Results from contact toxicity assay showed that fraction R₄ was more effective than the roots oil with LD₅₀ value of 6.65 %. These results suggests that essential oils of roots and aerial parts from I. graveolens may be explored as potential natural repellent and contact insecticides against T. castaneum in stored products.     

Use of Tanacetum tomentosum and Ta. dolichophyllum essential oils as botanical repellents and insecticidal agents against storage pest Tribolium castaneum (Coleoptera: Tenebrionidae)

In this study, repellent and fumigant activities of Tanacetum tomentosum and Ta. dolichophyllum essential oils were investigated against Tribolium castaneum. Results showed that both oils had effective fumigant and repellent activities. Tanacetum tomentosum oil emerged as more potent than Ta. dolichophyllum oil with LC₅₀ values of 6.85 and 4.32 μL/0.25 L air after 24 and 48 h of exposure, respectively. Ta. tomentosum oil also showed more repellent activity than Ta. dolichophyllum oil by exhibiting repellent activity in the range of 38.70–82.35%. Chemical analysis of both oils revealed that β‐bisabolene (50.0%) was the major component of Ta. tomentosum oil and that β‐eudesmol (31.4%) and α‐bisabolol (10.7%) were the major components of Ta. dolichophyllum oil. Doses of 40 and 80 μL/0.5 L air of both oils significantly protected 500 g of wheat grains for up to 6 months from insect infestation, without causing any side effects in the germination rates; the order of germination rates was Ta. tomentosum (89.45%) > Ta. dolichophyllum (86.78%). Thus, this study suggests that essential oils from Ta. tomentosum and Ta. dolichophyllum can be used as potential alternative botanical fumigants for controlling Tr. castaneum.     

Different effects of paternal trans-generational immune priming on survival and immunity in step and genetic offspring

Paternal trans-generational immune priming, whereby fathers provide immune protection to offspring, has been demonstrated in the red flour beetle Tribolium castaneum exposed to the insect pathogen Bacillus thuringiensis. It is currently unclear how such protection is transferred, as in contrast to mothers, fathers do not directly provide offspring with a large amount of substances. In addition to sperm, male flour beetles transfer seminal fluids in a spermatophore to females during copulation. Depending on whether paternal trans-generational immune priming is mediated by sperm or seminal fluids, it is expected to either affect only the genetic offspring of a male, or also their step offspring that are sired by another male. We therefore conducted a double-mating experiment and found that only the genetic offspring of an immune primed male show enhanced survival upon bacterial challenge, while phenoloxidase activity, an important insect immune trait, and the expression of the immune receptor PGRP were increased in all offspring. This indicates that information leading to enhanced survival upon pathogen exposure is transferred via sperm, and thus potentially constitutes an epigenetic effect, whereas substances transferred with the seminal fluid could have an additional influence on offspring immune traits and immunological alertness.     

Macromolecular and enzymatic abnormalities induced by a synthetic pyrethroid,Ripcord (Cypermethrin), in adult beetles of a stored grain pest,Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

The effects of a synthetic pyrethroid insecticide, cypermethrin, administered as a formulation Ripcord 25EC (emulsified concentrate), to adult beetles of a stored grain pest, Tribolium castaneum, have been studied, with an objective to ascertain its toxicity on enzymes such as carbohydrases, phosphatases, dehydrogenases, aminotransferases, and concentration of various biochemical components such as monosaccharides, glycogen, cholesterol, nucleic acids, urea, total lipids, and total proteins. Almost all the enzymes and biochemical components were sensitive to sublethal doses of Ripcord 25 EC and these effects were found to be dependent on the duration of treatment. All carbohydrate metabolizing enzymes (amylase, invertase, lactase, maltase, lactate dehydrogenase) were elevated, except for trehalase, which was also elevated up to day 3 but returned to normal levels subsequently. Phosphatases (alkaline as well as acidic) were increased first and decreased thereafter, while isocitrate dehydrogenase decreased throughout the experimental period. Transaminases (aspartate aminotransferase and alanine aminotransferase) showed a decreasing trend. Of the other biochemical components tested, glucose content decreased during the first 3 days but increased subsequently. Fructose content showed an increase, while the glycogen content decreased throughout the study. Total lipid content was not disturbed up to day 3 but increased thereafter. Cholesterol content was depleted by day 7. Total proteins started decreasing from day 3 onwards, while soluble proteins were not affected. DNA, RNA, and urea contents exhibited elevated levels, while uric acid showed a decreasing trend. Sublethal doses of Ripcord, therefore, resulted in extensive enzyme induction, and utilization of carbohydrates, proteins, and lipids, in the given order, perhaps to produce extra energy to combat insecticidal stress. Arch. Insect Biochem. Physiol. 39:144–154, 1998. © 1998 Wiley-Liss, Inc.     

Toxic and repellent properties of Xylopia aethiopica (Dunal) A. Richard on Tribolium castaneum Herbst infesting stored millets,Pennisetum glaucum (L.) R. Br.

The toxicity and repellency of Xylopia aethiopica seed extract was investigated in the laboratory against Tribolium castaneum Herbst. Concentration and days after treatment (DAT) caused a significant increase in T. castaneum adult mortality with an interaction effect of both on mortality when filter paper was impregnated with X. aethiopica extract. At 0.2 ml/60 cm² extract, significant mortality was observed at three–seven DAT when compared with one DAT. At 0.4 ml/60cm², 100% mortality was recorded at the lowest exposure period of one DAT. When 0.2 ml extract was applied to 5 g millet seeds, mortality at five–seven DAT was significantly higher than mortality observed in the control. Although repellency was dose-dependent, the percentage of T. castaneum that were repelled from treated filter paper was not significant. At 0.4 ml/60 cm², Class II repellency (26.7%) was observed. The results suggest that X. aethiopica can only effectively control T. castaneum populations that have infested millet but do not prevent cross-infestation via repellency.     

Taxonomic notes on Lasioglossum (Lasioglossum) subopacum (Smith) and L. (L.) okinawa Ebmer et Maeta (Hymenoptera, Halictidae) from Asia

Lasioglossum (Lasioglossum) subopacum (Smith) is recorded from the Korean Peninsula for the first time. Lasioglossum (Lasioglossum) okinawa Ebmer et Maeta from Japan is ranked to a subspecies of Lasioglossum (Lasioglossum) subopacum judging from the characteristics of the male. The male of Lasioglossum (Lasioglossum) subopacumokinawa is described for the first time. Some bionomical notes of both subspecies are presented.     

Antimycobacterial,antibacterial and antifungal activities of the methanol extract and compounds from Thecacoris annobonae (Euphorbiaceae)

This study was designed to evaluate the antimycobacterial, antibacterial and antifungal activities of the methanol extract from the stem bark of Thecacoris annobonae Pax & K. Hoffm, that of aristolochic acid I (1) and other isolated compounds. The microplate alamar blue assay (MABA) and the broth microdilution method were used to determine the minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of the above samples. The H⁺-ATPase-mediated proton pumping assay was used to evaluate a possible mechanism of action for both the methanol extract and aristolochic acid I. The results of the MIC determinations showed that the methanol extract and aristolochic acid I prevent the growth of all studied organisms. The results obtained in this study also showed that the methanol extract as well as aristolochic acid I inhibited the H⁺-ATPase activity. The overall results provided evidence that the methanol extract of T. annobonae might be a potential source of new antimicrobial drug against tuberculosis, and some bacterial and fungal diseases, but should be consumed with caution, bearing in mind that the main active component, aristolochic acid I is a potentially toxic compound.     

Fumigant toxicity of Laurus nobilis and Myrtus communis essential oils on larvae and adults of the Red flour beetle,Tribolium castaneum Herbst (Col.: Tenebrionidae)

Considering the invasion to food commodities by insects and harmful effect of chemical pesticides, essential oils are among the best known substances tested against stored product pests. These compounds may act as fumigants, contact insecticides, repellents or anti-feedants. In present study, fumigant toxicity of essential oils from Laurus nobilis L. and Myrtus communis L. was assessed on larvae and adults of Tribolium castaneum Herbst at 27?±?2?°C, 60?±?5% RH in darkness. Each essential oil was tested in five concentrations with three replicates. The LC₅₀ values of L. nobilis and M. communis against adults of beetle were calculated 243.78 and 56.11?μl/l and LC₉₅ values for them were 685.85 and 144.01?μl/l, respectively. For the larvae of T. castaneum, the LC₅₀ values for L. nobilis and M. communis were 211.64 and 69.63 and LC₉₅ values were 656.84 and 183.65?μl/l, respectively. Results showed that these essential oils may have potential as botanical control agents against larvae and adults of T. castaneum.