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Concept: Tenebrionidae


BACKGROUND: Coevolution with parasites and population size are both expected to influence the evolution of mating rates. To gain insights into the interaction between these dual selective factors, we used populations from a coevolution experiment with the red flour beetle, Tribolium castaneum, and its microsporidian parasite, Nosema whitei. We maintained each experimental population at two different population sizes. We assayed the mating behaviour of both males and females from coevolved and paired non-coevolved control populations after 24 generations of coevolution with parasites. RESULTS: Males from large, coevolved populations (i.e. ancestors were exposed to parasites) showed a reduced eagerness to mate compared to males from large, non-coevolved populations. But in small populations, coevolution did not lead to decreased male mating rates. Coevolved females from both large and small populations appeared to be more willing to accept mating than non-coevolved females. CONCLUSIONS: This study provides unique, experimental insights into the combined roles of coevolving parasites and population size on the evolution of mating rate. Furthermore, we find that males and females respond differently to the same environmental conditions. Our results show that parasites can be key determinants of the sexual behaviour of their hosts.

Concepts: Sexual intercourse, Male, Female, Population ecology, Fungus, Sex, Coevolution, Tenebrionidae


Coleoptera (beetles) is a massively successful order of insects, distinguished by their evolutionarily modified forewings called elytra. These structures are often presumed to have been a major driving force for the successful radiation of this taxon, by providing beetles with protection against a variety of harsh environmental factors. However, few studies have directly demonstrated the functional significance of the elytra against diverse environmental challenges. Here, we sought to empirically test the function of the elytra using Tribolium castaneum (the red flour beetle) as a model. We tested four categories of stress on the beetles: physical damage to hindwings, predation, desiccation, and cold shock. We found that, in all categories, the presence of elytra conferred a significant advantage compared to those beetles with their elytra experimentally removed. This work provides compelling quantitative evidence supporting the importance of beetle forewings in tolerating a variety of environmental stresses, and gives insight into how the evolution of elytra have facilitated the remarkable success of beetle radiation.

Concepts: Insect, Fungus, Beetle, Ground beetle, Insects, Tenebrionidae, Elytron, Endopterygota


During our screening program for agrochemicals from Chinese medicinal herbs and wild plants, the essential oils of Evodia calcicola and Evodia trichotoma leaves were found to possess strong repellency against the red flour beetle Tribolium castaneum adults, the cigarette beetle Lasioderma serricorne adults and the booklouse Liposcelis bostrychophila. The two essential oils obtained by hydrodistillation were investigated by GC-MS. The main components of E. calcicola essential oil were identified to be (-)-β-pinene (44.02%), β-phellandrene (20.93%), ocimene (16.49%), and D-limonene (9.87%). While the main components of the essential oil of E. trichotoma were D-limonene (69.55%), 1R-a-pinene (11.48%), caryophyllene (2.80%) and spathulenol (2.24%). Data showed that T. castaneum was the most sensitive than other two stored product insects. Compared with the positive control, DEET (N, N-diethyl-3-methylbenzamide), the two essential oils showed the same level repellency against the red flour beetle. However, the essential oil of E. trichotoma showed the same level repellency against the cigarette beetle, while E. calcicola essential oil possessed the less level repellency against L. serricorne, relative to the positive control, DEET. Moreover, the two crude oils also exhibited strong repellency against L. bostrychophila, but lesser level repellency than the positive control, DEET. Thus, the essential oils of E. calcicola and E. trichotoma may be potential to be developed as a new natural repellent in the control of stored product insects.

Concepts: Petroleum, Essential oil, Oil, Oils, Tenebrionidae, Anobiidae, Lasioderma serricorne, Lasioderma


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.

Concepts: Immune system, Reproduction, Bacteria, Immunology, Immunity, Spermatozoon, Semen, Tenebrionidae


The red flour beetle Tribolium castaneum is an emerging insect model organism representing the largest insect order, Coleoptera, which encompasses several serious agricultural and forest pests. Despite the ecological and economic importance of beetles, most insect olfaction studies have so far focused on dipteran, lepidopteran, or hymenopteran systems.

Concepts: Biology, Insect, Larva, Antenna, Insects, Pterygota, Tenebrionidae, Endopterygota


PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H(+)-ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.

Concepts: Protein, Signal transduction, Insect, Apoptosis, Beetle, Fruit, Legume, Tenebrionidae


Despite accumulating efforts to unveil the origin of insect wings, it remains one of the principal mysteries in evolution. Currently, there are two prominent models regarding insect wing origin: one connecting the origin to the paranotal lobe and the other to the proximodorsal leg branch (exite). However, neither hypothesis has been able to surpass the other. To approach this conundrum, we focused our analysis on vestigial (vg), a critical wing gene initially identified in Drosophila. Our investigation in Tribolium (Coleoptera) has revealed that, despite the well-accepted view of vg as an essential wing gene, there are two groups of vg-dependent tissues in the “wingless” first thoracic segment (T1). We show that one of these tissues, the carinated margin, also depends on other factors essential for wing development (such as Wingless signal and apterous), and has nubbin enhancer activity. In addition, our homeotic mutant analysis shows that wing transformation in T1 originates from both the carinated margin and the other vg-dependent tissue, the pleural structures (trochantin and epimeron). Intriguingly, these two tissues may actually be homologous to the two proposed wing origins (paranotal lobes and exite bearing proximal leg segments). Therefore, our findings suggest that the vg-dependent tissues in T1 could be wing serial homologs present in a more ancestral state, thus providing compelling functional evidence for the dual origin of insect wings.

Concepts: Evolution, Insect, Beetle, Thorax, Origin, Wing, Insect wing, Tenebrionidae


Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes - Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP - were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.

Concepts: Gene expression, Bacteria, Evolution, Molecular biology, RNA, Fungus, Beetle, Tenebrionidae


Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods.

Concepts: Male, Female, RNA, Insect, Sexual dimorphism, Gender, Sex, Tenebrionidae


RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle (Tribolium castaneum) and recalcitrant pea aphid (Acyrthosiphon pisum) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E (VTE) and inhibitor of apoptosis (IAP) genes. Injection and ingestion ofVTEandIAPdsRNAs resulted in up to 100% mortality ofT. castaneumlarvae and sustained suppression (>80%) of transcript levels. InA. pisum, injection ofVTEbut notIAPdsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) ofVTEtranscript levels. Feeding aphids onVTEdsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability inT. castaneumlarvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency.

Concepts: DNA, Gene, Virus, RNA, Insect, RNA interference, RNA polymerase, Tenebrionidae