Melittin-The principal toxin of honeybee venom-Is also produced in the honeybee fat body.

Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.

Steroid receptor coactivator TAIMAN is a new modulator of insect circadian clock.

TAIMAN (TAI), the only insect ortholog of mammalian Steroid Receptor Coactivators (SRCs), is a critical modulator of ecdysone and juvenile hormone (JH) signaling pathways, which govern insect development and reproduction. The modulatory effect is mediated by JH-dependent TAI's heterodimerization with JH receptor Methoprene-tolerant and association with the Ecdysone Receptor complex. Insect hormones regulate insect physiology and development in concert with abiotic cues, such as photo- and thermoperiod. Here we tested the effects of JH and ecdysone signaling on the circadian clock by a combination of microsurgical operations, application of hormones and hormone mimics, and gene knockdowns in the linden bug Pyrrhocoris apterus males. Silencing taiman by each of three non-overlapping double-strand RNA fragments dramatically slowed the free-running period (FRP) to 27-29 hours, contrasting to 24 hours in controls. To further corroborate TAIMAN's clock modulatory function in the insect circadian clock, we performed taiman knockdown in the cockroach Blattella germanica. Although Blattella and Pyrrhocoris lineages separated ~380 mya, B. germanica taiman silencing slowed the FRP by more than 2 hours, suggesting a conserved TAI clock function in (at least) some insect groups. Interestingly, the pace of the linden bug circadian clock was neither changed by blocking JH and ecdysone synthesis, by application of the hormones or their mimics nor by the knockdown of corresponding hormone receptors. Our results promote TAI as a new circadian clock modulator, a role described for the first time in insects. We speculate that TAI participation in the clock is congruent with the mammalian SRC-2 role in orchestrating metabolism and circadian rhythms, and that TAI/SRCs might be conserved components of the circadian clock in animals.

Physiological responses to honeybee venom poisoning in a model organism, the firebug Pyrrhocoris apterus.

In this study, the biochemical and physiological features of the firebug Pyrrhocoris apterus were investigated to understand the impact of the honeybee Apis mellifera venom on them using physiological methods (mortality, total level of metabolism), biochemical methods (ELISA, mass spectrometry, polyacrylamide gel electrophoresis, spectrophotometry) and molecular methods (real-time PCR). Together, the obtained findings suggest that venom injection increased the level of adipokinetic hormone (AKH) in the CNS of P. apterus, indicating that this hormone plays a key role in activating defence responses. Furthermore, histamine levels in the gut increased significantly after envenomation and did not seem to be modulated by AKH. In contrast, histamine levels in the haemolymph increased after treatment with AKH and AKH + venom. In addition, we found that vitellogenin levels in haemolymph decreased in both males and females after venom application. Lipids, which are the main energy metabolites used by Pyrrhocoris, were significantly exhausted from the haemolymph after the administration of venom and the co-application with AKH reversed this effect. However, we did not find much influence on the effect of digestive enzymes after the injection of venom. Our research has highlighted the noticeable effect of bee venom on P. apterus' body and provided new insights into the role of AKH in controlling defensive responses. However, it is also likely that there will be alternative defence mechanisms.

Sexual dimorphism of diapause regulation in the hemipteran bug Pyrrhocoris apterus.

Diapause is one of the major strategies for insects to prepare for and survive harsh seasons. In females, the absence of juvenile hormone (JH) is a hallmark of adult reproductive diapause, a developmental arrest, which is much less characterized in males. Here we show that juvenile hormone III skipped bisepoxide (JHSB3) titers in hemolymph remarkably differ between reproductive males and females of the linden bug Pyrrhocoris apterus, whereas no JH was detected in diapausing adults of both sexes. Like in females, ectopic application of JH mimic effectively terminated male diapause through the canonical JH receptor components, Methoprene-tolerant and Taiman. In contrast to females, long photoperiod induced reproduction even in males with silenced JH reception or in males with removed corpus allatum (CA), the JH-producing gland. JHSB3 was detected in the accessory glands (MAG) of reproductive males, unexpectedly, even in males without CA. If there is a source of JHSB3 outside CA or a long-term storage of JHSB3 in MAGs remains to be elucidated. These sex-related idiosyncrasies are further manifested in different dynamics of diapause termination in P. apterus by low temperature. We would like to propose that this sexual dimorphism of diapause regulation might be explained by the different reproductive costs for each sex.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Functional analysis and localisation of a thyrotropin-releasing hormone-type neuropeptide (EFLa) in hemipteran insects.

EFLamide (EFLa) is a neuropeptide known for a long time from crustaceans, chelicerates and myriapods. Recently, EFLa-encoding genes were identified in the genomes of apterygote hexapods including basal insect species. In pterygote insects, however, evidence of EFLa was limited to partial sequences in the bed bug (Cimex), migratory locust and a few phasmid species. Here we present identification of a full length EFLa-encoding transcript in the linden bug, Pyrrhocoris apterus (Heteroptera). We created complete null mutants allowing unambiguous anatomical location of this peptide in the central nervous system. Only 2-3 EFLa-expressing cells are located very close to each other near to the surface of the lateral protocerebrum with dense neuronal arborization. Homozygous null EFLa mutants are fully viable and do not have any visible defect in development, reproduction, lifespan, diapause induction or circadian rhythmicity. Phylogenetic analysis revealed that EFLa-encoding transcripts are produced by alternative splicing of a gene that also produces Prohormone-4. However, this Proh-4/EFLa connection is found only in Hemiptera and Locusta, whereas EFLa-encoding transcripts in apterygote hexapods, chelicerates and crustaceans are clearly distinct from Proh-4 genes. The exact mechanism leading to the fused Proh-4/EFLa transcript is not yet determined, and might be a result of canonical cis-splicing, cis-splicing of adjacent genes (cis-SAG), or trans-splicing.

First Comprehensive Study of a Giant Among the Insects, Titanus giganteus: Basic Facts from Its Biochemistry, Physiology, and Anatomy.

Titanus giganteus is one of the largest insects in the world, but unfortunately, there is a lack of basic information about its biology. Previous papers have mostly described Titanus morphology or taxonomy, but studies concerning its anatomy and physiology are largely absent. Thus, we employed microscopic, physiological, and analytical methods to partially fill this gap. Our study focused on a detailed analysis of the antennal sensilla, where coeloconic sensilla, grouped into irregularly oval fields, and sensilla trichoidea were found. Further, the inspection of the internal organs showed apparent degeneration of the gut and almost total absence of fat body. The gut was already empty; however, certain activity of digestive enzymes was recorded. The brain was relatively small, and the ventral nerve cord consisted of three ganglia in the thorax and four ganglia in the abdomen. Each testis was composed of approximately 30 testicular follicles filled with a clearly visible sperm. Chromatographic analysis of lipids in the flight muscles showed the prevalence of storage lipids that contained 13 fatty acids, and oleic acid represented 60% of them. Some of our findings indicate that adult Titanus rely on previously accumulated reserves rather than feeding from the time of eclosion.

Adipokinetic hormone activities in insect body infected by entomopathogenic nematode.

The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24h (from 20 to 51% in EPN vs. EPN+AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN+AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6times), AKH level in the corpora cardiaca (1.3times) and haemolymph (1.7times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the anti-stress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role.

Decrease in Methoprene tolerant and Taiman expression reduces juvenile hormone effects and enhances the levels of juvenile hormone circulating in males of the linden bug Pyrrhocoris apterus.

Juvenile hormone (JH) produced by the corpus allatum (CA) stimulates vitellogenesis and reduces the synthesis of hexamerin proteins in adult females of Pyrrhocoris apterus. At present it is unknown whether the signaling pathway involving the JH receptor gene Methoprene tolerant (Met) and its binding partner Taiman (Tai), regulates the synthesis of accessory gland proteins (ACPs) and hexamerin proteins or effects male survival. Knockdown of genes by injecting Met dsRNA or Tai dsRNA, reduced the amount of ACPs whilst enhancing the amount of hexamerin mRNA in the fat body and the release of hexamerin proteins into haemolymph, as occurs after the ablation of CA. Lifespan was enhanced by injecting Met but not Tai dsRNA. Diapause associated with the natural absence of JH had a stronger effect on all these parameters than the ablation of CA or the knockdown of genes. This indicates there is an additional regulating agent. Both Met and Tai dsRNA induced a several fold increase in JH (JH III skiped bisepoxide) but a concurrent loss of Met or Tai disabled its function. This supports the view that the Met/Tai complex functions as a JH receptor in the regulation of ACPs and hexamerins.