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In this study, we tested the hypothesis that a micro-serrated edge on the honey bee Apis mellifera stinger tip serves as a tool for more intensive crushing of cell membranes in the victim's tissues. This could have mechanical consequences as well as initiate metabolic pathways linked to cell membrane breakdown (e.g., production of biogenic amines). Accordingly, we found that hymenopteran species that use their stingers as an offensive or defensive weapon to do as much damage to the victim's body as possible had this cuticular microstructure. In parasitic hymenopterans, on the other hand, this structure was missing, as stingers are solely used to delicately transport venom to the victim's body in order to do little mechanical harm. We also demonstrated that the stinger lancets of the honey bee A. mellifera are living organs with sensilla innervated by sensory neurons and containing other essential tissues, rather than mere cuticular structures.

Structure and function of the honey bee, Apis mellifera stinger is very well known. Nevertheless, we described two novel ultrastructural details at the end of the stinger using scanning electron mi...

In classical neuroscience, Dale´s principle postulates that neuronal identity is conferred by the specific neurotransmitter that it releases. However, the brain might be more tractable to specific situations regardless of specific specialisation which may contradict this principle. Hence, this constrained approach of how we perceive and study the nervous system must be revisited and revised, specifically by studying the dopaminergic system. We presume a relatively flexible change in the dopaminergic system due to neuronal activity or environmental changes. While the parallel between the reward system of mammals and insects is generally well accepted, herein, we extend the idea that the insect nervous system might also possess incredible plasticity, similar to the mammalian system. In this review, we critically evaluate the available information about the reward system in vertebrates and invertebrates, emphasising the dopaminergic neuronal plasticity, a challenge to the classical Dale's principle. Thus, neurotransmitter switching significantly disrupts the static idea of neural network organisation and suggests greater possibilities for a dynamic response to the current life context of organisms.

The effects of the entomopathogenic nematode Steinernema carpocapsae on the Colorado potato beetle (CPB) Leptinotarsa decemlineata and the involvement of adipokinetic hormone (AKH) in the responsive reactions were examined in this study. It was observed that nematode application doubled the amount of AKH (Peram-CAH-I and Peram-CAH-II) in the central nervous system of L. decemlineata, indicating mobilization of anti-stress reactions in the body. Furthermore, the external co-application of Peram-CAH-II with the nematode significantly increased beetle mortality (5.6 and 1.8 times, 1 and 2 days after application, respectively). The mechanism underlying this phenomenon was investigated. As the effect on gut characteristics was equivocal, it was assumed that the nematodes profited from the observed mobilization of metabolites from the fat body into the Peram-CAH-II-induced hemolymph. This phenomenon supplied nematodes with a more nutrient-dense substrate on which they propagated. Furthermore, Peram-CAH-II lowered vitellogenin expression in the fat body, particularly in males, thus limiting the anti-pathogen defense capacity of the protein. However, there could be other possible mechanisms underpinning this chain of events. The findings could be theoretically intriguing but could also aid in developing real insect pest control methods in the future.

Animals and humans share similar reactions to the effects of addictive substances, including those of their brain networks to drugs. Our review focuses on simple invertebrate models, particularly the honeybee (Apis mellifera), and on the effects of drugs on bee behaviour and brain functions. The drug effects in bees are very similar to those described in humans. Furthermore, the honeybee community is a superorganism in which many collective functions outperform the simple sum of individual functions. The distribution of reward functions in this superorganism is unique - although sublimated at the individual level, community reward functions are of higher quality. This phenomenon of collective reward may be extrapolated to other animal species living in close and strictly organised societies, i.e. humans. The relationship between sociality and reward, based on use of similar parts of the neural network (social decision-making network in mammals, mushroom body in bees), suggests a functional continuum of reward and sociality in animals.

The role of adipokinetic hormone (Drome-AKH) in maintaining the levels of basic nutrients, under starvation conditions, was studied using Drosophila melanogaster mutants with AKH deficiency (Akh1) and AKH abundance (EE-Akh). Our results showed lipids as the main energy reserve in Drosophila, and their physiological level and metabolism were shown to be under the control of AKH. AKH abundance in the body resulted in lower levels of triacylglycerols and diacylglycerols than in the controls, probably due to a more intensive metabolism; interestingly, there was a disproportional representation of fatty acids in triacylglycerols and diacylglycerols in Drosophila. Lower level of glycogen and its partial control by AKH suggest its lesser role as the storage substance. However, maintenance of free carbohydrate level in Drosophila seemed to be critical; when glycogen stores are exhausted, carbohydrates are synthesized from other sources. Protein levels and their alterations, under starvation, did not seem controlled by AKH. AKH-deficient flies were more resistant while AKH-abundant flies were more sensitive to starvation; females were found to be more resistant than males, regardless of the AKH level, probably due to higher body mass and higher amount of nutrients. However, in accordance with the level of all nutrients, that of AKH also gradually decreased with prolonged starvation.

In honeybees (Apis mellifera), the rate of aging is modulated through social interactions and according to caste differentiation and the seasonal (winter/summer) generation of workers. Winter generation workers, which hatch at the end of summer, have remarkably extended lifespans as an adaptation to the cold season when the resources required for the growth and reproduction of colonies are limited and the bees need to maintain the colony until the next spring. In contrast, the summer bees only live for several weeks. To better understand the lifespan differences between summer and winter bees, we studied the fat bodies of honeybee workers and identified several parameters that fluctuate in a season-dependent manner. In agreement with the assumption that winter workers possess greater fat body mass, our data showed gradual increases in fat body mass, the size of the fat body cells, and Vg production as the winter season proceeded, as well as contrasting gradual decreases in these parameters in the summer season. The differences in the fat bodies between winter and summer bees are accompanied by respective increases and decreases in telomerase activity and DNA replication in the fat bodies. These data show that although the fat bodies of winter bees differ significantly from those of summer bees, these differences are not a priori set when bees hatch at the end of summer or in early autumn but instead gradually evolve over the course of the season, depending on environmental factors.

The fruit fly Drosophila melanogaster brain is the most extensively investigated model of a reward system in insects. Drosophila can discriminate between rewarding and punishing environmental stimuli and consequently undergo associative learning. Functional models, especially those modelling mushroom bodies, are constantly being developed using newly discovered information, adding to the complexity of creating a simple model of the reward system. This review aims to clarify whether its reward system also includes a hedonic component. Neurochemical systems that mediate the ‘wanting’ component of reward in the Drosophila brain are well documented, however, the systems that mediate the pleasure component of reward in mammals, including those involving the endogenous opioid and endocannabinoid systems, are unlikely to be present in insects. The mushroom body components exhibit differential developmental age and different functional processes. We propose a hypothetical hierarchy of the levels of reinforcement processing in response to particular stimuli, and the parallel processes that take place concurrently. The possible presence of activity-silencing and meta-satiety inducing levels in Drosophila should be further investigated.

Worker honey bees are subject to biochemical and physiological changes throughout the year. This study aimed to provide the reasons behind these fluctuations. The markers analysed included lipid, carbohydrate, and protein levels in the haemolymph; the activity of digestive enzymes in the midgut; the levels of adipokinetic hormone (AKH) in the bee central nervous system; the levels of vitellogenins in the bee venom and haemolymph; and the levels of melittin in the venom. The levels of all the main nutrients in the haemolymph peaked mostly within the period of maximal bee activity, whereas the activity of digestive enzymes mostly showed a two-peak course. Furthermore, the levels of AKHs fluctuated throughout the year, with modest but significant variations. These data suggest that the role of AKHs in bee energy metabolism is somewhat limited, and that bees rely more on available food and less on body deposits. Interestingly, the non-metabolic characteristics also fluctuated over the year. The vitellogenin peak reached its maximum in the haemolymph in winter, which is probably associated with the immunoprotection of long-lived winter bees. The analysis of bee venom showed the maximal levels of vitellogenin in autumn; however, it is not entirely clear why this is the case. Finally, melittin levels showed strong fluctuations, suggesting that seasonal control was unlikely.

It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. In this study we tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. We propose that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon we discuss two scenarios: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population.

The effect of Habrobracon hebetor venom and the role of the adipokinetic hormone (AKH) in poisoned adult females of the firebug Pyrrhocoris apterus were studied 24 and 48h after treatments. Venom application elicited total neuromuscular paralysis in firebugs, but the co-application of venom and Pyrap-AKH significantly reduced paralysis (up to 3.2 times) compared to the application of venom only. Although the mechanisms of their action are unknown, both agents might affect neuromuscular junctions. Venom application significantly increased the expression of both P. apterus Akh genes (Pyrap-Akh 5.4 times and Peram-Cah-II 3.6 times), as well as the level of AKHs in the central nervous system (2.5 times) and haemolymph (3.0 times). In the haemolymph, increased AKH levels might have led to the mobilization of stored lipids, which increased 1.9 times, while the level of free carbohydrates remained unchanged. Total metabolism, monitored by carbon dioxide production, significantly declined in paralysed P. apterus individuals (1.4 times and 1.9 times, 24 and 48h after the treatment, respectively), probably because of a malfunction of the muscular system. The results suggest an active role of AKH in the defence mechanism against the stress elicited by neuromuscular paralysis, and the possible involvement of this hormone in neuronal/neuromuscular signalling.

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.

This study describes defense functions of the insect neuropeptide sericotropin, which is recognized as an agent that stimulates silk production in some lepidopteran larvae. Sericotropin, expressed in brain tissue of the wax moth Galleria mellonella in all developmental stages, is not expressed in silk glands, indicating its tissue specificity. Fluorescence microscopy confirmed the presence of sericotropin in the brain-subesophageal complex being predominantly and densely distributed under the plasmatic membrane and in axonal parts of neurons. Injection of venom from Habrobracon hebetor and topical application of the entomopathogenic nematode (EPN) Steinernema carpocapsae with symbiotic bacteria Xenorhabdus spp. into or onto G. mellonella larvae resulted in upregulation of the sericotropin gene and peptide, suggesting a role for sericotropin in defense and immunity. Accordingly, two synthetic fragments of sericotropin killed entomotoxic Xenorhabdus spp. bacteria in a disc diffusion antimicrobial test. Further, total metabolism, monitored by carbon dioxide production, significantly decreased after application of either venom or EPN, probably because of muscle impairment by the venom and serious cell damage caused by EPN, especially in the midgut. Both venom and EPN upregulated expression of genes encoding antimicrobial peptides gallerimycin and galiomicin in Galleria brain; however, they downregulated prophenoloxidase and phenoloxidase activity in hemolymph. These results suggest that sericotropin is a multifunctional peptide that plays an important role in G. mellonella defense and immunity.

This study examined the effect of two natural toxins (a venom from the parasitic wasp Habrobracon hebetor and destruxin A from the entomopathogenic fungus Metarhizium anisopliae), and one pathogen (the entomopathogenic fungus Isaria fumosorosea) on the activity of basic digestive enzymes in the midgut of the cockroach Periplaneta americana. Simultaneously, the role of adipokinetic hormones (AKH) in the digestive processes was evaluated. The results showed that all tested toxins/pathogens elicited stress responses when applied into the cockroach body, as documented by an increase of AKH level in the central nervous system. The venom from H. hebetor showed no effect on digestive enzyme activities in the ceca and midgut in vitro. In addition, infection by I. fumosorosea caused a decrease in activity of all enzymes in the midgut and a variable decrease in activity in the ceca; application of AKHs did not reverse the inhibition. Destruxin A inhibited the activity of all enzymes in the midgut but none in the ceca in vitro; application of AKHs did reverse this inhibition, and no differences between both cockroach AKHs were found. Overall, the results demonstrated the variable effect of the tested toxins/pathogens on the digestive processes of cockroaches as well as the variable ability of AKH to counteract these effects.

International audience; AbstractWe tested the effect of Chlorella sorokiniana, a green, unicellular, freshwater alga, provided as a food supplement on several biological characteristics of the honey bee Apis mellifera. Chlorella was applied as (1) a moisturized powder, (2) a sugar-water solution, or (3) mixed with honey-sugar candy. All three applications were well accepted by the bees. We observed a positive effect of Chlorella on colony development, and also on basic aspects of metabolism, such as increased fat deposition and vitellogenin transcript levels, and a decrease in TOR and InR2 transcript levels. The effect of Chlorella on other characteristics was lower (protein levels) or even null (total fat body mass, level of adipokinetic hormone). Application of Chlorella modulated the hypopharyngeal gland size, and the activity of basic digestive enzymes in the bee midgut. Our observations suggest that the nutritional composition of Chlorella might be an appropriate dietary supplement for honey bees.

This study examined the expression and role of vitellogenin (Vg) in the body of the firebug Pyrrhocoris apterus (Heteroptera, Insecta) during the infection elicited by two entomopathogenic organisms, the nematode Steinernema carpocapsae and the fungus Isaria fumosorosea. Infection by S. carpocapsae significantly up-regulated Vg mRNA expression in the male body. The corresponding increase in Vg protein expression was also confirmed by electrophoretic and immunoblotting analyses. Remarkably, in females, the tendency was opposite. Nematodal infection significantly reduced both Vg mRNA and Vg protein expression levels in fat body and hemolymph, respectively. We speculate that infection of reproductive females reduces Vg expression to the level, which is still sufficient for defense, but insufficient for reproduction. This circumstance reduces energy expenditure and helps the individual to cope with the infection. Importantly, purified Vg significantly inhibited growth of Xenorhabdus spp., an entomotoxic bacteria isolated from S. carpocapsae. However, the effect of Vg against I. fumosorosea was not so obvious. The fungus significantly stimulated Vg gene expression in males, however, a similar increase was not recapitulated on the protein level. Nevertheless, in females, both mRNA and protein Vg levels were significantly reduced after the fungal infection. The obtained data demonstrate that Vg is likely an important defense protein, possibly with a specific activity. This considerably expands the known spectrum of Vg functions, as its primary role was thought to be limited to regulating egg development in the female body.

The adipokinetic hormones (AKHs) are known to be involved in insect immunity, thus their role in the cockroach Periplaneta americana infected with the entomopathogenic fungus Isaria fumosorosea was examined in this study. The application of I. fumosorosea resulted in a significant increase in both Akh gene expression and AKH peptide levels. Further, co-application of I. fumosorosea with Peram-CAH-II significantly enhanced cockroach mortality compared with the application of I. fumosorosea alone. The mechanism of AKH action could involve metabolic stimulation, which was indicated by a significant increase in carbon dioxide production; this effect can increase the turnover and thus efficacy of toxins produced by I. fumosorosea in the cockroach's body. I. fumosorosea treatment resulted in a significant decrease in haemolymph nutrients (carbohydrates and lipids), but co-application with Peram-CAH-II restored control level of lipids or even further increased the level of carbohydrates. Such nutritional abundance could enhance the growth and development of I. fumosorosea. Further, both I. fumosorosea and Peram-CAH-II probably affected oxidative stress: I. fumosorosea alone curbed the activity of catalase in the cockroach's gut, but co-application with Peram-CAH-II stimulated it. Interestingly, the hormone alone had no effect on catalase activity. Taken together, the results of the present study demonstrate the interactions between the fungus and AKH activity; understanding this relationship could provide insight into AKH action and may have practical implications for insect pest control in the future.

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.

The attrition of telomeres, the ends of eukaryote chromosomes, and activity of telomerase, the enzyme that restores telomere length, play a role in the ageing process and act as indicators of biological age. A notable feature of advanced eusocial insects is the longevity of reproductive individuals (queens and kings) compared to those from non-reproductive castes (workers and soldiers) within a given species, with a proposed link towards upregulation of telomerase activity in the somatic tissues of reproductive individuals. Given this, eusocial insects provide excellent model systems for research into ageing. We tested telomerase activity and measured telomere length in Bombus terrestris, which is a primitively eusocial insect species with several distinct features compared to advanced social insects. In somatic tissues, telomerase activity was upregulated only in the fat bodies of pre-diapause queens, and this upregulation was linked to heightened DNA synthesis. Telomere length was shorter in old queens compared to that in younger queens or workers. We speculate that (1) the upregulation of telomerase activity, together with DNA synthesis, is the essential step for intensifying metabolic activity in the fat body to build up a sufficient energy reserve prior to diapause, and that (2) the lifespan differences between B. terrestris workers and queens are related to the long diapause period of the queen. A possible relationship between telomere length regulation and TOR, FOXO, and InR as cell signaling components, was tested.

This study examined how adipokinetic hormone (AKH) and adenosine affect defense responses in Drosophila melanogaster larvae infected with entomopathogenic nematodes (EPN, Steinernema carpocapsae and Heterorhabditis bacteriophora). Three loss-of-function mutant larvae were tested: Akh1, AdoR1 (adenosine receptor), and Akh1 AdoR1. Mortality decreased in all mutants post-EPN infection compared with the control (w1118). Additionally, co-application of external AKH with EPN significantly increased mortality beyond rates observed in EPN-only treatment, while also elevating carbon dioxide production, a measure of metabolism. Furthermore trehalose levels increased in both w1118 and Akh1 larvae post-EPN infection, but the latter group exhibited a lower increase and total trehalose levels. Interestingly, baseline trehalose was relatively high in untreated AdoR1 and Akh1 AdoR1 mutants, with levels remaining unaffected by infection. Infection also elevated haemolymph lipid content overall, but the different mutations did not substantially influence this change. In contrast, haemolymph protein content dropped after EPN infection in all tested groups, but this decline was more intense among Akh1. In uninfected larvae mutations decreased antioxidative capacity in Akh1 and increased in AdoR1, however, its post-infection increases were similar in all mutants, suggesting that antioxidant response in Drosophila involves mechanisms also beyond AKH and adenosine. Furthermore, AKH application in w1118 larvae significantly increased movement distance and percentage of larval activity, but reduced velocity. Mutations of Akh and AdoR did not strongly affect locomotion.

Insects, like other organisms, must deal with a wide variety of potentially challenging environmental factors during the course of their life. An important example of such a challenge is the phenomenon of oxidative stress. This review summarizes the current knowledge on the role of adipokinetic hormones (AKH) as principal stress responsive hormones in insects involved in activation of anti-oxidative stress response pathways. Emphasis is placed on an analysis of oxidative stress experimentally induced by various stressors and monitored by suitable biomarkers, and on detailed characterization of AKH's role in the anti-stress reactions. These reactions are characterized by a significant increase of AKH levels in the insect body, and by effective reversal of the markers-disturbed by the stressors-after co-application of the stressor with AKH. A plausible mechanism of AKH action in the anti-oxidative stress response is discussed as well: this probably involves simultaneous employment of both protein kinase C and cyclic adenosine 3',5'-monophosphate pathways in the presence of extra and intra-cellular Ca(2+) stores, with the possible involvement of the FoxO transcription factors. The role of other insect hormones in the anti-oxidative defense reactions is also discussed.

Adipokinetic hormones (Akhs) are small peptides (8–10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. We employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh 1 mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene. The mutant flies were fully viable and compared to the control flies, had significantly low levels of hemolymph saccharides including trehalose and were resistant to starvation. These characteristics are similar to those obtained from the flies carrying targeted ablation of Akh-expressing neurons (reported earlier). We also found that the Akh 1 mutants are slightly heavy and had a slow metabolic rate. Furthermore, we showed that the ectopic expression of Dmel∖Akh reverses the Akh 1 phenotype and restores the wild-type characteristics. Our results confirmed that Akh is an important regulator of metabolic homeostasis in Drosophila .

Insect anti-stress responses, including those induced by insecticides, are controlled by adipokinetic hormones (AKHs). We examined the physiological consequences of Pyrap-AKH application on Tribolium castaneum adults (AKH-normal and AKH-deficient prepared by the RNAi technique) treated by two insecticides, pirimiphos-methyl and deltamethrin. Co-application of pirimiphos-methyl and/or deltamethrin with AKH significantly increased beetle mortality compared with application of the insecticides alone. This co-treatment was accompanied by substantial stimulation of general metabolism, as monitored by carbon dioxide production. Further, the insecticide treatment alone affected some basic markers of oxidative stress: it lowered total antioxidative capacity as well as the activity of superoxide dismutase in the beetle body ; in addition, it enhanced the activity of catalase and glutathione-S-transferase. However, these discrepancies in oxidative stress markers were eliminated/reduced by co-application with Pyrap-AKH. We suggest that the elevation of metabolism, which is probably accompanied with faster turnover of toxins, might be responsible for the higher mortality that results after AKH and insecticide co-application. Changes in oxidative stress markers are probably not included in the mechanisms responsible for increased mortality.

The ultrastructural and biochemical characteristics were studied in active and diapausing pupae of the horse chestnut leaf miner, Cameraria ohridella, collected in summer. The active pupae contained developing internal organs, while those in diapause mostly numerous non-differentiated cells packed with stored nutrients. These nutrients occurred in vacuoles as liquid material (lipids, proteins) or as reserve crystaloids (proteins). Triacylglycerols (TGs) were the main lipids stored by pupae and were more abundant in diapausing than active pupae. The amount of diacyglycerols (DGs) and phospholipids (PLs) was almost identical in both groups, which reflects the roles of these compounds in lipid transport and structure of cell membranes, respectively. A principal component analysis (PCA) indicated differences in the quality of the lipid compounds in both groups and that the tgs were mostly responsible for the dif- ference. Polyunsaturated linolenic acid (18 : 3) was the most abundant fatty acid in both active and diapausing pupae, nevertheless its content was significantly higher in the former; the level of 7 fatty acids reached values higher than 1%. Differences in other nutrients were similar to those of TGs; higher contents of both glycogen and proteins were recorded in diapausing than active pupae. The results revealed significant differences between the two summer pupal forms of C. ohridella and furthered our understanding of the develop- mental strategy of this successful, invasive pest insect.

Adipokinetic hormones (AKHs) are a group of insect metabolic neurohormones, synthesized and released from an endocrine retrocerebral gland, the corpus cardiacum (CC). Small amounts of AKH have also been identified in the brain, although their role in this organ is not clear. To address this gap in the knowledge about insect brain biology, we studied the nucleotide sequence, tissue distribution, and subcellular localization of AKHs in the brain and CC of the firebug Pyrrhocoris apterus. This insect expresses two AKHs; the octapeptides Pyrap-AKH and Peram-CAH-II, the presence of which was documented in the both studied organs. In situ hybridization and quantitative reverse-transcription (q-RT)-PCR revealed the expression of the genes encoding for both AKHs not only in the CC, but also in brain. Electron microscopy analysis of the brain revealed the presence of these hormones in specialized secretory granules localized predominantly in the cellular bodies of neurons. The hormones might be transported from the granules into the axons, where they could play a role in neuronal signaling. Under acute stress induced by the injection of 3 μmol KCl, the level of AKHs in the brain increased to a greater extent than that in the CC. These results might indicate an enhanced role of brain-derived AKHs in defence reaction under acute stress situations.

Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8-fold in the central nervous system (CNS) and by 3.8-fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co-injection of hydrogen peroxide with Pyrap-AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6-fold to control levels when hydrogen peroxide is co-injected with Pyrap-AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co-treatment of the CNS with hydrogen peroxide and Pyrap-AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap-AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.

Circadian rhythms are found in almost all organisms from cyanobacteria to humans, where most behavioral and physiological processes occur over a period of approximately 24 h in tandem with the day/night cycles. In general, these rhythmic processes are under regulation of circadian clocks. The role of circadian clocks in regulating metabolism and consequently cellular and metabolic homeostasis is an intensively investigated area of research. However, the links between circadian clocks and aging are correlative and only recently being investigated. A physiological decline in most processes is associated with advancing age, and occurs at the onset of maturity and in some instances is the result of accumulation of cellular damage beyond a critical level. A fully functional circadian clock would be vital to timing events in general metabolism, thus contributing to metabolic health and to ensure an increased “health-span” during the process of aging. Here, we present recent evidence of links between clocks, cellular metabolism, aging and oxidative stress (one of the causative factors of aging). In the light of these data, we arrive at conceptual generalizations of this relationship across the spectrum of model organisms from fruit flies to mammals.

The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.

The role of adipokinetic hormone (AKH) and adenosine in the anti-stress response was studied in Drosophila melanogaster larvae and adults carrying a mutation in the Akh gene (Akh(1)), the adenosine receptor gene (AdoR(1)), or in both of these genes (Akh(1) AdoR(1) double mutant). Stress was induced by starvation or by the addition of an oxidative stressor paraquat (PQ) to food. Mortality tests revealed that the Akh(1) mutant was the most resistant to starvation, while the AdoR(1) mutant was the most sensitive. Conversely, the Akh(1) AdoR(1) double mutant was more sensitive to PQ toxicity than either of the single mutants. Administration of PQ significantly increased the Drome-AKH level in w(1118) and AdoR(1) larvae; however, this was not accompanied by a simultaneous increase in Akh gene expression. In contrast, PQ significantly increased the expression of the glutathione S-transferase D1 (GstD1) gene. The presence of both a functional adenosine receptor and AKH seem to be important for the proper control of GstD1 gene expression under oxidative stress, however, the latter appears to play more dominant role. On the other hand, differences in glutathione S-transferase (GST) activity among the strains, and between untreated and PQ-treated groups were minimal. In addition, the glutathione level was significantly lower in all untreated AKH- or AdoR-deficient mutant flies as compared with the untreated control w(1118) flies and further declined following treatment with PQ. All oxidative stress characteristics modified by mutations in Akh gene were restored or even improved by 'rescue' mutation in flies which ectopically express Akh. Thus, the results of the present study demonstrate the important roles of AKH and adenosine in the anti-stress response elicited by PQ in a D. melanogaster model, and provide the first evidence for the involvement of adenosine in the anti-oxidative stress response in insects.