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Environmental conditions, especially related to winter, are crucial for shaping activity of insect immune system. However, our previous research clearly indicates differences in the immune system functioning when the cold stress was induced in the laboratory conditions and when the beetles were collected from natural environment during winter. This is probably related to the multiplication of observed effects by simultaneous presence of different stress factors characteristic of winter, including desiccation. For these reasons, our next step was analysis of the effects of short-term desiccation and recovery time on the functioning of immune system of burying beetle Nicrophorus vespilloides. Also, the effect of Tenmo–PVK-2 (tenebrionid periviscerokinin), member of the CAPA–PVK neuropeptide family, was investigated to better understand observed changes. Short-term desiccation decreases the phagocytic activity of burying beetle haemocytes, which is correlated with a reduction in their adhesive ability. On the other hand, there was a significant increase in phenoloxidase (PO) activity and the level of proPO expression, which may suggest sealing the cuticula by melanin deposition and prevention of water loss. Additionally, the elevated level of defensin expression may be associated with the cross-talk between mechanisms, which participate in insect response to environmental stress, including pathogen infection. After 1 h of recovery time, the activity of tested cellular and humoral mechanisms was mostly back to the control level. However, inhibition of the activity of PO and down-regulation of proPO were noted. These results also indicate importance of melanin deposition during water loss. Moreover, it suggests that some changes in immune system functioning during stress conditions do not have an immune function. Interestingly, part of the effects characteristic of recovery time were also observed after the application of Tenmo–PVK-2, mainly related to haemocyte morphology. These results indicate that CAPA–PVK neuropeptides may also influence on activity of burying beetle immune system. It should be also highlighted that, because of the study of the effects of CAPA–PVK neuropeptides, homologs of vertebrate neuromedin U, the results may be interesting for search evolutionary similarities in the functioning of the neuroendocrine system of insects and vertebrates.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Background Animal toxins can have medical and therapeutic applications. Principally, toxins produced by insects, arachnids, snakes and frogs have been characterized. Venomous mammals are rare, and their venoms have not been comprehensively investigated. Among shrews, only the venom of Blarina brevicauda has been analysed so far, and blarina toxin has been proven to be its main toxic component. It is assumed that Neomys fodiens employs its venom to hunt larger prey. However, the toxic profile, properties and mode of action of its venom are largely unknown. Therefore, we analysed the cardio-, myo- and neurotropic properties of N. fodiens venom and saliva of non-venomous Sorex araneus (control tests) in vitro in physiological bioassays carried out on two model organisms: beetles and frogs. For the first time, we fractionated N. fodiens venom and S. araneus saliva by performing chromatographic separation. Next, the properties of selected compounds were analysed in cardiotropic bioassays in the Tenebrio molitor heart. Results The venom of N. fodiens caused a high decrease in the conduction velocity of the frog sciatic nerve, as well as a significant decrease in the force of frog calf muscle contraction. We also recorded a significant decrease in the frog heart contractile activity. Most of the selected compounds from N. fodiens venom displayed a positive chronotropic effect on the beetle heart. However, one fraction caused a strong decrease in the T. molitor heart contractile activity coupled with a reversible cardiac arrest. We did not observe any responses of the insect heart and frog organs to the saliva of S. araneus. Preliminary mass spectrometry analysis revealed that calmodulin-like protein, thymosin β-10, hyaluronidase, lysozyme C and phospholipase A2 are present in the venom of N. fodiens, whereas thymosin β4, lysozyme C and β-defensin are present in S. araneus saliva. Conclusion Our results showed that N. fodiens venom has stronger paralytic properties and lower cardioinhibitory activity. Therefore, it is highly probable that N. fodiens might use its venom as a prey immobilizing agent. We also confirmed that S. araneus is not a venomous mammal because its saliva did not exhibit any toxic effects. Electronic supplementary material The online version of this article (10.1186/s12983-017-0230-0) contains supplementary material, which is available to authorized users.

The usage of insects as model organisms is becoming more and more common in toxicological, pharmacological, genetic and biomedical research. Insects, such as fruit flies (Drosophila melanogaster), locusts (Locusta migratoria), stick insects (Baculum extradentatum) or beetles (Tenebrio molitor) are used to assess the effect of different active compounds, as well as to analyse the background and course of certain diseases, including heart disorders. The goal of this study was to assess the influence of secondary metabolites extracted from Solanaceae and Brassicaceae plants: Potato (Solanum tuberosum), tomato (Solanum lycopersicum), black nightshade (Solanum nigrum) and horseradish (Armoracia rusticana), on T. molitor beetle heart contractility in comparison with pure alkaloids. During the in vivo bioassays, the plants glycoalkaloid extracts and pure substances were injected at the concentration 10&minus, 5 M into T. molitor pupa and evoked changes in heart activity. Pure glycoalkaloids caused mainly positive chronotropic effects, dependant on heart activity phase during a 24-h period of recording. Moreover, the substances affected the duration of the heart activity phases. Similarly, to the pure glycoalkaloids, the tested extracts also mainly accelerated the heart rhythm, however S. tuberosum and S. lycopersicum extracts slightly decreased the heart contractions frequency in the last 6 h of the recording. Cardioacceleratory activity of only S. lycopersicum extract was higher than single alkaloids whereas S. tubersoum and S. nigrum extracts were less active when compared to pure alkaloids. The most cardioactive substance was chaconine which strongly stimulated heart action during the whole recording after injection. A. rusticana extract which is composed mainly of glucosinolates did not significantly affect the heart contractions. Obtained results showed that glycoalkaloids were much more active than glucosinolates. However, the extracts depending on the plant species might be more or less active than pure substances.

Insect overwintering is one of the most astonishing phases of the insect life cycle. Despite vast amounts of knowledge available about the physiological mechanisms of this phenomenon, the impact of stress factors on insect immune system functioning during the winter is still unknown. The aim of this study is to analyze how low temperatures influence the immune system of the beetle Nicrophorus vespilloides. The results show that the beetle's immune system is differently modulated by cold induced in laboratory settings than that which occurs in natural conditions. Among beetles cultured in conditions similar to summer, low temperatures, did not influence the number of circulating haemocytes, phenoloxidase activity, haemocytes morphology, and percentage ratio of haemocyte types. In these beetles, differences were noted only in the ability of haemocytes to perform phagocytosis. Individuals acclimated in natural conditions in autumn had a higher level of humoral response and a different percentage ratio of haemocyte types. During the winter period, the number of haemocytes in the beetles decreased, but the percentage ratio of phagocytic haemocytes increased. Furthermore, we noted an increase of phenoloxidase activity. Our study also showed mitotic divisions of haemocytes in haemolymph collected from burying beetles after cold exposure and from burying beetles collected from natural conditions during autumn and winter. Differences in response to low temperatures in laboratory conditions and the natural environment suggest that the simultaneous presence of other stress factors during winter such as desiccation and starvation have a significant influence on the activity of burying beetle's immune system.

Model organisms are often used in biological, medical and environmental research. Among insects, Drosophila melanogaster, Galleria mellonella, Apis mellifera, Bombyx mori, Periplaneta americana, and Locusta migratoria are often used. However, new model organisms still appear. In recent years, an increasing number of insect species has been suggested as model organisms in life sciences research due to their worldwide distribution and environmental significance, the possibility of extrapolating research studies to vertebrates and the relatively low cost of rearing. Beetles are the largest insect order, with their representative - Tribolium castaneum - being the first species with a completely sequenced genome, and seem to be emerging as new potential candidates for model organisms in various studies. Apart from T. castaneum, additional species representing various Coleoptera families, such as Nicrophorus vespilloides, Leptinotarsa decemlineata, Coccinella septempunctata, Poecilus cupreus, Tenebrio molitor and many others, have been used. They are increasingly often included in two major research aspects: biomedical and environmental studies. Biomedical studies focus mainly on unraveling mechanisms of basic life processes, such as feeding, neurotransmission or activity of the immune system, as well as on elucidating the mechanism of different diseases (neurodegenerative, cardiovascular, metabolic, or immunological) using beetles as models. Furthermore, pharmacological bioassays for testing novel biologically active substances in beetles have also been developed. It should be emphasized that beetles are a source of compounds with potential antimicrobial and anticancer activity. Environmental-based studies focus mainly on the development and testing of new potential pesticides of both chemical and natural origin. Additionally, beetles are used as food or for their valuable supplements. Different beetle families are also used as bioindicators. Another important research area using beetles as models is behavioral ecology studies, for instance, parental care. In this paper, we review the current knowledge regarding beetles as model organisms and their practical application in various fields of life science.

Neuropeptides control the functioning of the nervous system of insects, and they are the most diverse signalling molecules in terms of structure and function. Allatostatins are pleiotropic neuropeptides that are considered potent myoinhibitors of muscle contractions in insects. We investigated the effects caused by three distinct allatostatins, Dippu-AST1 (LYDFGL-NH2 from Diploptera punctata), Grybi-MIP1 (GWQDLNGGW-NH2 from Gryllus bimaculatus) and Trica-ASTC (pESRYRQCYFNPISCF-OH from Tribolium castaneum) on contractile activity of the myocardium, oviduct and hindgut of two tenebrionid beetles, Tenebrio molitor and Zophobas atratus. Studies showed that all three peptides exerted myostimulatory effects on the oviduct and hindgut of the beetles, however they did not cause any effect on myocardium. The effects of Dippu-AST1, Grybi-MIP1 and Trica-ASTC were dose-dependent and tissue and species specific. The highest stimulatory effect was caused by Trica-ASTC, showing stimulation of approximately 82% at a 10-12 M concentration and 76% at a 10-11 M concentration for T. molitor and Z. atratus, respectively. The oviduct of T. molitor was more susceptible to allatostatins than that of Z. atratus. Dippu-AST1 showed the maximum stimulating effect at 10-11 M (57%), whereas Grybi-MIP 1 at 10-10 M caused a 41% stimulation. Trica-ASTC, in both species, showed a myostimulatory effect over the whole range of tested concentrations but was most potent at a 10-12 M concentration and caused a 54% and 31.9% increase in the frequency of contractions in the oviduct of T. molitor and Z. atratus, respectively. The results suggest that allatostatins may affect the regulation of egg movement within the oviducts and movement of food in the digestive tract of beetles and do not regulate directly the activity of heart, thus being good candidate compounds in neuropeptides based pest control agents in future research.

In insects, two types of the immune responses, cellular and humoral, constitute a defensive barrier against various parasites and pathogens. In response to pathogens, insects produce a wide range of immune agents that act on pathogens directly, such as cecropins or lysozyme, or indirectly by the stimulation of hemocyte migration or by increasing phenoloxidase (PO) activity. Recently, many new immunologically active substances from insects, such as peptides and polypeptides, have been identified. Nevertheless, in the most cases, their physiological functions are not fully known. One such substance is yamamarin – a pentapeptide isolated from the silk mothAntheraea yamamai. This yamamarin possesses strong antiproliferative properties and is probably involved in diapause regulation. Here, we examined the immunotropic activity of yamamarin by testing its impact on selected functions of the immune system in heterologous bioassays with the beetleTenebrio molitor, commonly known as a stored grains pest. Our results indicate that the pentapeptide affects the activity of immune processes in the beetle. We show that yamamarin induces changes in both humoral and cellular responses. The yamamarin increases the activity of PO, as well as causes changes in the hemocyte cytoskeleton and stimulates phagocytic activity. We detected an increased number of apoptotic hemocytes, however after the yamamarin injection, no significant variations in the antibacterial activity in the hemolymph were observed. The obtained data suggest that yamamarin could be an important controller of the immune system inT. molitor.

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nicve-MS), myoinhibitory peptide (Trica-MIP-5) and the short neuropeptide F (Nicve-sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 × 10-8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve-MS: IC50 = 5.20 × 10-5 mol/L; Trica-MIP-5: IC50 = 5.95 × 10-6 mol/L; Nicve-sNPF: IC50 = 4.08 × 10-5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin-immunolabeled neurons only in VNC were observed. Moreover, MIP-immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve-MS (QDVDHVFLRFa) and six isoforms of Nicve-MIP (Nicve-MIP-1-DWNRNLHSWa; Nicve-MIP-2-AWQNLQGGWa; Nicve-MIP-3-AWQNLQGGWa; Nicve-MIP-4-AWKNLNNAGWa; Nicve-MIP-5-SEWGNFRGSWa; Nicve-MIP-6- DPAWTNLKGIWa; and Nicve-sNPF-SGRSPSLRLRFa).

Insects are the largest and the most widely distributed group of animals in the world. Their diversity gives rise to an incredible variety of different mechanisms for the regulation of life processes. There are many agents that regulate immunology, reproduction, growth and development or metabolism. Hence, it seems that insects may be a source of numerous substances with utility for the treatment human diseases. Of particular importance in the regulation of insect physiology are peptides, including neuropeptides, peptide hormones and antimicrobial peptides. There are two main ways in which these peptides may be useful. 1) Peptides isolated from insects may become potential therapeutic drugs for different diseases. 2) Many insect peptide hormones show structural or functional homology to mammalian peptide hormones, and comparative studies may provide a new perspective on human disorders. In our review, we focused on three groups of insect-derived peptides: 1) immune-active peptides, 2) peptide hormones and 3) peptides present in venoms. In our review, we show the considerable potential of insect peptides in the search for new solutions for mammalian diseases treatment. We summarize knowledge about the properties of insect peptides against different virulent agents, and their anti-inflammatory or anti-nociceptive properties as well as compare the insect and mammalian/vertebrate peptide endocrine systems to determine the usefulness of knowledge about insect peptide hormones in drug design. The field of possible uses of insect-delivered peptides for the treatment of various human diseases has still not been sufficiently explored. Undoubtedly, more attention should be paid to insects in the search for new drugs.

Glycoalkaloids, the biologically active secondary metabolites produced by Solanaceae plants, are natural defenses against animals, insects and fungi. In this paper, the effects of glycoalkaloids present in extracts of Solanaceae plants (potato, tomato and black nightshade) or pure commercial glycoalkaloids on the coleopteran Zophobas atratus F. were evaluated by in vitro and in vivo bioassays using heart experimental models. Each tested extract induced a dose-dependent cardioinhibitory effect. The perfusion of Zophobas atratus semi-isolated heart using the highest potato and tomato extract concentration (1 mmol/L) caused irreversible cardiac arrests, while extract from black nightshade produced fast but reversible arrests. Pure commercial glycoalkaloids caused similar but less evident effects compared with extracts. Our results showed that the bioactivity of tested compounds depended on their structure and suggested the existence of synergistic interactions when combinations of the main glycoalkaloids of potato and black nightshade were used for trials. Surprisingly, injection of tomato and potato extracts in 1-day-old pupae of Zophobas atratus induced reversible positive chronotropic effects and decreased the duration of the both phases (anterograde and retrograde) of the heart contractile activity. Furthermore, these extracts affected the amplitude of the heart contractions.

Necrophagous beetles of the genus Nicrophorus have developed various defence mechanisms that reduce the negative effects of adverse environmental conditions. However, many physiological and ecological aspects, including the functioning of the immune system in burying beetles, are still unknown. In this study, we show developmental changes in cellular and humoral responses of larvae, pupae, and adults of Nicrophorus vespilloides. We assessed changes in total haemocyte count, phenoloxidase activity, and phagocytic ability of haemocytes. We found that during larval development there is a progressive increase in humoral and cellular activities, and these responses are correlated with alterations of total haemocyte counts in the haemolymph. In the pupal stage, a sharp drop in the number of phagocytic haemocytes and an increase in phenoloxidase activity were observed. In adults, cellular and humoral responses remained at a lower level. It is probable that high lytic activity of anal and oral secretions produced by parents supports a lower response of the immune system in the initial phase of larval development. In the studied stages, we also observed differences in polymerisation of F-actin cytoskeleton of haemocytes, number of haemocytes forming filopodia, and filopodia length. These results suggest that the differences in immune responses during various stages of development of N. vespilloides are associated with a dynamically changing environment and different risks of infection. For the first time a detailed analysis of stage-specific alterations in immune system activity during development of the burying beetle is presented.

Melittin (MEL) is a basic polypeptide originally purified from honeybee venom. MEL exhibits a broad spectrum of biological activity. However, almost all studies on MEL activity have been carried out on vertebrate models or cell lines. Recently, due to cheap breeding and the possibility of extrapolating the results of the research to vertebrates, insects have been used for various bioassays and comparative physiological studies. For these reasons, it is valuable to examine the influence of melittin on insect physiology. Here, for the first time, we report the immunotropic and cardiotropic effects of melittin on the beetle Tenebrio molitor as a model insect. After melittin injection at 10−7 M and 10−3 M, the number of apoptotic cells in the haemolymph increased in a dose-dependent manner. The pro-apoptotic action of MEL was likely compensated by increasing the total number of haemocytes. However, the injection of MEL did not cause any changes in the percent of phagocytic haemocytes or in the phenoloxidase activity. In an in vitro bioassay with a semi-isolated Tenebrio heart, MEL induced a slight chronotropic-positive effect only at a higher concentration (10−4 M). Preliminary results indicated that melittin exerts pleiotropic effects on the functioning of the immune system and the endogenous contractile activity of the heart. Some of the induced responses in T. molitor resemble the reactions observed in vertebrate models. Therefore, the T. molitor beetle may be a convenient invertebrate model organism for comparative physiological studies and for the identification of new properties and mechanisms of action of melittin and related compounds.

Only a small percentage of insect species are pests. However, pest species cause significant losses in agricultural and forest crops, and many are vectors of diseases. Currently, many scientists are focused on developing new tools to control insect populations, including secondary plant metabolites, e.g., alkaloids, glycoalkaloids, terpenoids, organic acids and alcohols, which show promise for use in plant protection. These compounds can affect insects at all levels of biological organization, but their action generally disturbs cellular and physiological processes, e.g., by altering redox balance, hormonal regulation, neuronal signalization or reproduction in exposed individuals. Secondary plant metabolites cause toxic effects that can be observed at both lethal and sublethal levels, but the most important effect is repellence. Plants from the Solanaceae family, which contains numerous economically and ecologically important species, produce various substances that affect insects belonging to most orders, particularly herbivorous insects and other pests. Many compounds possess insecticidal properties, but they are also classified as molluscides, acaricides, nematocides, fungicides and bactericides. In this paper, we present data on the sublethal and lethal toxicity caused by pure metabolites and crude extracts obtained from Solanaceae plants. Pure substances as well as water and/or alcohol extracts cause lethal and sublethal effects in insects, which is important from the economical point of view. We discuss the results of our study and their relevance to plant protection and management.

Neuropeptides and peptide hormones from non-neuronal tissues play important roles in the regulation of insect life. In recent years, the rapid development of analytical techniques has contributed to the discovery of more than 30 families of peptide neurohormones that differ structurally and functionally. Although the discovery of the first neuropeptide occurred almost forty years ago, our knowledge about their full mode of activities, primary structures, synthesis, interactions with receptors or places of action increases gradually and there is still much to unravel. However, one thing is certain. Neuropeptides perform an extremely diverse range of activities. One neuropeptide can affect physiology in different ways. The neuropeptides can act as neurotransmitters, co-transmitters as well as neuromodulators. Most of these molecules have diverse pleiotropic activities on different tissues and organs. Their mode of action includes allatotropic, myotropic, cardiotropic or gonadotropic effects. Activity of some of them is conserved among most of insect species, indicating crucial roles in insect physiology and age of these systems. On the other hand, activity of other neuropeptides and peptide hormones is highly diverse, depending on species or even stages of development. This may indicate that some compounds have taken over the function of others. Insect heart work is regulated in a very complex manner. Myocardium activity undergoes regulation both, by nervous and hormonal way. What is important is that these same compounds can influent on heart as both nervous and hormonal factors. For that reason, the regulation of myocardium is still unclear. In this paper, we summarize the existing knowledge regarding cardioactivity and the involvement of insect neurohormones and some peptide hormones from non-neural tissues to regulation of insect myocardium.

Seeds of 3 commercial China aster (Callistephus chinensis Nees) lots were divided by hand into 3 grades with different colours: dark brown, brown and light brown, and 2 grades with a different size: length below 3.9 mm (small) and above 3.9 mm (large). The colour grading was done based on the Royal Horticultural Society Colour Chart and size grading was done by hand for each seed. Then, seeds were routinely germinated based on the International Seed Testing Association (ISTA) rules. The size of seeds had no effect on their germination. The dark brown seeds germinated better than the light brown ones. Removing light brown seeds from the China aster seed lot improved their germination.

The peptides proctolin, crustacean cardioactive peptide (CCAP) and FMR- Famide, which are known to modulate insect muscle contractions, were assayed for their action on oviduct contractions in Hylobius abietis. A video microscopy technique and computer-based method of data acquisition and analysis were used to investigate the ef- fects of theses peptides on spontaneous contractions of continuously perfused oviducts. All three peptides tested stimulate spontaneous contraction activity of the pine weevil oviduct, increasingthefrequencyandamplitudeofphasiccontractionsinadose-dependentmanner. Proctolin is more potent as a stimulator than CCAP. For proctolin a threshold response of oviduct muscles is at concentration of peptide 10 −11 -10 −10 mol/L and for CCAP at concentration range 10 −10 -10 −9 mol/L. FMRFamide exerts a weak stimulatory effect on the oviduct, and at higher concentrations of the peptide (above 10 −8 mol/L). The peptides exert different responses on oviduct contractions and they may play a role as functional regulators in such processes as egg movement and oviposition.

Five proctolin analogues modified in position 2 of the peptide chain by (4-NH2)- (I), (4-NO2)- (II), (4-NMe2)- (III), (4-OMe)-L-phenylalanine (IV) and β-(cyclohexyl-4-OMe)-L-alanine (V) were synthesized by conventional liquid phase method. Biological activity of the obtained peptides was investigated in cardioexcitatory test on two insect species, cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor L. Peptides I–IV exhibited higher activity than proctolin itself.

Five proctolin analogues modified in position 1 of the peptide chain by Cit (I), Lys (II), His (III), Phe(p-NH2) (IV) and γ-Abu (V) were synthesized by conventional liquid phase method. Biological activity of the obtained peptides was investigated by the cardioexcitatory test on two insect species, cockroach, Periplaneta americana L. and yellow mealworm, Tenebrio molitor L.

Protective mechanisms against cold stress are well studied in terrestrial and polar insects; however, little is known about these mechanisms in tropical insects. In our study, we tested if a tropical cockroach Gromphadorhina coquereliana, possesses any protective mechanisms against cold stress. Based on the results of earlier studies, we examined how short-term (3 h) cold (4°C) influences biochemical parameters, mitochondrial respiration activity, and the level of HSPs and aquaporins expression in the fat body and leg muscles of G. coquereliana. Following cold exposure, we found that the level of carbohydrates, lipids and proteins did not change significantly. Nevertheless, we observed significant changes in mitochondrial respiration activity. The oxygen consumption of resting (state 4) and phosphorylating (state 3) mitochondria was altered following cold exposure. The increase in respiratory rate in state 4 respiration was observed in both tissues. In state 3, oxygen consumption by mitochondria in fat body was significantly lower compared to control insects, whereas there were no changes observed for mitochondria in muscle tissue. Moreover, there were cold-induced changes in UCP protein activity, but the changes in activity differed in fat body and in muscles. Additionally, we detected changes in the level of HSP70 and aquaporins expression. Insects treated with cold had significantly higher levels of HSP70 in fat body and muscles. On the other hand, there were lower levels of aquaporins in both tissues following exposure to cold. These results suggest that fat body play an important role in protecting tropical insects from cold stress.

Insects cope with thermal stressors using mechanisms such as rapid cold hardening and acclimation. These mechanisms have been studied in temperate insects, but little is known about their use by tropical insects in response to cold stress. Here, we investigated whether cold stress (1×8 h and 3×8 h at 4°C) triggers a metabolic response in the Madagascar cockroach Gromphadorhina coquereliana. We examined the effects of cold on the levels of selected metabolites in the fat body tissue of G. coquereliana. After cold exposure, we found that the quantity of total protein increased significantly in the insect fat body, whereas glycogen decreased slightly. Using antibodies, we observed upregulation of AQP-like proteins and changes in the HSP70 levels in the fat body of G. coquereliana when exposed to cold. We also examined the content and nature of the free sugars in the G. coquereliana hemolymph and discovered an increase in the levels of polyols and glucose in response to cold stress. These results suggest an important role of the fat body tissue of tropical insects upon cold exposure.

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus . Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae , CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria . The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10 −9 –10 −6 M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus . CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10 −9 –10 −6 M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor , however, the CavTKs induced no significant effect on the heart. Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut. In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain. Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6–10]-Mas-MT-I, whereas the [4–10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln1]- and [Gly1]-Any-GS also show a strong cardioinhibitory effect. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X1-Tyr-Leu-Pro-Thr-OH, where X1 = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3'-NH2) (III), Tyr (3'-NO2) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-L-butyric acid), Afb (p-NH2) (VI), Afb (p-NO2) (VII), and the tetrapeptide Tyr (3'-NH2)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10(-9)-10(-7) M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10(-7) M. Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.

A hypertrehalosemic neuropeptide from the corpora cardiaca of the two tenebrionid beetle species, Tenebrio molitor and Zophobas rugipes , was purified by high performance liquid chromotography, and its sequence determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. Additionally, the C-terminus of the peptide was blocked as shown by the lack of breakdown using carboxypeptidase. In both species an identical octapeptide, designated Tem-HrTH, with the following amino acid sequence, was found: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH 2 . This primary sequence has an 88% homology with the hypertrehalosemic hormone I (Pea-CAH-I) from the American cockroach as well as with the red pigment-concentrating hormone (RPCH) of prawns. Injection of the synthetic peptide into larvae or young adults of T. molitor or adult Z. rugipes increases the hemolymph carbohydrate levels in a dose-dependent manner. Thin layer chromatography identified the elevated sugar component of the hemolymph as the disaccharide trehalose. Carbohydrate release from larval fat body in vitro was also shown upon administration of a low concentration of synthetic Tem-HrTH.

Six proctolin analogues (I-VI) modified in position 1 of the peptide chain by the following amino acids: homo-Arg, Gac, Gav, Gap, Phe (p-guanidino) and Orn, were synthesized by conventional liquid phase method. The myotropic activity of the obtained peptides was investigated in cardioexcitatory test on two insect species, cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor.

The corpus cardiacum-corpus allatum complex of Tenebrio molitor contains factors causing hyperglycaemia, stimulating carbohydrate release from the fat body in vitro, and accelerating heart beat in the donor insect. However, no adipokinetic response can be demonstrated upon injection of extracts of this complex in larvae or young imagines. The carbohydrate elevation in the haemolymph is time- and dose-dependent, with a maximal effect about 90–180 min after injection. About 0.03–0.05 gland equivalents are needed to produce a significant increase in the larval and imaginal bioassays; maximal responses in both systems were reached with about 0.5 gland equivalents. Fat body treated in vitro with an extract of 0.5 gland equivalents releases about 130% more carbohydrate into 1 ml physiological saline after 1 h than paired tissue controls. The characteristic response of a semi-isolated adult heart to the endogenous myoactive factor(s) of the corpus cardiacum-allatum extract was an increase in heart beat frequency and a decrease in the amplitude of contraction. The response was dose-dependent and 0.05 gland equivalents were needed to exert a stimulatory effect on the myocardium. Fractionation of a methanolic extract of endocrine complex from Tenebrio molitor by RP-HPLC shows that the hyperglycaemic activity is confined to two regions. A myoactive factor appears to elute just after the second hyperglycaemic material, whereas a cardio-inhibitory factor has more hydrophobic properties. Synthetic neuropeptides adipokinetic hormone I and red pigment concentrating hormone injected as a dose of 8 pmol produce a weak hyperglycaemic effect, whereas myoactive factors I and II and hypertrehalosaemic factor II cause no hyperglycaemia. No adipokinetic response was shown upon injection of any of the neuropeptides tested. A very small stimulation of the adult heart beat frequency was observed upon administration of 20 pmol adipokinetic hormone I and myoactive factor I; all other peptides were without effect. In conclusion, the bioassays with Tenebrio appear to show some species-specificity and the hyperglycaemic/myoactive factors present in the corpus cardiacum-allatum neurosecretory complex may possess distinctive properties compared with adipokinetic hormone I, red pigment concentrating hormone, myoactive factors I and II, and hypertrehalosaemic factor II.

We have identified and characterized an uncoupling protein in mitochondria isolated from leg muscle and from fat body, an insect analogue tissue of mammalian liver and adipose tissue, of the cockroach Gromphadorhina coquereliana (GcUCP). This is the first functional characterization of UCP activity in isolated insect mitochondria. Bioenergetic studies clearly indicate UCP function in both insect tissues. In resting (non-phosphorylating) mitochondria, cockroach GcUCP activity was stimulated by the addition of micromolar concentrations of palmitic acid and inhibited by the purine nucleotide GTP. Moreover, in phosphorylating mitochondria, GcUCP activity was able to divert energy from oxidative phosphorylation. Functional studies indicate a higher activity of GcUCP-mediated uncoupling in cockroach muscle mitochondria compared to fat body mitochondria. GcUCP activation by palmitic acid resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of UCPs in insects. GcUCP protein was immunodetected using antibodies raised against human UCP4 as a single band of around 36 kDa. GcUCP protein expression in cockroach muscle mitochondria was significantly higher compared to mitochondria isolated from fat body. LC-MS/MS analyses revealed 100% sequence identities for peptides obtained from GcUCP to UCP4 isoforms from D. melanogaster (the highest homology), human, rat or other insect mitochondria. Therefore, it can be proposed that cockroach GcUCP corresponds to the UCP4 isoforms of other animals.

Essential role in the regulation of all physiological processes and maintenance of homeostasis in the vast majority of animal organisms plays a neuro- endocrine system. So far, very well known to features are neuro-hormonal mediators regulating life processes of human. Over the last decade attention of researches has focused on identification and evaluation functions of insect neuropeptides and genes encoding them. In the present review, the structure and functions of insect neuro-endocrine system are characterized, the current state of knowledge on the difficulties and strategies in the neuropeptidomics research of these animals, with special emphasis on the use of mass spectrometry and the current rules of neuropeptides classification. Moreover, a list of known neuropeptidome insect species is presented, functional similarities between insect neuropeptides and their homologues occurring in vertebrates are defined and potential applications of research in the field of neuropeptidomics ewaluated. Available literature data suggest that neuropeptides so far identified in the insect model organism such as Drosophila melanogaster, may be used in the future to design a new generation of pharmaceuticals and bioinsecticides limiting the number of harmful insects from medical and economic point of view.

Background The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown. Results We tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. Conclusion Neb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.