Search

In recent years, many new immunologically active peptides from insects have been identified. Unfortunately, in most cases, their physiological functions are not fully known. One example is yamamarin, a pentapeptide isolated from the caterpillars of the Antheraea yamamai moth. This peptide has strong antiproliferative properties and is probably involved in the regulation of diapause. Additionally, antiviral activity was discovered. The results of the research presented in this paper are, to our knowledge, the first attempt to characterize the biological effects of yamamarin on the functioning of the reproductive processes and embryonic development of insects using a model species, the beetle Tenebrio molitor, a commonly known pest of grain storage. Simultaneously, we tested the possible activity of the molecule in an in vivo system. In this research, we present the multifaceted effects of yamamarin in this beetle. We show that yamamarin influences ovarian growth and development, maturation of terminal oocytes, level of vitellogenin gene transcript, the number of laid eggs, duration of embryonic development, and larval hatching. In experiments with palmitic acid-conjugated yamamarin (C16-yamamarin), we also showed that this peptide is a useful starting molecule for the synthesis of biopharmaceuticals or new peptidomimetics with gonadotropic activity and effects on embryonic development. The data obtained additionally provide new knowledge about the possible function of yamamarin in insect physiology, pointing to the important role of this pentapeptide as a regulator of reproductive processes and embryonic development in a heterologous bioassay with T. molitor.

Neuropeptides of short neuropeptides F family (sNPF) have been identified in various arthropods. They are pleiotropic neuromolecules which so far have been mainly associated with regulation of feeding and metabolism, as well as growth and development, locomotion, circadian rhythm or learning and memory. Here, we describe the effects of Tenebrionid sNPF peptide (SGRSPSLRLRFa) on various aspects of the male reproductive physiology in the Tenebrio molitor beetle. We identified in silico the putative sNPF receptor Tenmo-sNPFR. Based on RT-PCR technique, it was shown that the receptor might be present in the male reproductive tissues of this beetle. The analysis of receptor amino acid sequence showed that it is similar to other beetle sNPFRs, as well as other insect species, and belongs rhodopsin-like G-protein-coupled receptors (GPCRs). Injections of Trica-sNPF and its shorter form Trica-sNPF(4–11) caused differentiated effects in T. molitor male reproductive tissues. After 24 h post injections, the peptides decreased the concentration of the soluble protein fraction in testes of 4- and 8-day-old beetles as well as the dry mass of these organs but only in 8-day-old individuals. The same effects were shown with regard to accessory glands. Both peptides decrease the concentration of the soluble protein fraction but do not affect the dry mass of this organ. Furthermore, injections of Trica-sNPF at the 10–7 M concentration decrease the total sperm number in the reproductive system. Surprisingly, the same concentration of the shorter form, Trica-sNPF(4–11) increased the sperm number. It was also shown that both peptides in different manner influence contractions of ejaculatory duct. The data presented in this article give new evidence that sNPFs are involved in the regulation of reproductive events in beetles, which might be the part of a larger neuropeptide network combining feeding, growth and development with the physiology of reproduction. Electronic supplementary material The online version of this article (10.1007/s00360-020-01296-z) contains supplementary material, which is available to authorized users.

Bee pollen, because of its high content of nutrients, is a very valuable medicinal and nutritional product. However, since its composition is not completely studied, the consumption of this product may cause adverse effects, including allergic reactions. Therefore, this study aimed to discover and characterize the bioactive proteins of bee pollen collected in Poland, focusing mainly on the allergens. For this purpose, the purified and concentrated pollen aqueous solutions were analyzed using the nanoLC-MALDI-TOF/TOF MS analytical platform. As a result of the experiments, 197 unique proteins derived from green plants (Viridiplantae) and 10 unique proteins derived from bees (Apis spp.) were identified. Among them, potential plant allergens were discovered. Moreover, proteins belonging to the group of hypothetical proteins, whose expression had not been confirmed experimentally before, were detected. Because of the content of bioactive compounds—both beneficial and harmful—there is a critical need to develop guidelines for standardizing bee pollen, especially intended for consumption or therapeutic purposes. This is of particular importance because awareness of the allergen content of bee pollen and other bee products can prevent health- or life-threatening incidents following the ingestion of these increasingly popular “superfoods”.

to comprehensively characterize honeybee venom, royal jelly, propolis, and pollen, by applying advanced analytical and bioinformatics methodologies. Honeybee products (HBP) contain many bioactive components with both beneficial and harmful effects on the human organism. Nevertheless, the overall composition of the HBP remains not fully investigated. Thus, this research is focused on complementary proteomic and metabolomic characterization of biologically active compounds derived from HBP, regarding their toxicological and pharmacological properties. The objectives of the study will be achieved by the application of up to date mass spectrometry techniques. Due to increasing interest in using of HBP in medicine, this project will contribute to improving the safety of HBP‑derived dietary supplements and drugs.

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

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.

Despite much research on the insect immune system, hormonal regulation of its activity is not well-understood. Previous research on insect neuroendocrinology suggests that neuropeptides may play an important role in the regulation of the insect immune system. Especially recent studies dealing for example with adipokinetic hormones, bursicon or insulin-like peptides provided deeper insights on this issue showing that neuropeptides can modulate various aspects of insect immune responses, both at the molecular and cellular level. The presented review summarizes the current knowledge about the role of neuropeptides regulating the insect immune system activity. Based on structural and functional homology of some vertebrate and insect neuropeptide families, several propositions of insect neuropeptides that might also possess immunotropic activities, but have not been examined for this aspect, are discussed.

BACKGROUND In mammals, the cholinergic nervous system plays a crucial role in neuronal regulation of physiological processes. It acts on cells by two types of receptors - nicotinic and muscarinic receptors. Both signal transmission pathways also operate in the central and peripheral cholinergic nervous system of insects. METHOD In our pharmacological experiments, we studied the effects of two muscarinic agonists (carbachol, pilocarpine) and two muscarinic antagonists (atropine, scopolamine) on the muscle contractile activity of visceral organs in the beetle, Tenebrio molitor. RESULTS Both antagonists, when injected to haemolymph at concentration 10-5 M, caused delayed and prolonged cardioinhibitory effects on heart contractility in ortho- and antidromic phases of heart activity in T. molitor pupa what was observed as negative chrono- and inotropic effects. Agonist of muscarinic receptors - carbachol evoked opposite effect and increased contraction rate but only in antidromic phase. Pilocarpine, the second agonist induced weak negative chronotropic effects in the antiand orthodromic phases of heart activity. However, neither agonists had an effect on semi-isolated beetle heart in vitro. Only atropine at the highest tested concentrations slightly decreased the frequency of myocardial contractions. These suggest the regulation of heart activity by muscarinic system indirectly. The tested compounds also affected the contractility of the oviduct and hindgut, but the responses of these organs were varied and depended on the concentration of the applied compounds. CONCLUSION These pharmacological experiments suggest the possible modulation of insect visceral muscle contractility by the cholinergic nervous system and indirectly indicate the presence of muscarinic receptor(s) in the visceral organs of the beetle T. molitor.

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.

The evolutionary success of insects is undoubtedly related to a well-functioning immune system. This is especially apparent during insect development by the adaptation of individuals to the changing risk of infection. In addition, current studies show that the insect immune system is characterized by some specificity in response to natural pathogens (for example, bacteria, viruses or fungi) and artificial challengers (for example, latex beads or nylon filaments). However, developmental changes and the specificity of immune system reactions simultaneously have not been analysed. Thus, the aim of the present research was to determine changes in haemocyte morphology in response to attenuated Staphylococcus aureus and latex beads across each developmental stage of the beetle Tenebrio molitor. The results of the present research clearly showed differences in the morphology of T. molitor haemocytes during development. The haemocytes of larvae and 4-day-old adult males were characterized by the highest adhesion ability, which was expressed as the largest average surface area, filopodia length and number of filopodia. In contrast, the haemocytes of pupae and 30-day-old adult males had a significantly lower value for these morphological parameters, which was probably related to metamorphosis (pupae) and immunosenescence (30-day-old adults). The haemocytes of the tested individuals reacted differently to the presence of S. aureus and latex beads. The presence of S. aureus led to a significant decrease in all previously mentioned morphological parameters in larvae and in both groups of adult individuals. In these groups, incubation of haemocytes with latex beads caused only a slight decrease in surface area and filopodia length and number. This morphological response of haemocytes to biotic and artificial challengers might be related to an increase in the migration abilities of haemocytes during infection. However, the differences in haemocyte reactivity towards S. aureus and latex beads might be explained by differences in pathogen recognition. Conversely, increased adhesive abilities of pupal haemocytes were also observed, which might be related to the specificity of metamorphosis and the hormonal titre during this developmental stage.

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 10−5 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.

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH-1L and/or CuH-2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH-1L, CuH-2L and CuH-3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat-SK-1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat-SK-1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat-SK-1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone-treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat-SK-1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy-dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat-SK-1-induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.

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.

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

Insect sulfakinins are multifunctional neuropeptides homologous to vertebrate gastrin/cholecystokin (CCK) neuropeptide hormones. We investigated the action of the nonsulfated sulfakinin Zopat-SK-1 (pETSDDYGHLRFa) on the levels of chosen metabolites in the Zophobas atratus beetle fat body. Samples of fat body were collected 2h and 24h after hormone injection. The administration of 20pmol of Zopat-SK-1 to feeding larvae significantly increased concentrations of lipids and proteins and decreased the content of glycogen in fat body tissue in the 24h experimental group. In contrast, the only increase in total lipid concentration in prepupal fat bodies was observed 24h after Zopat-SK-1 treatment. Simultaneously, changes in the quality and quantity of free sugars in the hemolymph were measured. In larval hemolymph, a marked increase in free sugar concentration and a decrease in glucose content were observed 24h and 2h after Zopat-SK-1 application, respectively. No changes in the prepupal stage were observed. For the first time we show potent metabolic activity of sulfakinin in the fat body tissue of an insect. Our findings imply a physiological function of the nonsulfated form of sulfakinin in energy storage and release processes in fat body tissue of larvae and prepupae was indicated. We suggest a role for sulfakinin signaling in the regulation of energy metabolism in insect tissues.

The nineteenth amino acid peptide Neb-colloostatin SIVPLGLPVPIGPIVVGPR and its analogue with point mutation (P4A) were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. The amine-N nitrogen atoms of serine residues were found to be the primary metal binding sites of both peptides. These binding modes provide the deprotonation and coordination of two amide nitrogens of the Neb-colloostatin and the formation of the 3N {NH2,2N−,CO} complex at pH 7.5–8. The proline residue P4 is the breakpoint in the coordination of copper(II) ions and the 4N {NH2,3N−} complex was not observed. At pH 8 the analogue (P4A) of Neb-colloostatin forms the copper(II) complex with 4N {NH2,3N−} binding site. The potentiometric and spectroscopic results did not reveal the stabilizing role of the oxygen donors of side chains of seryl residues (S1) in copper(II) complexes. At pH 8 the copper(II) ions change the secondary structures of both peptides in comparison to those without copper(II) ions. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The copper(II) complexes of Neb-colloostatin and its analogue exert stronger pro-apoptotic activity on insect hemocytes in comparison to those of peptides without metal ions.

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.

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A 1 GVSGH 6 GQH 9 GVH 12 G (Allo1A) and (H9A) H 1 GVSGH 6 GQA 9 GVH 12 G (Allo9A) have been studied by potentiometric, UV–visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H 6 ,H 12 ) leads to the formation of the CuL complex with 4N {NH 2 ,N Im –H 1 ,N Im –H 6 ,N Im –H 12 } binding site in wide pH range (5–8). For the Cu(II)–Allo1A system, the results demonstrated that at physiological pH 7.4 the predominant complex the CuH − 1 L consists of the 3N {NH 2 ,N − ,CO,N Im } coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)–Allo9A complex formed at pH 7.4. The results may suggest that the N-terminal-His 1 and His 6 residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

In insects, neuropeptide adipokinetic hormone (AKH) released from the corpora cardiaca mobilizes lipids and carbohydrates in the fat body. We examined the developmental differences in the action of Tenmo-AKH, a bioanalogue belonging to the adipokinetic/hypertrahelosemic family (AKH/HrTH), on the lipid composition of larval and pupal fat bodies in the beetle Zophobas atratus . Tenmo-AKH was administered to the beetle larvae and pupae either as a single dose or as two doses of 20 pmol during a 24 h interval. Extracts of fat bodies were used to analyse the lipid composition by gas chromatography (GC) combined with mass spectrometry (GC–MS). Control extracts were analyzed using the same method. Fatty acids (FA) and fatty acid methyl esters (FAME) were the most abundant compounds in the fat bodies from both developmental stages. We observed significant differences in their concentrations following hormonal treatment. Tenmo-AKH also induced a distinct increase in larval sterols, fatty alcohols and benzoic acid.

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.

MALDI-TOF MS and MS/MS techniques were used for the isolation and identification of neuropeptides from the ventral nerve cord (VNC) of two beetle species Tenebrio molitor and Zophobas atratus. Two peptides, proctolin and myosuppressin (Zopat-MS), with well-established myotropic properties were identified as well as Trica-NVPL-4trunc. The presence of proctolin and myosuppressin was confirmed by immunocytochemical studies in adults and larvae of both beetles. In addition, the myosuppressin gene in Z. atratus was sequenced and expression analyses showed that it is present in all parts of the beetle central nervous system. Results suggest that the identified peptides act as neurotransmitters/neuromodulators in beetles, regulate visceral muscle contractions and indirectly influence important physiological processes such as feeding and reproduction.

We investigated the influence of adipokinetic hormone (AKH), an insect neurohormone, on uncoupling protein 4 (ZaUCP4) expression and activity in larval and pupal fat body mitochondria of the beetle Zophobas atratus in relation to intermediary metabolism. Homologous Tenmo-AKH was administered to the beetle larvae and pupae as either a single dose or as two doses of 20 pmol during a 24 h interval. In the larval and pupal fat bodies, downregulation of ZaUCP4 expression at the mRNA and protein levels was observed 24 h and 48 h after AKH treatment, respectively. In both developmental stages, ZaUCP4 activity was lowered in fat body mitochondria 48 h after AKH treatment. In the AKH-injected larvae, changes in ZaUCP4 expression were accompanied by the mobilization of carbohydrate reserves, no change in the concentration of total lipids and an increase in the free fatty acid level. In contrast, AKH had no effect on carbohydrate metabolism in the pupal fat body but induced lipid mobilization. It seems that AKH influences ZaUCP4 expression by triggering multiple events and that it has different physiological roles in controlling intermediary metabolism in the fat body of the beetle larvae and pupae.

Neb -colloostatin (SIVPLGLPVPIGPIVVGPR), an insect oostatic factor found in the ovaries of the flesh fly Neobellieria bullata , strongly induces apoptosis in insect haemocytes. To explain the role of Ser 1 and Pro 4 residues of Neb -colloostatin in the pro-apoptotic activity of this peptide, the synthesis of a series of analogs was performed, such as: [Ac-Ser 1 ]- (1), [ d -Ser 1 ]- (2), [Thr 1 ]- (3), [Asp 1 ]- (4), [Glu 1 ]- (5), [Gln 1 ]- (6), [Ala 1 ]- (7), [Val 1 ]- (8), [ d -Pro 4 ]-(9), [Hyp 4 ]- (10), [Acp 4 ]- (11), [Ach 4 ]- (12), [Ala 4 ]- (13), [Ile 4 ]- (14), and [Val 4 ]-colloostatin (15). All peptides were bioassayed in vivo for the pro-apoptotic action on haemocytes of Tenebrio molitor . Additionally, the structural properties of Neb -colloostatin and its analogs were examined by the circular dichroism in water and methanol. Peptides 1, 4, 5, 7, 8, 10, 12, 14, and 15 strongly induce T. molitor haemocytes to undergo apoptosis and they show about 120–230% of the Neb -colloostatin activity at a dose of 1 nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

The subject of this paper is a search for new biological properties of alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) and a series of its analogues. The studies on structure/activity relationship in alloferon, the synthesis of a series of 28 analogues were performed. The analogues were modified at position 1 or 6, and other were oligopeptides with a shortened peptide sequence. Biological effects of the peptides were evaluated by the pro-apoptotic action in vivo on haemocytes of Tenebrio molitor and in the cardiotropic test in vitro on the heart of T. molitor and Zophobas atratus. In the in vivo bioassays, new biological activities of alloferon and its analogues were discovered. In haemocytotoxic bioassay, alloferon strongly induces T. molitor haemocytes to undergo apoptosis at a dose of 10 nM. Moreover, [Phe(p-NH2)(1)]-, [Tyr(6)]- and [1-10]-alloferon exhibit a two-fold increase of caspases activation in comparison with the alloferon. However, alloferon and its analogues show a weak cardiostimulatory activity in Z. atratus but the heart of T. molitor is not sensitive to these peptides. The results obtained here suggest that alloferon plays pleiotropic functions in insects.

Effects of Led-NPF-I peptide hormone (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and its analogues were tested on the contractile activity of the oviduct of two beetle species Tenebrio molitor and Zophobas atratus. The influence of Led-NPF-I on the developmental processes of T. molitor was also examined. The unmodified Led-NPF-I molecule exerts inhibitory effects on contractions of the oviduct in both beetles; however, the efficiency of inhibition was stronger in T. molitor females. Structure–activity studies showed that replacement of Arg residue in positions 2, 7 and 9 in the amino acid sequence by His, D-Arg or Lys led to changes in myoinhibitory properties of almost all of the analogues in both species. In the majority of the analogues, the effect was weaker when compared to the native molecule. In the developmental bioassays, injections of Led-NPF-I peptide delayed the moulting process of T. molitor larvae and accelerated pupal moult. It also increased the growth in pre-starved and non-starved la...

In insects, the majority of studies have been conducted on the hormonal regulation of female reproduction. Thus far, little is known about the regulation of male reproductive physiology, especially by peptide hormones. We report here, for the first time in insects, the effects of three peptides, Neb-colloostatin (SIVPLGLPVPIGPIVVGPR), Neb-TMOF (NPTNLH) and Lepde-NPF-I (ARGPQLRLRFa), on various aspects of reproduction in male Tenebrio molitor beetles. All three tested peptides increased the soluble protein concentration in the testes and the dry mass of the beetle's testes. They also significantly changed the protein profiles of the testes. Injection of these peptides also significantly changed the number of sperm cells in the testes. However, the observed effects were age specific. The most prominent changes were observed in 4-day-old males. Neb-colloostatin and Neb-TMOF decreased the number of sperm cells, whereas Lepde-NPF-I increased the number of spermatocytes. Moreover, in vitro experiments revealed that Neb-TMOF and Lepde-NPF-I increased the contractility of the ejaculatory duct of T. molitor males. The results obtained suggest that different reproductive processes in males might be regulated by complex mechanisms.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

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.

article i nfo Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.

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.

Three neuropeptides Zopat-MS-2 (pEDVDHVFLRFa), Zopat-SK-1 (pETSDDYGHLRFa) and Zopat-NVPL-4trunc. (GRWGGFA), recently isolated from the neuroendocrine system of the Zophobas atratus beetle, were tested for their myotropic and hyperglycaemic activities in this species. These peptides exerted differentiated dose-dependent and tissue specific physiological effects. Zopat-MS-2 inhibited contractions of the isolated heart, ejaculatory duct, oviduct and hindgut of adult beetles and induced bimodal effects in the heart contractile activity of pupae in vivo. It also increased the haemolymph free sugar level in larvae of this species, apart from myotropic activity. Zopat-SK-1 showed myostimulatory action on the isolated hindgut of the adult beetles, but it decreased contractions of the heart, ejaculatory duct and oviduct. Injections of this peptide at a dose of 2 μg also caused delayed cardioinhibitory effects on the heartbeat of the pupae. Together with the ability to increase free sugar level in the haemolymph of larvae these were new physiological activities of sulfakinins in insects. Zopat-NVPL-4trunc. inhibited the muscle contractions of the two organs: hindgut and ejaculatory duct but it was inactive on the oviduct and the heart of the adult beetles. This peptide also increased free sugar level concentration in the haemolymph of Z. atratus larvae. These physiological actions are the first biological activities discovered for this group of the insect peptides. The present work showed pleiotropic activity of three neuropeptides and indicates that the visceral muscle contractions and the haemolymph sugar homeostasis in Z. atratus are regulated by complex mechanisms.

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.

The physiological processes that occur during the aging of insects are poorly understood. The aim of this study was to describe the changes in contractile activity and hemodynamic parameters of the heart that take place as the coleopteran beetle, Tenebrio molitor, ages. The frequency of heart contractions in beetles that had just undergone metamorphosis (median 24.7 beats/min) was significantly lower than the frequency of heart contractions in older beetles. In 56% of beetles that were

Two myotropic peptides, proctolin and FMRFamide, were tested for their action on the frequency of contractions of the the male ejaculatory duct and female oviduct of two beetle species: Tenebrio molitor and Zophobas atratus. The tested peptides caused changes in endogenous contraction frequencies of the reproductive tracts in both species. Proctolin exerted a strong stimulatory effect on spontaneous muscular contractions of the ejaculatory ducts and oviducts of T. molitor and Z. atratus. FMRFamide action, however, differed between species and organ tested. At high doses (10−7–10−5 M) it stimulated oviduct contractions in Z. atratus but inhibited the T. molitor oviduct contractility. In T. molitor, the peptide had a stimulatory effect on oviduct contractions at low doses (10−11–10−9 M). In contrast to proctolin, FMRFamide is a weak inhibitor of the ejaculatory ducts of both beetles at high concentrations (10−6–10−5 M). Differentiated responses of the ejaculatory ducts and oviducts of T. molitor an...

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.

We have examined the effects of the Led-NPF-I peptide (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and a series of ten analogues on the heart contractile activity of Tenebrio molitor and Zophobas atratus, and the structure–activity relationships for cardioactive action of Led-NPF-I were established. A video microscopy technique and computer-based method of data acquisition and analysis were used to study the action of the peptides on continuously perfused heart preparations. Cardiac activity was progressively inhibited by Led-NPF-I when the peptide concentrations were increased from 10−9 to 10−5M. Substitution of the L-proline residue at position 4 of the native peptide with hydroxyproline, valine or D-proline caused a loss of cardioinhibitory activity. Also, replacement of arginine residues at all three positions 2, 7 and 9 with another basic amino acid histidine, reduces cardioinhibitory action of Led-NPF-I. Some modifications of the C-terminal residues, as the Phe(4-NO2)-, Phe(4-NH2)- and Phe(4-NMe2)-analogues, resulted in agonistic peptides with biological activity similar to that of the native peptide. However, three other C-terminal analogues tested [Tyr10]-, [D-Phe10]-Led-NPF-I, and Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-OH were inactive in the heart bioassay, which suggests that this end of the amino acid chain may play an important role in bioactivity and interaction of the native peptide with its receptor on the myocardium. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Insect sulfakinins (SKs) are multifunctional neuropeptides structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK). It has been proposed that SKs play a role in modulating energy management in insects by interacting with adipokinetic hormone (AKH), the principle hormone controlling insect intermediary metabolism. To exclude head factors (including AKH) that influence the activity of the nonsulfated sulfakinin Zopat-SK-1 in the larvae of the beetle Zophobas atratus, ligature and in vitro bioassays were used. Our study showed that in the neck-ligated larvae, Zopat-SK-1 evoked a much more pronounced glycogenolytic effect in fat body tissue and a significantly higher hypertrahelosemic effect in hemolymph than in larvae without ligation. We found that the concentration of the sugar trehalose increased under hormonal treatment but no changes in glucose levels were observed. Under in vitro conditions, the maximal glycogenolytic effect of Zopat-SK-1 in fat body was observed at 10 pmol of hormone. Ligature and in vitro bioassays indicated that Zopat-SK-1 activity in the Z. atratus larvae is modulated by head signals and/or factors from the gastrointestinal tract. Our data indicate the existence of a brain–gastrointestinal axis that has a role in controlling of energy (carbohydrate) metabolism in the insect body. Moreover, these results, together with immunological evidence of a cholecystokinin-like (sulfakinin) receptor in the Z. atratus fat body, help us to better understand the SK signaling pathways and its physiological role in insect biology.

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.

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.