Proteins from toad's parotoid macroglands: do they play a role in gland functioning and chemical defence?

BACKGROUND: Parotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands. RESULTS: Applying a proteomic approach we identified 162 proteins in the extract from toad's parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings. CONCLUSION: This finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

Effects of Environmental Conditions on the Individual Architectures and Photosynthetic Performances of Three Species in Drosera.

The aim of this study was to determine the environmental conditions, individual architectures, and photosynthetic efficiencies of three sundew species: Drosera rotundifolia, D. anglica, and D. intermedia, found in well-preserved peatlands and sandy lake shores in NW Poland. Morphological traits and chlorophyll a fluorescence (Fv/Fm) were measured in 581 individuals of Drosera. D. anglica occupies the best-lit and warmest habitats, and also those that are the most heavily hydrated and the richest in organic matter; its rosettes are larger under conditions of higher pH, less organic matter, and less well-lit habitats. D. intermedia occupies substrates with the highest pH but the lowest conductivity, the poorest level of organic matter, and the least hydration. It is highly variable in terms of individual architecture. D. rotundifolia occupies habitats that are the most diverse, and that are often poorly lit, with the lowest pH but the highest conductivity. It is the least variable in terms of individual architecture. The value of the Fv/Fm ratio in Drosera is low (0.616 ± 0.137). The highest photosynthetic efficiency is achieved by D. rotundifolia (0.677 ± 0.111). It is significant for all substrates, indicating its high phenotypic plasticity. The other species have lower and similar Fv/Fm values (D. intermedia, 0.571 ± 0.118; D. anglica, 0.543 ± 0.154). Due to its very low photosynthetic efficiency, D. anglica avoids competition by occupying highly hydrated habitats. D. intermedia has adapted to the occupation of highly variable habitats in terms of hydration, while D. rotundifolia is primarily adapted to variable light conditions.

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L.

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10-8 and 10-6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10-8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10-6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.

Neuropeptidomes of Tenebrio molitor L. and Zophobas atratus Fab. (Coleoptera, Polyphaga: Tenebrionidae).

Neuropeptides are signaling molecules that regulate almost all physiological processes in animals. Around 50 different genes for neuropeptides have been described in insects. In Coleoptera, which is the largest insect order based on numbers of described species, knowledge about neuropeptides and protein hormones is still limited to a few species. Here, we analyze the neuropeptidomes of two closely related tenebrionid beetles: Tenebrio molitor and Zophobas atratus─both of which are model species in physiological and pharmacological research. We combined transcriptomic and mass spectrometry analyses of the central nervous system to identify neuropeptides and neuropeptide-like and protein hormones. Several precursors were identified in T. molitor and Z. atratus, of which 50 and 40, respectively, were confirmed by mass spectrometry. This study provides the basis for further functional studies of neuropeptides as well as for the design of environmentally friendly and species-specific peptidomimetics to be used as biopesticides. Furthermore, since T. molitor has become accepted by the European Food Safety Authority as a novel food, a deeper knowledge of the neuropeptidome of this species will prove useful for optimizing production programs at an industrial scale.

Innovative System for Evaluation and Rehabilitation of Human Imbalance.

<b>Introduction:</b> Mobile posturography is based on wearable inertial sensors; it allows to test static stability (static posturography) and gait disturbances. </br> </br> <b>Aim:</b> The aim of this work was to present the results of research on the innovative MEDIPOST system used for diagnosis and rehabilitation of balance disorders. </br> </br> <b>Material and methods:</b> Fourteen articles published in influenced foreign journals were presented and discussed. The deve-lopment and construction of the device was preceded by a literature review and methodological work. The Dizziness Handi-cap Inventory (DHI) questionnaire was translated and validated. The methodology of posturography with head movements with a frequency of 0.3 Hz was also developed in the group with chronic vestibular disorders. Simultaneous measurements were performed (static posturogrphy vs. MEDIPOST) in the CTSIB-M (Modified Clinical Test of Sensory Interaction in Balance) test in healthy subjects and patients with unilateral peripheral dysfunction.</br> </br> <b>Results:</b> In the posturography with head movements the improvement of sensitivity (67 to 74%) and specificity (65 to 71%) was noted. In the CTSIB-M test the intraclass correlation coefficients for both methods were 0.9. The greatest differences between examinations were observed for the mean angular velocity in the tests on the foam (trials no. 3 and 4), in particular on the foam with eyes closed (trial no. 4 - sensitivity 86.4%, specificity 87.7%). Two functional tests were analyzed: the Swap Seats test and the 360 degree turn test. In the former, the results are studied from 6 sensors - 86% of the true positives and 73% of the true negatives for the fall/ no-fall group classification. The second test differentiates people with vestibular impairment and healthy people. It can be analyzed with 1 (sensitivity 80%) and 6 sensors (sensitivity 86%, specificity 84%). Currently, the MEDIPOST device is in the development and certification phase.

Solanaceae glycoalkaloids: α-solanine and α-chaconine modify the cardioinhibitory activity of verapamil.

CONTEXT: Solanaceae glycoalkaloids (SGAs) possess cardiomodulatory activity. OBJECTIVE: This study investigated the potential interaction between verapamil and glycoalkaloids. MATERIAL AND METHODS: The cardioactivity of verapamil and glycoalkaloids (α-solanine and α-chaconine) was tested in adult beetle (Tenebrio molitor) myocardium in vitro using microdensitometric methods. The myocardium was treated with pure substances and mixtures of verapamil and glycoalkaloids for 9 min with saline as a control. Two experimental variants were used: simultaneous application of verapamil and glycoalkaloids or preincubation of the myocardium with one of the compounds followed by perfusion with a verapamil solution. We used 9 × 10-6-5 × 10-5 M and 10-9-10-5 M concentration for verapamil and glycoalkaloids, respectively. RESULTS: Verapamil, α-solanine and α-chaconine showed cardioinhibitory activity with IC50 values equal to 1.69 × 10-5, 1.88 × 10-7 and 7.48 × 10-7 M, respectively. When the glycoalkaloids were applied simultaneously with verapamil, an antagonistic effect was observed with a decrease in the maximal inhibitory effect and prolongation of t50 and the recovery time characteristic of verapamil. We also confirmed the expression of two transcript forms of the gene that encodes the α1 subunit of L-type calcium channels in the myocardium and brain with equal transcription levels of both forms in the myocardium and significant domination of the shorter form in the brain of the insect species tested. DISCUSSION AND CONCLUSIONS: The results show that attention to the composition of the daily diet during therapy with various drugs is particularly important. In subsequent studies, the nature of interaction between verapamil and SGAs on the molecular level should be checked, and whether this interaction decreases the efficiency of cardiovascular therapy with verapamil in humans.

A new, widespread venomous mammal species: hemolytic activity of Sorex araneus venom is similar to that of Neomys fodiens venom.

BACKGROUND: Venom production has evolved independently many times in the animal kingdom, although it is rare among mammals. Venomous shrews produce toxins in their salivary glands and use their venoms to hunt and store prey. Thus far, the toxicity and composition of shrew venoms have been studied only in two shrew species: the northern short-tailed shrew, Blarina brevicauda, and the Eurasian water shrew, Neomys fodiens. Venom of N. fodiens has potent paralytic activity which enables hunting and storing prey in a comatose state. Here, we assayed the hemolytic effects of extracts from salivary glands of N. fodiens and the common shrew, Sorex araneus, in erythrocytes of Pelophylax sp. frogs. We identified toxins in shrew venom by high-performance liquid chromatography coupled to tandem mass spectrometry. RESULTS: Our results prove, confirming a suggestion made four centuries ago, that S. araneus is venomous. We also provide the first experimental evidence that shrew venoms produce potent hemolysis in frog erythrocytes. We found significant concentration-dependent effects of venoms of N. fodiens and S. araneus on hemolysis of red blood cells evaluated as hemoglobin release. Treatment of erythrocytes with N. fodiens venom at concentrations of 1.0 and 0.5 mg/ml and with S. araneus venom at concentration of 1.0 mg/ml caused an increased release of hemoglobin. Our findings confirm that hemolytic effects of N. fodiens venom are stronger than those produced by S. araneus venom. We identified four toxins in the venom of N. fodiens: proenkephalin, phospholipase A2 (PLA2), a disintegrin and metalloproteinase domain-containing protein (ADAM) and lysozyme C, as well as a non-toxic hyaluronidase. In the venom of S. araneus we found five toxins: proenkephalin, kallikrein 1-related peptidase, beta-defensin, ADAM and lysozyme C. PLA2 and ADAMs are likely to produce hemolysis in frog erythrocytes. CONCLUSIONS: Our results clearly show that shrew venoms possess hemolytic action that may allow them to hunt larger prey. Since a member of the numerous genus Sorex is venomous, it is likely that venom production among shrews and other eulipotyphlans may be more widespread than it has previously been assumed.

Detection of balance disorders using rotations around vertical axis and an artificial neural network.

Vestibular impairments affect patients' movements and can result in difficulties with daily life activities. The main aim of this study is to answer the question whether a simple and short test such as rotation about a vertical axis can be an objective method of assessing balance dysfunction in patients with unilateral vestibular impairments. A 360˚ rotation test was performed using six MediPost devices. The analysis was performed in three ways: (1) the analytical approach based only on data from one sensor; (2) the analytical approach based on data from six sensors; (3) the artificial neural network (ANN) approach based on data from six sensors. For approaches 1 and 2 best results were obtained using maximum angular velocities (MAV) of rotation and rotation duration (RD), while approach 3 used 11 different features. The following sensitivities and specificities were achieved: for approach 1: MAV-80% and 60%, RD-69% and 74%; for approach 2: 61% and 85% and RD-74% and 56%; for approach 3: 88% and 84%. The ANN-based six-sensor approach revealed the best sensitivity and specificity among parameters studied, however one-sensor approach might be a simple screening test used e.g. for rehabilitation purposes.

Utility of the Novel MediPost Mobile Posturography Device in the Assessment of Patients with a Unilateral Vestibular Disorder.

Balance disorders are a growing problem worldwide. Thus, there is an increasing need to provide an inexpensive and feasible alternative to standard posturographic platforms (SP) used for the assessment of balance and to provide a possible solution for telemonitoring of patients. A novel mobile posturography (MP) MediPost device was developed to address these issues. This prospective study used a Modified Clinical Test of Sensory Interaction on Balance to evaluate healthy individuals and patients with a unilateral vestibular disorder through SP and MP simultaneously. The control group included 65 healthy volunteers, while the study group included 38 patients diagnosed with a unilateral vestibular deficit. The angular velocity values obtained from both methods were compared by intraclass correlation coefficients (ICC) and Bland−Altman plot analysis. Diagnostic capabilities were measured in terms of sensitivity and specificity. The ICC between the two methods for conditions 2−4 was indicative of excellent reliability, with the ICC > 0.9 (p < 0.001), except for Condition 1 (standing stance, eyes open) ICC = 0.685, p < 0.001, which is indicative of moderate reliability. ROC curve analysis of angular velocity for condition 4 represents the most accurate differentiating factor with AUC values of 0.939 for SP and 0.953 for MP. This condition also reported the highest sensitivity, specificity, PPV, and NPV values with 86.4%, 87.7%, 80%, and 90.5% for SP, and 92.1%, 84.6%, 77.8%, and 94.8% for MP, respectively. The newly developed MediPost device has high sensitivity and specificity in distinguishing between healthy individuals and patients with a unilateral vestibular deficit.

Insects as a New Complex Model in Hormonal Basis of Obesity.

Nowadays, one of the biggest problems in healthcare is an obesity epidemic. Consumption of cheap and low-quality energy-rich diets, low physical activity, and sedentary work favor an increase in the number of obesity cases within many populations/nations. This is a burden on society, public health, and the economy with many deleterious consequences. Thus, studies concerning this disorder are extremely needed, including searching for new, effective, and fitting models. Obesity may be related, among other factors, to disrupting adipocytes activity, disturbance of metabolic homeostasis, dysregulation of hormonal balance, cardiovascular problems, or disorders in nutrition which may lead to death. Because of the high complexity of obesity, it is not easy to find an ideal model for its studies which will be suitable for genetic and physiological analysis including specification of different compounds' (hormones, neuropeptides) functions, as well as for signaling pathways analysis. In recent times, in search of new models for human diseases there has been more and more attention paid to insects, especially in neuro-endocrine regulation. It seems that this group of animals might also be a new model for human obesity. There are many arguments that insects are a good, multidirectional, and complex model for this disease. For example, insect models can have similar conservative signaling pathways (e.g., JAK-STAT signaling pathway), the presence of similar hormonal axis (e.g., brain-gut axis), or occurrence of structural and functional homologues between neuropeptides (e.g., neuropeptide F and human neuropeptide Y, insulin-like peptides, and human insulin) compared to humans. Here we give a hint to use insects as a model for obesity that can be used in multiple ways: as a source of genetic and peptidomic data about etiology and development correlated with obesity occurrence as well as a model for novel hormonal-based drug activity and their impact on mechanism of disease occurrence.

A comparison of head movements tests in force plate and accelerometer based posturography in patients with balance problems due to vestibular dysfunction.

This study compares HS posturography on inertial sensors (MediPost) with force platform posturography in patients with unilateral vestibular dysfunction. The study group included 38 patients (age 50.6; SD 11.6) with unilateral vestibular weakness (UV) and 65 healthy volunteers (48.7; SD 11.5). HS tests were performed simultaneously on the force plate and with MediPost sensor attached at L4. Four conditions applied: eyes open/closed, firm/foam. The tests were performed twice, with the head moving at the frequency of 0.3 Hz (HS 0.3) and 0.6 Hz (HS 0.6). Mean sway velocity was significantly lower for MediPost than force plate in 4th condition both in UV and healthy group. For HS 0.3 the differences between devices were marginal; the highest sensitivity (87%) and specificity (95%) were in 4th condition. For HS 0.6 MediPost revealed lower sensitivity than force plate although the surface parameter improved results. MediPost IMU device and force platform posturography revealed a similar ability to differentiate between patients with balance problems in course of vestibular pathology and healthy participants, despite the differences observed between measuring methods. In some tests surface parameter may be more appropriate than sway velocity in improving MediPost sensitivity.

Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

Insulin-Like Peptides and Cross-Talk With Other Factors in the Regulation of Insect Metabolism.

The insulin-like peptide (ILP) and insulin-like growth factor (IGF) signalling pathways play a crucial role in the regulation of metabolism, growth and development, fecundity, stress resistance, and lifespan. ILPs are encoded by multigene families that are expressed in nervous and non-nervous organs, including the midgut, salivary glands, and fat body, in a tissue- and stage-specific manner. Thus, more multidirectional and more complex control of insect metabolism can occur. ILPs are not the only factors that regulate metabolism. ILPs interact in many cross-talk interactions of different factors, for example, hormones (peptide and nonpeptide), neurotransmitters and growth factors. These interactions are observed at different levels, and three interactions appear to be the most prominent/significant: (1) coinfluence of ILPs and other factors on the same target cells, (2) influence of ILPs on synthesis/secretion of other factors regulating metabolism, and (3) regulation of activity of cells producing/secreting ILPs by various factors. For example, brain insulin-producing cells co-express sulfakinins (SKs), which are cholecystokinin-like peptides, another key regulator of metabolism, and express receptors for tachykinin-related peptides, the next peptide hormones involved in the control of metabolism. It was also shown that ILPs in Drosophila melanogaster can directly and indirectly regulate AKH. This review presents an overview of the regulatory role of insulin-like peptides in insect metabolism and how these factors interact with other players involved in its regulation.

Fully Automatic Fall Risk Assessment Based on a Fast Mobility Test.

This paper presents a fall risk assessment approach based on a fast mobility test, automatically evaluated using a low-cost, scalable system for the recording and analysis of body movement. This mobility test has never before been investigated as a sole source of data for fall risk assessment. It can be performed in a very limited space and needs only minimal additional equipment, yet provides large amounts of information, as the presented system can obtain much more data than traditional observation by capturing minute details regarding body movement. The readings are provided wirelessly by one to seven low-cost micro-electro-mechanical inertial measurement units attached to the subject's body segments. Combined with a body model, these allow segment rotations and translations to be computed and for body movements to be recreated in software. The subject can then be automatically classified by an artificial neural network based on selected values in the test, and those with an elevated risk of falls can be identified. Results obtained from a group of 40 subjects of various ages, both healthy volunteers and patients with vestibular system impairment, are presented to demonstrate the combined capabilities of the test and system. Labelling of subjects as fallers and non-fallers was performed using an objective and precise sensory organization test; it is an important novelty as this approach to subject labelling has never before been used in the design and evaluation of fall risk assessment systems. The findings show a true-positive ratio of 85% and true-negative ratio of 63% for classifying subjects as fallers or non-fallers using the introduced fast mobility test, which are noticeably better than those obtained for the long-established Timed Up and Go test.

Short neuropeptide F signaling regulates functioning of male reproductive system in Tenebrio molitor beetle.

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.

Individual variation in cardiotoxicity of parotoid secretion of the common toad, Bufo bufo, depends on body size - first results.

Anurans secrete a wide diversity of toxins from skin glands to defend themselves against predators and pathogens. Bufonids produce potent poison in parotoid macroglands located in the postorbital region. Parotoid secretion is a rich source of bioactive compounds with cardiotoxic, cytotoxic and hemolytic activity. Poison content and toxicity may vary between species, populations, and among conspecifics inhabiting the same area. In the present paper, we pre-analyzed the individual variation in cardiotoxicity of parotoid extract of common toads (Bufo bufo Linnaeus, 1758) and impact of body mass (BM), snout to vent length (SVL), and body condition (BC) of toad on the poison toxicity. We hypothesized that large toads produce poison with higher cardiotoxicity than smaller ones. Parotoid extract was fractionated by reverse phase chromatography, and then in vitro physiological bioassays were carried out on the semi-isolated hearts of the mealworm beetle (Tenebrio molitor Linnaeus, 1758) to determine cardiotoxicity of the whole poison and separated fractions. Generalized linear mixed models were used to determine effects of BM, SVL, and BC on the poison toxicity. We recorded significant changes in the insect heart contractility after treatment with the whole poison and separated fractions. We found an individual variation in cardiotoxicity of the parotoid extract which was explained by the body size of toad. Poison of smaller toads displayed a negative, whereas poison of larger toads positive, chronotropic effect on the heart contractility. Thus, we conclude that the effectiveness of parotoid secretion in repelling predators may vary depending on the toad individual size.

FMRFamide-Related Peptides Signaling Is Involved in the Regulation of Muscle Contractions in Two Tenebrionid Beetles.

Peptidergic signaling regulates various physiological processes in insects. Neuropeptides are important messenger molecules that act as neurotransmitters, neuromodulators or hormones. Neuropeptides with myotropic properties in insects are known as FMRFamide-like peptides (FaLPs). Here, we describe the myotropic effects of the endogenous FaLPs in the regulation of contractile activity of the heart, ejaculatory duct, oviduct and the hindgut in two beetle species, Tenebrio molitor and Zophobas atratus. A putative receptor was identified in silico in both species. Using RT-PCR these putative FaLPs receptors were found in the various tissues of both beetles, including visceral organs. Analysis of the amino acid sequence of the receptor indicated that it is similar to other insect FaLPs receptors and belongs to G-protein coupled receptors. A synthetic FaLP (NSNFLRFa) found as the bioanalogue of both species demonstrated concentration-dependent and organ-specific myoactive properties. The peptide had species-specific cardioactivity, in that it stimulated Z. atratus heart contractions, while slightly inhibiting that of T. molitor and had mainly myostimulatory effect on the examined visceral organs of both beetle species, with the lowest activity in the ejaculatory duct of these beetles. The peptide was the most active in the hindgut of both species, but only at high concentration of 10-5 M. The results suggest that FaLPs are potent modulators of endogenous contractile activity of the visceral muscles in beetles and may indirectly affect various physiological processes.

Role of the Insect Neuroendocrine System in the Response to Cold Stress.

Insects are the largest group of animals. They are capable of surviving in virtually all environments from arid deserts to the freezing permafrost of polar regions. This success is due to their great capacity to tolerate a range of environmental stresses, such as low temperature. Cold/freezing stress affects many physiological processes in insects, causing changes in main metabolic pathways, cellular dehydration, loss of neuromuscular function, and imbalance in water and ion homeostasis. The neuroendocrine system and its related signaling mediators, such as neuropeptides and biogenic amines, play central roles in the regulation of the various physiological and behavioral processes of insects and hence can also potentially impact thermal tolerance. In response to cold stress, various chemical signals are released either via direct intercellular contact or systemically. These are signals which regulate osmoregulation - capability peptides (CAPA), inotocin (ITC)-like peptides, ion transport peptide (ITP), diuretic hormones and calcitonin (CAL), substances related to the general response to various stress factors - tachykinin-related peptides (TRPs) or peptides responsible for the mobilization of body reserves. All these processes are potentially important in cold tolerance mechanisms. This review summarizes the current knowledge on the involvement of the neuroendocrine system in the cold stress response and the possible contributions of various signaling molecules in this process.

Identification, Localization in the Central Nervous System and Novel Myostimulatory Effect of Allatostatins in Tenebrio molitor Beetle.

Allatostatins (ASTs) are pleiotropic insect neuropeptides that are potent myoinhibitors of muscle contractions. In this study, we identified and immunolocalized peptides from the MIP/AST and PISCF/AST families in the nervous system of a model beetle, Tenebrio molitor. Neurons containing MIPs were immunolocalized in the brains of adults and the ventral nerve cords of larvae, pupae and imagines of this species as well as in the retrocerebral complex. PISCFs were immunolocalized in the ventral nerve cord of all stages as well as the brain of the adult beetle. Faint signals were also observed in the corpus allatum but not in the corpus cardiacum. The results allowed us to deduce the sequences of three neuropeptides belonging to MIP/ASTs, Tenmo-MIP4-NWGQFGXWa, Tenmo-MIP5-SKWDNFRGSWa and Tenmo-MIP6-EPAWSNLKGIWa, and one peptide from the PISCF/AST family, QSRYXQCYFNPISCX. Furthermore, we showed for the first time myostimulatory action of endogenous MIP/ASTs. Tenmo-MIP5 caused dose-dependent stimulation of the contractile activity of the beetle oviduct muscles, showing a sigmoidal curve up to 81.20% at the 10-8 M concentration, and the EC50 value for the myostimulatory effect of this peptide was 8.50 × 10-12 M. This is the first report of myostimulatory action of an endogenous myoinhibitory peptide in insect muscles.