Mitochondrial metabolism and oxidative stress in the tropical cockroach under fluctuating thermal regimes.

The cockroach Gromphadorhina coquereliana can survive at low temperatures under extensive periods of cold stress. To assess energy management and insect adaptation in response to cold, we measured mitochondrial activity and oxidative stress in muscle and fat body tissues from G. coquereliana under a fluctuating thermal regime (FTR; stressed at 4°C for 3 h on 3 consecutive days, with or without 24 h recovery). Compared with our earlier work showing that a single exposure to cold significantly affects mitochondrial parameters, here, repeated exposure to cold triggered an acclimatory response, resulting in unchanged mitochondrial bioenergetics. Immediately after cold exposure, we observed an increase in the overall pool of ATP and a decrease in typical antioxidant enzyme activity. We also observed decreased activity of uncoupling protein 4 in muscle mitochondria. After 24 h of recovery, we observed an increase in expression of antioxidant enzymes in muscles and the fat body and a significant increase in the expression of UCP4 and HSP70 in the latter. This indicates that processes related to energy conversion and disturbance under cold stress may trigger different protective mechanisms in these tissues, and that these mechanisms must be activated to restore insect homeostasis. The mitochondrial parameters and enzymatic assays suggest that mitochondria are not affected during FTR but oxidative stress markers are decreased, and a 24 h recovery period allows for the restoration of redox and energy homeostasis, especially in the fat body. This confirms the crucial role of the fat body in intermediary metabolism and energy management in insects and in the response to repeated thermal stress.

Characterization of Gromphadorhina coquereliana hemolymph under cold stress.

Low temperatures in nature occur together with desiccation conditions, causing changes in metabolic pathways and cellular dehydration, affecting hemolymph volume, water content and ion homeostasis. Although some research has been conducted on the effect of low temperature on Gromphadorhina coquereliana, showing that it can survive exposures to cold or even freezing, no one has studied the effect of cold on the hemolymph volume and the immune response of this cockroach. Here, we investigated the effect of low temperature (4 °C) on the abovementioned parameters, hemocyte morphology and total number. Cold stress affected hemocytes and the immune response, but not hemolymph volume. After stress, the number of circulating hemocytes decreased by 44.7%, but the ratio of apoptotic cells did not differ significantly between stressed and control individuals: 8.06% and 7.18%, respectively. The number of phagocyting hemocytes decreased by 16.66%, the hemocyte morphology drastically changed, and the F-actin cytoskeleton differed substantially in cold-stressed insects compared to control insects. Moreover, the surface area of the cells increased from 393.69 µm2 in the control to 458.38 µm2 in cold-treated animals. Together, our results show the links between cold stress and the cellular immune response, which probably results in the survival capability of this species.

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.

Thermal stress causes DNA damage and mortality in a tropical insect.

Cold tolerance is considered an important factor determining the geographic distribution of insects. We have previously shown that despite its tropical origin, the cockroach Gromphadorinha coquereliana is capable of surviving exposures to cold. However, the freezing tolerance of this species had not yet been examined. Low temperature is known to alter membrane integrity in insects, but whether chilling or freezing compromises DNA integrity remains a matter of speculation. In the present study, we subjected the G. coquereliana adults to freezing to determine their supercooling point (SCP) and evaluated whether the cockroaches were capable of surviving partial and complete freezing. Next, we conducted single cell gel electrophoresis (SCGE) assays to determine whether heat, cold and freezing altered hemocyte DNA integrity. The SCP of this species was high and around -4.76°C, which is within the typical range of freezing-tolerant species. Most cockroaches survived to 1 day after partial ice formation (20% mortality), but died progressively in the next few days after cold stress (70% mortality after 4 days). One day after complete freezing, most insects died (70% mortality), and after 4 days, 90% of them had succumbed. The SCGE assays showed substantial levels of DNA damage in hemocytes. When cockroaches were heat-stressed, the level of DNA damage was similar to that observed in the freezing treatment, though all heat-stressed insects survived. The present study shows that G. coquereliana can be considered as moderately freeze-tolerant, and that extreme low temperature stress can affect DNA integrity, suggesting that this cockroach may possess an efficient DNA repair system.

The Influence of Bee Venom Melittin on the Functioning of the Immune System and the Contractile Activity of the Insect Heart-A Preliminary Study.

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.

Beetles as Model Organisms in Physiological, Biomedical and Environmental Studies - A Review.

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.

Insect Peptides - Perspectives in Human Diseases Treatment.

BACKGROUND: Insects are the largest and the most widely distributed group of animals in the world. Their diversity is a source of incredible variety of different mechanisms of life processes regulation. 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 useful in human diseases treatment. Especially important in the regulation of insect physiology are peptides, like neuropeptides, peptide hormones or antimicrobial peptides. There are two main aspects where they can be helpful, 1) Peptides isolated from insects may become potential drugs in therapy of different diseases, 2) A lot of insect peptide hormones show structural or functional homology to mammalian peptide hormones and the comparative studies may give a new look on human disorders. In our review we focused on three group of insect derived peptides: 1) immune-active peptides, 2) peptide hormones and 3) peptides present in venoms. CONCLUSION: In our review we try to show the considerable potential of insect peptides in searching for new solutions for mammalian diseases treatment. We summarise the knowledge about properties of insect peptides against different virulent agents, anti-inflammatory or anti-nociceptive properties as well as compare insect and mammalian/vertebrate peptide endocrine system to indicate usefulness of knowledge about insect peptide hormones in drug design. The field of possible using of insect delivered peptide to therapy of various human diseases is still not sufficiently explored. Undoubtedly, more attention should be paid to insects due to searching new drugs.

The physiological role of fat body and muscle tissues in response to cold stress in the tropical cockroach Gromphadorhina coquereliana.

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.

Cardioregulatory Functions of Neuropeptides and Peptide Hormones in Insects.

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.

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect.

In the present study, we describe the existence of mitochondrial ATP-dependent K(+) channel (mitoKATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoKATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoKATP in insect mitochondria. MitoKATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.