Antheraea peptide and its analog: Their influence on the maturation of the reproductive system, embryogenesis, and early larval development in Tenebrio molitor L. beetle.

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.

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.

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.

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.

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.

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.

Cholinergic Agonists and Antagonists Have an Effect on the Metabolism of the Beetle Tenebrio Molitor.

Synthetic insecticides are still widely used in plant protection. The main target for their action is the nervous system, in which the cholinergic system plays a vital role. Currently available insecticides have low selectivity and act on the cholinergic systems of invertebrates and vertebrates. Acetylcholine, a cholinergic system neurotransmitter, acts on cells by two types of receptors: nicotinic and muscarinic. In mammals, the role of muscarinic acetylcholine receptors (mAChRs) is quite well-known but in insects, is still not enough. Based on data indicating that the muscarinic cholinergic system strongly affects mammalian metabolism, we investigated if it similarly occurs in insects. We investigated the influence of agonists (acetylcholine, carbachol, and pilocarpine) and antagonists (tropane alkaloids: atropine and scopolamine) of mAChRs on the level of selected metabolites in Tenebrio molitor beetle trophic tissues. We analyzed the glycogen content in the fat body and midgut, the total free sugar concentration in the hemolymph and the lipid amount in the fat body. Moreover, we analyzed the levels of insulin-like peptides in the hemolymph. The tested compounds significantly influenced the mentioned parameters. They increased the glycogen content in the fat body and midgut but decreased the concentration of free sugars in the hemolymph. The observed effects were tissue-specific, and were also time- and dose-dependent. We used nonligated and neck-ligated larvae (to eliminate the influence of head factors on tissue metabolism) to determine whether the observed changes are the result of direct or indirect impacts on tissues. The obtained data suggest that the cholinergic system affects the fat body and midgut indirectly and directly and a pleiotropic role for mAChRs exists in the regulation of energy metabolism in insects. Moreover, tested compounds significantly affected the level of insulin-like peptides in hemolymph. Our studies for the first time showed that mAChRs are involved in regulation of insect metabolism of trophic tissues, and act on them directly and indirectly. Improved knowledge about insect cholinergic system may help in searching more selective and environment-friendly solutions in pest management.

Heart mechanical and hemodynamic parameters of a beetle, Tenebrio molitor, at selected ages.

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 < 1 week of age, a pattern of contractile activity with alternating periods of higher and lower contraction frequency was detected, suggesting that some posteclosion developmental processes occur during the first week of adulthood. All beetles that were 1 week of age showed a regular rhythm of heart contractions (median 72 beats/min). In older beetles, abnormalities such as heart arrhythmias or heart arrest were observed. The incidence of arrhythmia as well as the arrhythmicity index was highest in beetles that were 8-18 weeks old. The calculated stroke volume (SV) was also found to increase from eclosion to 12 weeks of age, and then decreased as adults aged further. Interestingly, cardiac output increased gradually, but the ejection fraction did not change significantly with age.

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.

A microsatellite polymorphism in IGF1 gene promoter and timing of natural menopause in Caucasian women.

BACKGROUND: Genes involved in the IGF-1 aging pathways in the human ovary can be considered strong candidates for predictors of the natural menopause timing. This study evaluates the association between a cytosine-adenine (CA) microsatellite polymorphism in the IGF1 gene promoter P1 and age at natural menopause. METHODS: Genomic DNA was extracted from the peripheral blood, PCR was performed using primers designed to amplify the polymorphic (CA) n repeat of the human IGF1 gene, an allele dose effect for the most common (CA)19 repeats allele, Cox proportional hazard regression models and the Kaplan-Meier cumulative survivorship method with the log-rank test were used to determine statistical significance of studied associations in a sample of 257 Polish women aged 40-58 years. RESULTS: Crude Cox proportional hazard regression analysis confirmed the association between the IGF1 gene polymorphism and the menopause timing (p=0.038). This relationship remained statistically significant after controlling for other menopause confounders in multivariate modelling. Out of the input variables, the (CA)n polymorphism in the IGF1 gene promoter, age at menarche and smoking status were independent covariates of the natural menopause timing (χ2=12.845; df=3; p=0.034). The onset of menopause at a younger age was likely associated with the IGF1 genotype variant not carrying the (CA)19 repeats allele, menarche before the age of 12 and a current cigarette smoker status (HR=1.6). CONCLUSION: This study provides evidence that a common cytosine-adenine (CA) microsatellite repeat polymorphism in the P1 promoter region of the IGF1 gene is an independent predictive factor for age at natural menopause in Caucasian women also after adjusting for other menopause covariates.

Identification and localisation of selected myotropic neuropeptides in the ventral nerve cord of tenebrionid beetles.

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.

New myotropic and metabotropic actions of pyrokinins in tenebrionid beetles.

Pyrokinins are a large family of insect neuropeptides exhibiting pleiotropic activity, but are predominantly myostimulatory hormones. In this study, four pyrokinins Tenmo-PK-1 (HVVNFTPRLa), Tenmo-PK-2 (SPPFAPRLa), Tenmo-PK-3 (HLSPFSPRLa) and Zopat-PK-1 (LPHYPRLa) from the neuro-endocrine system of two tenebrionid beetles, Tenebrio molitor and Zophobas atratus, were tested in homologous bioassays to evaluate their putative myotropic and glycaemic actions. The four investigated bioassays systems (the heart, oviduct, ejaculatory duct and hindgut) revealed species-specific and organ-specific myotropic actions for the pyrokinins tested. In most bioassays with both beetles, the peptides showed myostimulatory properties with different efficacy. However, the T. molitor heart is not sensitive to Tenmo-PK-1, Tenmo-PK-2 and Tenmo-PK-3, and one of the peptides Tenmo-PK-1, is myoinhibitory on the oviduct. Tenmo-PK-2, which is also present in Z. atratus, exerted an inhibitory effect on the contractions of the heart and ejaculatory duct muscles in this beetle. Such myoinhibitory properties of pyrokinins in insects are shown here for the first time. Only one of the peptides tested, Tenmo-PK-2, stimulated a hyperglycaemic response in the haemolymph of larvae of T. molitor and Z. atratus, and this effect suggests a possible additional metabotropic function of this peptide in beetles. The differences in the myotropic and glycaemic responses to pyrokinins suggest that these peptides modulate contractions of muscles from visceral organs and free sugar levels in the haemolymph of the beetles, through complex and species-specific mechanisms.

Analysis of the coding sequence and expression of the coiled-coil α-helical rod protein 1 gene in normal and neoplastic epithelial cervical cells.

The role of the CCHCR1 (coiled-coil α-helical rod protein 1) protein in the cell is poorly understood. It is thought to be engaged in processes such as proliferation and differentiation of epithelial cells, tissue-specific gene transcription and steroidogenesis. It is supposed to participate in keratinocyte transformation. It has also been found that this protein interacts with the E2 protein of human papilloma virus type 16 (HPV16). The oncogenic HPV forms, such as HPV16, are known to be necessary but not sufficient agents in the development of cervical carcinoma. In the present study, the CCHCR1 gene coding sequence and its expression was analyzed in normal, precancerous and cervical cancer cells. Changes in the non-coding region were found in 20.3% of the examined probes from women with cervical cancer or precancerous lesions and in 16.67% of the control probes. Most of the detected changes were single nucleotide polymorphisms (SNPs). Changes in the coding region were found in 22.8% of the probes with cervical cancer and in 16.67% of the control probes and all of them were SNPs. The level of CCHCR1 transcripts was determined using the real-time PCR method and the highest gene expression was detected in the H-SIL group and slightly decreased in the cervical carcinoma cells, compared with the control probes. It suggests that CCHCR1 could have a role in the process of cervical epithelial cell transformation, but this suggestion must be confirmed experimentally.

Proliferation of cells and expression of RARs, RXRs and HPV viral E6 and E7 proteins in cervical cancer cell lines after treatment with ATRA.

All-trans retinoic acid (ATRA) is considered to be a potential chemotherapeutic drug due to its capability to regulate cell growth and differentiation. The effects of ATRA on the proliferation of cells and gene regulation are mediated by retinoid receptors (RAR and RXR), which belong to the nuclear receptor superfamily of ligand- inducible transcription factors. ATRA can act either as a growth inhibitor or growth promoter, according to the functional state of retinoic receptors. Thus, we have established the effect of ATRA on the proliferation of cervical cancer cells line HeLa and CaSki and expression of retinoids receptors as well as the viral HPV oncogenic proteins E6 and E7. ATRA had no effect on proliferation CaSki cells, but it stimulated the growth of HeLa cells, which depended on the incubation time and the concentration of ATRA in cell culture. The overexpression of RAR alpha in HeLa cells after the administration of 10(-7) mM ATRA was also observed 72 hours, and the decrease of CaSki by 60-90%. In the study of cervical cancer cell lines, the very low levels of other endogenous RAR and RXR receptors were observed. ATRA does not repress the expression of two viral oncoproteins E6 and E7 HPV16/18, which play a key role in carcinogenesis of the cervix. Our results support the suggestions that the cell response to vitamin A, and other retinoids in the diet, may depend on cell type, and that the cancer cells are differentially resistant to retinoids. Thus, despite the important biological functions of retinoids, the effects of retinoids in a supplementation in supra-physiological doses as well as their physiological action are difficult to define.

Multiplex PCR for identification of herpes virus infections in adolescents.

The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.

[Molecular and physiological characterization of the pyrokinin insect neuropeptide family]. / Charakterystyka molekularna i fizjologiczna neuropeptydów owadzich z rodziny pirokinin.

Peptides from the pyrokinin (PK) family are a large, structurally and functionally diverse group of the insect neuropeptides produced by neurosecretory cells of the insect nervous system. This family contains short and long peptides which share C-terminal -FXPRLa amino acid sequence. Pyrokinins regulate the visceral muscle contractions, pheromone biosynthesis, pupariation and diapause duration in insects. They are encoded by two genes PBAN and capa, which are mainly expressed in the suboesophageal ganglion. Peptides are then transported to the retrocerebral complex and released into haemolymph. Recent studies are focused on application of pyrokinins as biopesticides in the regulation of insect pests growth and development.

HPV genotypes in the oral cavity/oropharynx of children and adolescents: cross-sectional survey in Poland.

UNLABELLED: Human papillomaviruses (HPVs) are a very complex group of pathogenic viruses, with more than 80 types, causing human infection. Given the prevalence of HPV infection and its relationship with the development of cervical and many other cancers, HPV vaccine development has been a major public health initiative worldwide in the last decade. The aim of the presented study was to identify HPV DNA by MY-PCR in 4,150 school children and adolescents, aged 10-18 years in the Wielkopolska region, Poland. All individuals were asked to fill in extensive questionnaires; further normal, oral squamous cells were collected from each pupil. Cellular DNA was isolated and used as a MY-PCR template to estimate the incidence of HPV-active infection. Forty five subjects (1.08% of the sample) were carriers of oropharyngeal HPVs. HPV status and variables of interest, such as age, gender, socioeconomical status, and risk factors (smoking and sexual intercourse history, alcohol consumption) were not correlated. The presence of HPVs in the oral cavity was cumulated in several schools of the region. DNA sequencing of MY-PCR products revealed only four HPV genotypes. The most frequent genotype was HPV11 (38/45 HPV-positive cases), while other more rare genotypes were HPV6 (3/45), HPV12 (3/45), and HPV57 (1/45). CONCLUSION: Our findings presented herein, reveal a relatively low prevalance of oropharyngeal HPVs in Polish adolescents and fill an important gap in the knowledge of oral HPV infections of children above 10 years and adolescents.

Serum and tissue levels of insulin-like growth factor-I in women with dysplasia and HPV-positive cervical cancer.

The ectocervical epithelium is the target of the oncogenic human papillomavirus (HPV), which acts as an etiological agent in the development of cervical carcinoma. However, the HPV-mediated transformation of human epithelial cells is a multi-step process dependant on unknown factors additional to the virus, which is a necessary but, in and of itself, insufficient catalyst. In the present study, we characterized the role of insulin-like growth factor-I (IGF-I) in this process. IGF-I is an endocrine hormone with an autocrine and paracrine role in many tissues. Our data demonstrated that autocrine secretion of IGF-I can contribute to HPV-induced carcinogenesis of the epithelium. An immunohistochemical study showed that IGF-I was present in the nuclei of the reproductive layer of the paraepidermal epithelium in 89% of cases of intraepithelial neoplasia (CIN I-III) and 60% of cases of invasive cervical cancer. The presence of IGF-I at a nuclear localization in the cells studied suggests that it may also have intranuclear actions.