Solanum Nigrum Fruit Extract Increases Toxicity of Fenitrothion-A Synthetic Insecticide, in the Mealworm Beetle Tenebrio Molitor Larvae.

Synthetic insecticides are widely used for crop protection both in the fields and in the food stored facilities. Due to their toxicity, and assumptions of Integrated Pest Management, we conducted two independent experiments, where we studied the influence of Solanum nigrum unripe fruit extract on the toxicity of an organophosphorus insecticide fenitrothion. In the first variant of the experiment, Tenebrio molitor larvae were fed with blended fenitrothion (LC50) and the extract in four concentrations (0.01, 0.1, 1 and 10%) in ratio 1:1 for 3 days. In the second variant, a two-day application of fenitrothion (LC40) was preceded by a one-day extract treatment. The first variant did not show any increase in lethality compared to fenitrothion; however, ultrastructure observations exhibited swollen endoplasmic reticulum (ER) membranes in the midgut and nuclear and cellular membranes in the fat body, after application of blended fenitrothion and extract. An increased amount of heterochromatin in the fat body was observed, too. In the second variant, pre-treatment of the extract increased the lethality of larvae, decreased the level of glycogen and lipids in the fat body and disrupted integrity of midgut cellular membranes. S. nigrum extract, applied prior to fenitrothion treatment can be a factor increasing fenitrothion toxicity in T. molitor larvae. Thus, this strategy may lead to decreased emission of synthetic insecticides to the environment.

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.

Sublethal Effects of Solanum nigrum Fruit Extract and Its Pure Glycoalkaloids on the Physiology of Tenebrio molitor (Mealworm).

BACKGROUND: Solanaceae plants produce glycoalkaloids (GAs) that affect various physiological processes of herbivorous insects and they are being tested as potential alternatives for synthetic pesticides. They cause lethal and sublethal effects. Nevertheless, their mode of action remains unclear. Therefore, we examined the effects of Solanum nigrum fruit extracts and pure glycoalkaloids on a model beetle, Tenebrio molitor. METHODS: Plant extracts or pure alkaloids were added to the food of the larvae for three days. The lipid, glycogen, and protein content in the fat body and the midgut were determined, and the contractility of the heart, hindgut, and oviduct muscles was tested using the video-microscopy technique. Finally, the ultrastructure of the fat body and the midgut was observed using electron microscopy. RESULTS: No lethal effects were noted. Sublethal changes were observed in the content of biomolecules, malformations of organelles, chromatin condensation, and heart and oviduct contractility. The observed effects differed between the tested glycoalkaloids and the extract. CONCLUSIONS: Both the extract and pure GAs have a wide range of effects that may result in impaired development, food intake, and reproduction. Some early effects may be used as bioindicators of stress. The effects of the extract and pure alkaloids suggest that the substances produced by the plant may act additively or synergistically.

Insecticidal properties of Solanum nigrum and Armoracia rusticana extracts on reproduction and development of Drosophila melanogaster.

Plant-derived substances, because of high biological activity, arouse interest of many scientists. Thus, plant extracts and pure substances are intensively studied on various insects as potential insecticides. In such studies, D. melanogaster is one of the most important model organisms. In our studies, we analysed the contents of two plant extracts and tested the activity of their main components against fruit flies and compared observed effects to effects caused by crude extracts. Then, we assessed the development of the next, unexposed generation. The chemical analysis of extracts revealed the presence of numerous glycoalkaloids and glucosinolates in Solanum nigrum and Armoracia rusticana extracts. These extracts, as well as their main components, revealed lethal and sublethal effects, such as the altered developmental time of various life stages and malformations of imagoes. Interestingly, the results for the extracts and pure main compounds often varied. Some of the results were also observed in the unexposed generation. These results confirm that the tested plants produce a range of substances with potential insecticidal effects. The different effects of extracts and pure main components suggest the presence of minor compounds, which should be tested as insecticides.

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.

Plant-Derived Substances Used Against Beetles-Pests of Stored Crops and Food-and Their Mode of Action: A Review.

Plants are sources of numerous active substances that are used to protect crops. Currently, due to the limitations of using synthetic insecticides, plant products have attracted increasing attention as possible pesticides. In this review, we discuss some of the most interesting plant products (for example, Solanaceae, or Asteraceae extracts, Artemisia absinthium or Citrus spp. essential oils, and single compounds like α-chaconine, or α-solanine) that exhibit insecticidal activity against beetles that are pests of stored food products. Next, we describe and discuss the mode of action of these products, including lethal and sublethal effects, such as antifeedant or neurotoxic activity, ultrastructural malformation, and effects on prooxidant/antioxidant balance. Furthermore, the methods of application of plant-derived substances in food storage areas are presented.

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.

Peptide hormones regulate the physiological functions of reproductive organs in Tenebrio molitor males.

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.

Ultrastructural and developmental toxicity of potato and tomato leaf extracts to beet armyworm, Spodoptera exigua (lepidoptera: noctuidae).

Beet Armyworm, Spodoptera exigua is a herbivorous moth and a serious pest of many economically important plants, which are used as food sources. Because of rigorous standards of food quality, usage of synthetic insecticides in crop protection, against pests, is limited. Solanaceae plant extracts may be a relatively cheap source of efficient natural insecticides that can limit usage of synthetic substances. Their biological activity is not fully known. In particular, ultrastructural studies, using transmission electron microscopy, are not usual. In the present article we describe the effects of sublethal concentrations of tomato and potato leaf extracts against S. exigua. Acute lethal effects were not observed. Both extracts exerted similar effects within midgut and fat body cells. Midgut cells were not significantly altered while fat body cells showed prominent swelling of nuclear envelope and endoplasmic reticulum, vacuolization of mitochondria and fusion of fat droplets. These changes were much more intensive within groups exposed to potato than tomato extracts at highest concentration at least. Light microscopy was used to observe and document developmental alterations of S. exigua exposed to potato and tomato leaf extracts. Potato leaf extracts significantly decreased hatching success and caused morphological malformations of imagoes. Among them, malformations of wings were the most prominent. Interestingly, these effects were not observed within populations exposed to tomato extracts at highest concentration at least.

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.

[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.