Effect of Short-Term Desiccation, Recovery Time, and CAPA–PVK Neuropeptide on the Immune System of the Burying Beetle Nicrophorus vespilloides

Environmental conditions, especially related to winter, are crucial for shaping activity of insect immune system. However, our previous research clearly indicates differences in the immune system functioning when the cold stress was induced in the laboratory conditions and when the beetles were collected from natural environment during winter. This is probably related to the multiplication of observed effects by simultaneous presence of different stress factors characteristic of winter, including desiccation. For these reasons, our next step was analysis of the effects of short-term desiccation and recovery time on the functioning of immune system of burying beetle Nicrophorus vespilloides. Also, the effect of Tenmo–PVK-2 (tenebrionid periviscerokinin), member of the CAPA–PVK neuropeptide family, was investigated to better understand observed changes. Short-term desiccation decreases the phagocytic activity of burying beetle haemocytes, which is correlated with a reduction in their adhesive ability. On the other hand, there was a significant increase in phenoloxidase (PO) activity and the level of proPO expression, which may suggest sealing the cuticula by melanin deposition and prevention of water loss. Additionally, the elevated level of defensin expression may be associated with the cross-talk between mechanisms, which participate in insect response to environmental stress, including pathogen infection. After 1 h of recovery time, the activity of tested cellular and humoral mechanisms was mostly back to the control level. However, inhibition of the activity of PO and down-regulation of proPO were noted. These results also indicate importance of melanin deposition during water loss. Moreover, it suggests that some changes in immune system functioning during stress conditions do not have an immune function. Interestingly, part of the effects characteristic of recovery time were also observed after the application of Tenmo–PVK-2, mainly related to haemocyte morphology. These results indicate that CAPA–PVK neuropeptides may also influence on activity of burying beetle immune system. It should be also highlighted that, because of the study of the effects of CAPA–PVK neuropeptides, homologs of vertebrate neuromedin U, the results may be interesting for search evolutionary similarities in the functioning of the neuroendocrine system of insects and vertebrates

Cholinergic Agonists and Antagonists Have an Effect on the Metabolism of the Beetle <i>Tenebrio Molitor</i>

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

Evaluation of the physiological activity of venom from the Eurasian water shrew Neomys fodiens

Abstract Background Animal toxins can have medical and therapeutic applications. Principally, toxins produced by insects, arachnids, snakes and frogs have been characterized. Venomous mammals are rare, and their venoms have not been comprehensively investigated. Among shrews, only the venom of Blarina brevicauda has been analysed so far, and blarina toxin has been proven to be its main toxic component. It is assumed that Neomys fodiens employs its venom to hunt larger prey. However, the toxic profile, properties and mode of action of its venom are largely unknown. Therefore, we analysed the cardio-, myo- and neurotropic properties of N. fodiens venom and saliva of non-venomous Sorex araneus (control tests) in vitro in physiological bioassays carried out on two model organisms: beetles and frogs. For the first time, we fractionated N. fodiens venom and S. araneus saliva by performing chromatographic separation. Next, the properties of selected compounds were analysed in cardiotropic bioassays in the Tenebrio molitor heart. Results The venom of N. fodiens caused a high decrease in the conduction velocity of the frog sciatic nerve, as well as a significant decrease in the force of frog calf muscle contraction. We also recorded a significant decrease in the frog heart contractile activity. Most of the selected compounds from N. fodiens venom displayed a positive chronotropic effect on the beetle heart. However, one fraction caused a strong decrease in the T. molitor heart contractile activity coupled with a reversible cardiac arrest. We did not observe any responses of the insect heart and frog organs to the saliva of S. araneus. Preliminary mass spectrometry analysis revealed that calmodulin-like protein, thymosin β-10, hyaluronidase, lysozyme C and phospholipase A2 are present in the venom of N. fodiens, whereas thymosin β4, lysozyme C and β-defensin are present in S. araneus saliva. Conclusion Our results showed that N. fodiens venom has stronger paralytic properties and lower cardioinhibitory activity. Therefore, it is highly probable that N. fodiens might use its venom as a prey immobilizing agent. We also confirmed that S. araneus is not a venomous mammal because its saliva did not exhibit any toxic effects

Alkaloids - natural cardioactive substances

Choroby kardiologiczne należą do najbardziej rozpowszechnionych schorzeń. Poznanie i opisanie jak największej liczby substancji modyfikujących oraz normalizujących aktywność serca, stanowi zatem podstawę rozwoju wiedzy dotyczącej profilaktyki i leczenia chorób serca. Do substancji takich należą alkaloidy. Wykazują one szerokie spektrum aktywności fizjologicznej. Prowadzone do tej pory badania pozwoliły scharakteryzować ponad 10 000 różnych rodzajów surowców zawierających alkaloidy, z których ponad sto znalazło zastosowanie w farmacji jako substancje przeciwbólowe, pobudzające, rozluźniające, analeptyczne, czy wreszcie wpływające na pracę serca. W artykule przedstawiono najważniejsze z medycznego punktu widzenia alkaloidy oraz ich działanie na organizm człowieka. Przedstawiono także wynik badań nad działaniem wybranych alkaloidów i ich mieszanin na miokardium owada, jako modelu w badaniach nad farmakologicznym działaniem testowanych substancji.Cardiovascular diseases belong to the most common ones in humans.Therefore, broad knowledge of substances able to modify and standardize activity of the heart, both of the natural and synthetic origin, provides the basis for the development of prophylaxis and treatment of cardiac ailments. In this regard, alkaloids seem to be of a great importance, as they exhibit a wide range of physiological activity. Investigations carried out until now, have led to identification of over 10 000 different types of materials containing alkaloids, of which more than one hundred were used in pharmacy as analgetics, stimulants, relaxants, analeptics and influencing heart activity. The paper describes the most important alkaloids from the medical point of view and their effects on the human body. Also results of experimental studies on the effects exerted by selected alkaloids and mixtures thereof on the insect's myocardium are presented, as a model in studies on the pharmacological effects of the tested substances

Biological evaluation of analogues of an insect neuropeptide proctolin.

Continuing our studies on proctolin (Arg-Tyr-Leu-Pro-Thr) we performed the synthesis and biological evaluation of 52 analogues substituted in position 2, 3, 4, and 5 of the peptide chain. The peptides were bioassayed for cardiotropic activity in vitro on Tenebrio molitor and myotropic activity on foregut of Schistocerca gregaria. Twenty analogues retained 20-80% of proctolin activity

Owady - alternatywne organizmy modelowe do badań chorób człowieka

W ostatniej dekadzie nastąpił gwałtowny wzrost zainteresowania wykorzystaniem bezkręgowców, w tym owadów, jako organizmów modelowych w badaniach chorób człowieka. Opublikowano liczne prace, w których wykazano możliwość wykorzystania owadzich modeli w badaniach chorób neurodegeneracyjnych, cukrzycy, otyłości, czy chorób serca. Szybki rozwój technik biologii molekularnej, biotestów fizjologicznych i farmakologicznych, a także poznanie genomów muszki Drosophila melanogaster, chrząszcza Tribolium castaneum, jedwabnika Bombyx mori i pszczoły Apis mellifera dodatkowo stanowią silne wsparcie metodologiczne tego kierunku badań. Poznano szereg genów owadów, będących ortologami genów ludzkich, odpowiedzialnych za rozwój różnych chorób, zidentyfikowano wiele białek kodowanych przez te geny, scharakteryzowano ich fenotypy morfologiczne i fizjologiczne, a także opisano działanie u owadów niektórych leków stosowanych w chorobach neurodegeneracyjnych i kardiologicznych człowieka.Over the last decade the interest of using invertebrates, including insects, as model organisms in studies of human diseases has rapidly increased. Until now, hundreds of papers which demonstrate the possibility of using insect models in studies on human neurodegenerative diseases, diabetes, obesity or heart diseases were published. The rapid development of molecular biology techniques, physiological and pharmacological bioassays, as well as genome sequencing of fruit fly Drosophila melanogaster, red flour beetle Tribolium castaneum, silkworm Bombyx mori and honey bee Apis mellifera also provides a strong methodological support for this research approach. As a result of these studies a number of insect genes, orthologs of human genes known to be responsible for the development of various diseases, and many proteins encoded by these genes were identified. Also morphological and physiological phenotypes of different genes and the action of some drugs applied in neurodegenerative and cardiac diseases in humans have been characterized in insects

New proctolin analogues:Synthesis and biological investigation in insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X1-Tyr-Leu-Pro-Thr-OH, where X1 = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I-IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I-IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII-IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.</p

New proctolin analogues:Synthesis and biological investigation in insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X1-Tyr-Leu-Pro-Thr-OH, where X1 = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I-IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I-IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII-IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.</p

Further proctolin analogues modified in the position 2 of the peptide chain and their myotropic effects in insects Tenebrio molitor and Schistocerca gregaria

We have extended our studies on the structure-activity relationship in neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) by evaluating the effects of a series of proctolin analogues modified in position 2 of the peptide chain, including: [Phe(p-Cl)2]-(1), [D-Phe(p-Cl)2]-(2), [N-Me-Tyr2]-(3), [D-Phe(p-NH2)2]-(4), [D-Phe(p-N,N-di-Me)2]-(5), [N-Me-Tyr(OMe)]-(6), [D-3-Pal2]-(7), [L-Nal2]-(8), [D-Nal2]-(9), [Lys(Nic)2]-(10), [D-Lys(Nic)2]-(11), [D-Phe-(p-NO2)2]-(12). These peptides were evaluated for myotropic activity on the heart of Tenebrio molitor and contractile activity of the foregut of Schistocerca gregaria. Analogues 1-5, 7-9, and 12 retained a weak cardiotropic activity in Tenebrio molitor while peptides 1, 8 and 12 preserved 15-25% of the locust-gut contracting activity of proctolin. Peptides 2, 4 and 7 showed weak inhibitory activity in Schistocerca gregaria foregut, whereas only peptides 2, 4 and 7 reduced the maximum response to appllied proctolin by 64% and 49% respectively, at the 10-6 M concentration.</p