Sulfakinins influence lipid composition and insulin-like peptides level in oenocytes of Zophobas atratus beetles.

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

The myosuppressin structure-activity relationship for cardiac contractility and its receptor interactions support the presence of a ligand-directed signaling pathway in heart.

The structural conservation and activity of the myosuppressin cardioinhibitory peptide across species suggests it plays an important role in physiology, yet much remains unknown regarding its signaling. We previously reported Drosophila melanogaster myosuppressin (dromyosuppressin, DMS; TDVDHVFLRF-NH2) decreases cardiac contractility through a G protein-coupled receptor, DMS-R2. Our study showed the DMS N-terminus amino acids influence its structure-activity relationship (SAR), yet how they act is not established. We predicted myosuppressin N-terminal amino acids played a role in signaling. Here, we tested our hypothesis in the beetle, Zophobas atratus, using a semi-isolated heart bioassay to explore SAR in a different Order and focus on cardiac signaling. We generated a series of myosuppressin truncated analogs by removing the N-terminal residue and measuring the activity of each structure on cardiac contractility. While DVDHVFLRF-NH2 decreased cardiac contractility, we found VDHVFLRF-NH2, DHVFLRF-NH2, and HVFLRF-NH2 increased activity. In contrast, VFLRF- NH2 decreased activity and FLRF-NH2 was inactive. Next, we analyzed molecular docking data and found the active truncated analogs interacted with the 3-6 lock in DMS-R2, the myosuppressin cardiac receptor, disrupting the salt bridge between H114 and E369, and K289 and Q372. Further, the docking results showed the inhibitory effect on contractility may be associated with contact to Y78, while the analogs that increased contractility lacked this interaction. The data from our study demonstrated N-terminal amino acids played a role in myosuppressin activity and signaling suggesting the cardiac receptor can be targeted by biased agonists. Our myosuppressin cardiac contractility data and predicted receptor interactions describe the presence of functional selectivity in a ligand-directed signaling pathway in heart.

A possible role of tachykinin-related peptide on an immune system activity of mealworm beetle, Tenebrio molitor L.

Tachykinin-related peptides (TRPs) are important neuropeptides. Here we show that they affect the insect immune system, especially the cellular response. We also identify and predict the sequence and structure of the tachykinin-related peptide receptor (TRPR) and confirm the presence of expression of gene encoding TRPR on Tenebrio molitor haemocytes. After application of the Tenmo-TRP-7 in T. molitor the number of circulating haemocytes increased and the number of haemocytes participating in phagocytosis of latex beads decreased in a dose- and time-dependent fashion. Also, Tenmo-TRP-7 affects the adhesion ability of haemocytes. Six hours after injection of Tenmo-TRP-7, a decrease of haemocyte surface area was observed under both tested Tenmo-TRP-7 concentrations (10-7 and 10-5 M). The opposite effect was reported 24 h after injection, which indicates that the influence of Tenmo-TRP-7 on modulation of haemocyte behaviour differs at different stages of stress response. Tenmo-TRP-7 application also resulted in increased phenoloxidase activity 6 and 24 h after injection. The assessment of DNA integrity of haemocytes showed that the injection of Tenmo-TRP-7 at 10-7 M led to a decrease in DNA damage compared to control individuals. This effect was only visible 6 h after Tenmo-TRP-7 application. After 24 h, Tenmo-TRP-7 injection increased DNA damage. We also confirmed the expression of immune-related genes in nervous tissue of T. molitor. Transcripts for genes encoding receptors participating in pathogen recognition processes and antimicrobial peptides were detected in T. molitor brain, retrocerebral complex and ventral nerve cord. These results may indicate a role of the insect nervous system in pathogen recognition and modulation of immune response similar to vertebrates. Taken together, our results support the notion that tachykinin-related peptides probably play an important role in the regulation of the insect immune system. Moreover, some resemblances with action of tachykinin-related peptides and substance P showed that insects can be potential model organisms for analysis of hormonal regulation of conserved innate immune mechanisms.

Identification of sulfakinin receptors (SKR) in Tenebrio molitor beetle and the influence of sulfakinins on carbohydrates metabolism.

Sulfakinins (SKs) are pleiotropic neuropeptides commonly found in insects, structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK) neuropeptides. SKs together with sulfakinin receptors (SKRs) are involved in sulfakinin signaling responsible for variety of biological functions, including food intake or fatty acid metabolism. In the present study, we determined the distribution of SKRs in Tenebrio molitor larvae and characterized the impact of nonsulfated and sulfated SKs on carbohydrates and insulin-like peptides (ILPs) level in beetle hemolymph. Our results indicate the presence of both sulfakinin receptors, SKR1 and SKR2, in the nervous system of T. molitor. The distribution of SKR2 in peripheral tissues was more widespread than SKR1, and their transcripts have been found in fat body, gut and hemolymph. This is also the first evidence for SKRs presence in insect hemocytes indicating immunotropic activity of SKs. Moreover, in the present study, we have demonstrated that SKs regulate ILPs and carbohydrates level in insect hemolymph, and that sulfation is not crucial for peptides activity. Our study confirms the role of SKs in maintaining energy homeostasis in beetles.

The activity of the nonsulfated sulfakinin Zopat-SK-1 in the neck-ligated larvae of the beetle Zophobas atratus.

Insect sulfakinins (SKs) are multifunctional neuropeptides structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK). It has been proposed that SKs play a role in modulating energy management in insects by interacting with adipokinetic hormone (AKH), the principle hormone controlling insect intermediary metabolism. To exclude head factors (including AKH) that influence the activity of the nonsulfated sulfakinin Zopat-SK-1 in the larvae of the beetle Zophobas atratus, ligature and in vitro bioassays were used. Our study showed that in the neck-ligated larvae, Zopat-SK-1 evoked a much more pronounced glycogenolytic effect in fat body tissue and a significantly higher hypertrahelosemic effect in hemolymph than in larvae without ligation. We found that the concentration of the sugar trehalose increased under hormonal treatment but no changes in glucose levels were observed. Under in vitro conditions, the maximal glycogenolytic effect of Zopat-SK-1 in fat body was observed at 10 pmol of hormone. Ligature and in vitro bioassays indicated that Zopat-SK-1 activity in the Z. atratus larvae is modulated by head signals and/or factors from the gastrointestinal tract. Our data indicate the existence of a brain-gastrointestinal axis that has a role in controlling of energy (carbohydrate) metabolism in the insect body. Moreover, these results, together with immunological evidence of a cholecystokinin-like (sulfakinin) receptor in the Z. atratus fat body, help us to better understand the SK signaling pathways and its physiological role in insect biology.

Synthesis, cardiostimulatory, and cardioinhibitory effects of selected insect peptides on Tenebrio molitor.

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain. Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6-10]-Mas-MT-I, whereas the [4-10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln(1)]- and [Gly(1)]-Any-GS also show a strong cardioinhibitory effect.