Arkadiusz Urbański

Developmental changes in cellular and humoral responses of the burying beetle Nicrophorus vespilloides (Coleoptera, Silphidae).

Necrophagous beetles of the genus Nicrophorus have developed various defence mechanisms that reduce the negative effects of adverse environmental conditions. However, many physiological and ecological aspects, including the functioning of the immune system in burying beetles, are still unknown. In this study, we show developmental changes in cellular and humoral responses of larvae, pupae, and adults of Nicrophorus vespilloides. We assessed changes in total haemocyte count, phenoloxidase activity, and phagocytic ability of haemocytes. We found that during larval development there is a progressive increase in humoral and cellular activities, and these responses are correlated with alterations of total haemocyte counts in the haemolymph. In the pupal stage, a sharp drop in the number of phagocytic haemocytes and an increase in phenoloxidase activity were observed. In adults, cellular and humoral responses remained at a lower level. It is probable that high lytic activity of anal and oral secretions produced by parents supports a lower response of the immune system in the initial phase of larval development. In the studied stages, we also observed differences in polymerisation of F-actin cytoskeleton of haemocytes, number of haemocytes forming filopodia, and filopodia length. These results suggest that the differences in immune responses during various stages of development of N. vespilloides are associated with a dynamically changing environment and different risks of infection. For the first time a detailed analysis of stage-specific alterations in immune system activity during development of the burying beetle is presented.

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.

Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H1 and H6 or H9 or H12) binding sites Structure Stability and Biological Activity

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

Impact of cold on the immune system of burying beetle, Nicrophorus vespilloides (Coleoptera: Silphidae).

Insect overwintering is one of the most astonishing phases of the insect life cycle. Despite vast amounts of knowledge available about the physiological mechanisms of this phenomenon, the impact of stress factors on insect immune system functioning during the winter is still unknown. The aim of this study is to analyze how low temperatures influence the immune system of the beetle Nicrophorus vespilloides. The results show that the beetles immune system is differently modulated by cold induced in laboratory settings than that which occurs in natural conditions. Among beetles cultured in conditions similar to summer, low temperatures, did not influence the number of circulating haemocytes, phenoloxidase activity, haemocytes morphology, and percentage ratio of haemocyte types. In these beetles, differences were noted only in the ability of haemocytes to perform phagocytosis. Individuals acclimated in natural conditions in autumn had a higher level of humoral response and a different percentage ratio of haemocyte types. During the winter period, the number of haemocytes in the beetles decreased, but the percentage ratio of phagocytic haemocytes increased. Furthermore, we noted an increase of phenoloxidase activity. Our study also showed mitotic divisions of haemocytes in haemolymph collected from burying beetles after cold exposure and from burying beetles collected from natural conditions during autumn and winter. Differences in response to low temperatures in laboratory conditions and the natural environment suggest that the simultaneous presence of other stress factors during winter such as desiccation and starvation have a significant influence on the activity of burying beetles immune system.

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.

Myotropic activity and immunolocalization of selected neuropeptides of the burying beetle Nicrophorus vespilloides (Coleoptera: Silphidae): Burying beetles’ myoactive neuropeptides

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nicve‐MS), myoinhibitory peptide (Trica‐MIP‐5) and the short neuropeptide F (Nicve‐sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 × 10−8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve‐MS: IC50 = 5.20 × 10−5 mol/L; Trica‐MIP‐5: IC50 = 5.95 × 10−6 mol/L; Nicve‐sNPF: IC50 = 4.08 × 10−5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin‐immunolabeled neurons only in VNC were observed. Moreover, MIP‐immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve‐MS (QDVDHVFLRFa) and six isoforms of Nicve‐MIP (Nicve‐MIP‐1—DWNRNLHSWa; Nicve‐MIP‐2—AWQNLQGGWa; Nicve‐MIP‐3—AWQNLQGGWa; Nicve‐MIP‐4—AWKNLNNAGWa; Nicve‐MIP‐5—SEWGNFRGSWa; Nicve‐MIP‐6— DPAWTNLKGIWa; and Nicve‐sNPF—SGRSPSLRLRFa).

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

HIGHLIGHTSEffects of three peptides on male reproductive tract of beetle were analysed.All peptides increased soluble proteins concentrations in testes and their dry mass.Lepde‐NPF‐I influence on spermatocytes number and contractility of ejaculatory duct.Neb‐TMOF decreased spermatocytes number but increased ejaculatory duct contractility.Neb‐colloostatine increased spermatocytes number only in 8‐day‐old males. ABSTRACT 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 beetles 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.

High stability and biological activity of the copper(II) complexes of alloferon 1 analogues containing tryptophan

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH-1L and/or CuH-2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH-1L, CuH-2L and CuH-3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

Developmental changes in haemocyte morphology in response to Staphylococcus aureus and latex beads in the beetle Tenebrio molitor L.

The evolutionary success of insects is undoubtedly related to a well-functioning immune system. This is especially apparent during insect development by the adaptation of individuals to the changing risk of infection. In addition, current studies show that the insect immune system is characterized by some specificity in response to natural pathogens (for example, bacteria, viruses or fungi) and artificial challengers (for example, latex beads or nylon filaments). However, developmental changes and the specificity of immune system reactions simultaneously have not been analysed. Thus, the aim of the present research was to determine changes in haemocyte morphology in response to attenuated Staphylococcus aureus and latex beads across each developmental stage of the beetle Tenebrio molitor. The results of the present research clearly showed differences in the morphology of T. molitor haemocytes during development. The haemocytes of larvae and 4-day-old adult males were characterized by the highest adhesion ability, which was expressed as the largest average surface area, filopodia length and number of filopodia. In contrast, the haemocytes of pupae and 30-day-old adult males had a significantly lower value for these morphological parameters, which was probably related to metamorphosis (pupae) and immunosenescence (30-day-old adults). The haemocytes of the tested individuals reacted differently to the presence of S. aureus and latex beads. The presence of S. aureus led to a significant decrease in all previously mentioned morphological parameters in larvae and in both groups of adult individuals. In these groups, incubation of haemocytes with latex beads caused only a slight decrease in surface area and filopodia length and number. This morphological response of haemocytes to biotic and artificial challengers might be related to an increase in the migration abilities of haemocytes during infection. However, the differences in haemocyte reactivity towards S. aureus and latex beads might be explained by differences in pathogen recognition. Conversely, increased adhesive abilities of pupal haemocytes were also observed, which might be related to the specificity of metamorphosis and the hormonal titre during this developmental stage.

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH2)1]‐ and [Phe(p‐OMe)1]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.