Elżbieta Czarniewska

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.

The pro-apoptotic action of the peptide hormone, Neb -colloostatin, on insect haemocytes

SUMMARY The gonadoinhibitory peptide hormone Neb-colloostatin was first isolated from ovaries of the flesh fly Neobellieria bullata. This 19-mer peptide is thought to be a cleaved product of a collagen-like precursor molecule that is formed during remodelling of the extracellular matrix. In this study, we report that upon injection of picomolar and nanomolar doses, this peptide exerts a pro-apoptotic action on haemocytes of Tenebrio molitor adults, as visualized by changes in morphology and viability. The F-actin cytoskeleton was found to aggregate into distinctive patches. This may be responsible for the observed inhibition of adhesion of haemocytes and for the stimulation of filopodia formation. However, Neb-colloostatin injection did not induce the formation of autophagic vacuoles. Our results suggest that physiological concentrations of Neb-colloostatin play an important role in controlling the quantity and activity of haemocytes in insect haemolymph. They also suggest that during periods in which Neb-colloostatin is released, this peptide may cause a weakening of the insects’ immune system. This is the first report that exposure to a peptide hormone causes apoptosis in insect haemocytes.

Mono- and polynuclear copper(II) complexes of alloferons 1 with point mutations (H6A) and (H12A): stability structure and cytotoxicity.

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity.

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A(1)GVSGH(6)GQH(9)GVH(12)G (Allo1A) and (H9A) H(1)GVSGH(6)GQA(9)GVH(12)G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H(6),H(12)) leads to the formation of the CuL complex with 4N {NH2,NIm-H(1),NIm-H(6),NIm-H(12)} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH-1L consists of the 3N {NH2,N(-),CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His(1) and His(6) residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

Novel biological effects of alloferon and its selected analogues: structure-activity study.

The subject of this paper is a search for new biological properties of alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) and a series of its analogues. The studies on structure/activity relationship in alloferon, the synthesis of a series of 28 analogues were performed. The analogues were modified at position 1 or 6, and other were oligopeptides with a shortened peptide sequence. Biological effects of the peptides were evaluated by the pro-apoptotic action in vivo on haemocytes of Tenebrio molitor and in the cardiotropic test in vitro on the heart of T. molitor and Zophobas atratus. In the in vivo bioassays, new biological activities of alloferon and its analogues were discovered. In haemocytotoxic bioassay, alloferon strongly induces T. molitor haemocytes to undergo apoptosis at a dose of 10 nM. Moreover, [Phe(p-NH2)(1)]-, [Tyr(6)]- and [1-10]-alloferon exhibit a two-fold increase of caspases activation in comparison with the alloferon. However, alloferon and its analogues show a weak cardiostimulatory activity in Z. atratus but the heart of T. molitor is not sensitive to these peptides. The results obtained here suggest that alloferon plays pleiotropic functions in insects.

Large eggs and ploidy of green frog populations in Central Europe

Green frogs of Central Europe consist of three taxa: Pelophylax ridibundus, P. lessonae and their natural hybridogenetic hybrid, P. esculentus, which forms as a rule mixed populations with its parental species. We examined 659 095 eggs from P. ridibundus (48 females), P. lessonae (133 females) and P. esculentus (170 females) originating from 39 populations in Austria, Denmark, Germany and Poland. Some females of each taxon laid eggs that fell into discrete size classes (small, medium and large). Large eggs were mostly diploid gametes from which triploids developed. They were found in P. esculentus (25 233 large eggs in 152 spawns), P. lessonae (81 in 10 spawns) andP. ridibundus (7 in 3 spawns). The main purpose of the paper was to demonstrate that the numbers of large eggs were clearly associated with triploid P. esculentus frogs. In pure hybrid (esculentus) populations large eggs comprised between 2.44-40.96% of all ova, while triploid adult frogs constituted between 13.9-73.2% of all individuals, in mixed ridibundus-esculentus populations the large eggs and triploid frogs ranged between 0.85-36.6% and 9.2-56.2%, respectively. However, in mixed lessonae-esculentus populations large eggs comprised only 1.74% of the spawns, whereas triploid frogs represented 2.1% of the adults in the population.

Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

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.

The pro-apoptotic action of new analogs of the insect gonadoinhibiting peptide Neb-colloostatin: Synthesis and structure–activity studies

Neb-colloostatin (SIVPLGLPVPIGPIVVGPR), an insect oostatic factor found in the ovaries of the flesh fly Neobellieria bullata, strongly induces apoptosis in insect haemocytes. To explain the role of Ser(1) and Pro(4) residues of Neb-colloostatin in the pro-apoptotic activity of this peptide, the synthesis of a series of analogs was performed, such as: [Ac-Ser(1)]- (1), [d-Ser(1)]- (2), [Thr(1)]- (3), [Asp(1)]- (4), [Glu(1)]- (5), [Gln(1)]- (6), [Ala(1)]- (7), [Val(1)]- (8), [d-Pro(4)]-(9), [Hyp(4)]- (10), [Acp(4)]- (11), [Ach(4)]- (12), [Ala(4)]- (13), [Ile(4)]- (14), and [Val(4)]-colloostatin (15). All peptides were bioassayed in vivo for the pro-apoptotic action on haemocytes of Tenebrio molitor. Additionally, the structural properties of Neb-colloostatin and its analogs were examined by the circular dichroism in water and methanol. Peptides 1, 4, 5, 7, 8, 10, 12, 14, and 15 strongly induce T. molitor haemocytes to undergo apoptosis and they show about 120-230% of the Neb-colloostatin activity at a dose of 1nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.

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.