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Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

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.

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.

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.

Insect sulfakinins are multifunctional neuropeptides homologous to vertebrate gastrin/cholecystokin (CCK) neuropeptide hormones. We investigated the action of the nonsulfated sulfakinin Zopat-SK-1 (pETSDDYGHLRFa) on the levels of chosen metabolites in the Zophobas atratus beetle fat body. Samples of fat body were collected 2h and 24h after hormone injection. The administration of 20pmol of Zopat-SK-1 to feeding larvae significantly increased concentrations of lipids and proteins and decreased the content of glycogen in fat body tissue in the 24h experimental group. In contrast, the only increase in total lipid concentration in prepupal fat bodies was observed 24h after Zopat-SK-1 treatment. Simultaneously, changes in the quality and quantity of free sugars in the hemolymph were measured. In larval hemolymph, a marked increase in free sugar concentration and a decrease in glucose content were observed 24h and 2h after Zopat-SK-1 application, respectively. No changes in the prepupal stage were observed. For the first time we show potent metabolic activity of sulfakinin in the fat body tissue of an insect. Our findings imply a physiological function of the nonsulfated form of sulfakinin in energy storage and release processes in fat body tissue of larvae and prepupae was indicated. We suggest a role for sulfakinin signaling in the regulation of energy metabolism in insect tissues.

The nineteenth amino acid peptide Neb-colloostatin SIVPLGLPVPIGPIVVGPR and its analogue with point mutation (P4A) were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. The amine-N nitrogen atoms of serine residues were found to be the primary metal binding sites of both peptides. These binding modes provide the deprotonation and coordination of two amide nitrogens of the Neb-colloostatin and the formation of the 3N {NH2,2N−,CO} complex at pH 7.5–8. The proline residue P4 is the breakpoint in the coordination of copper(II) ions and the 4N {NH2,3N−} complex was not observed. At pH 8 the analogue (P4A) of Neb-colloostatin forms the copper(II) complex with 4N {NH2,3N−} binding site. The potentiometric and spectroscopic results did not reveal the stabilizing role of the oxygen donors of side chains of seryl residues (S1) in copper(II) complexes. At pH 8 the copper(II) ions change the secondary structures of both peptides in comparison to those without copper(II) ions. 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 copper(II) complexes of Neb-colloostatin and its analogue exert stronger pro-apoptotic activity on insect hemocytes in comparison to those of peptides without metal ions.

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 {NH 2 ,N Im –H 1 ,N Im –H 6 ,N Im –H 12 } binding site in wide pH range (5–8). For the Cu(II)–Allo1A system, the results demonstrated that at physiological pH 7.4 the predominant complex the CuH − 1 L consists of the 3N {NH 2 ,N − ,CO,N Im } coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 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 pH 7.4. 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.

In insects, neuropeptide adipokinetic hormone (AKH) released from the corpora cardiaca mobilizes lipids and carbohydrates in the fat body. We examined the developmental differences in the action of Tenmo-AKH, a bioanalogue belonging to the adipokinetic/hypertrahelosemic family (AKH/HrTH), on the lipid composition of larval and pupal fat bodies in the beetle Zophobas atratus . Tenmo-AKH was administered to the beetle larvae and pupae either as a single dose or as two doses of 20 pmol during a 24 h interval. Extracts of fat bodies were used to analyse the lipid composition by gas chromatography (GC) combined with mass spectrometry (GC–MS). Control extracts were analyzed using the same method. Fatty acids (FA) and fatty acid methyl esters (FAME) were the most abundant compounds in the fat bodies from both developmental stages. We observed significant differences in their concentrations following hormonal treatment. Tenmo-AKH also induced a distinct increase in larval sterols, fatty alcohols and benzoic acid.

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.

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.

We investigated the influence of adipokinetic hormone (AKH), an insect neurohormone, on uncoupling protein 4 (ZaUCP4) expression and activity in larval and pupal fat body mitochondria of the beetle Zophobas atratus in relation to intermediary metabolism. Homologous Tenmo-AKH was administered to the beetle larvae and pupae as either a single dose or as two doses of 20 pmol during a 24 h interval. In the larval and pupal fat bodies, downregulation of ZaUCP4 expression at the mRNA and protein levels was observed 24 h and 48 h after AKH treatment, respectively. In both developmental stages, ZaUCP4 activity was lowered in fat body mitochondria 48 h after AKH treatment. In the AKH-injected larvae, changes in ZaUCP4 expression were accompanied by the mobilization of carbohydrate reserves, no change in the concentration of total lipids and an increase in the free fatty acid level. In contrast, AKH had no effect on carbohydrate metabolism in the pupal fat body but induced lipid mobilization. It seems that AKH influences ZaUCP4 expression by triggering multiple events and that it has different physiological roles in controlling intermediary metabolism in the fat body of the beetle larvae and pupae.

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 1 nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.

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.

article i nfo Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus . Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae , CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria . The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10 −9 –10 −6 M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus . CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10 −9 –10 −6 M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor , however, the CavTKs induced no significant effect on the heart. Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut. In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.

The corpus cardiacum-corpus allatum complex of Tenebrio molitor contains factors causing hyperglycaemia, stimulating carbohydrate release from the fat body in vitro, and accelerating heart beat in the donor insect. However, no adipokinetic response can be demonstrated upon injection of extracts of this complex in larvae or young imagines. The carbohydrate elevation in the haemolymph is time- and dose-dependent, with a maximal effect about 90–180 min after injection. About 0.03–0.05 gland equivalents are needed to produce a significant increase in the larval and imaginal bioassays; maximal responses in both systems were reached with about 0.5 gland equivalents. Fat body treated in vitro with an extract of 0.5 gland equivalents releases about 130% more carbohydrate into 1 ml physiological saline after 1 h than paired tissue controls. The characteristic response of a semi-isolated adult heart to the endogenous myoactive factor(s) of the corpus cardiacum-allatum extract was an increase in heart beat frequency and a decrease in the amplitude of contraction. The response was dose-dependent and 0.05 gland equivalents were needed to exert a stimulatory effect on the myocardium. Fractionation of a methanolic extract of endocrine complex from Tenebrio molitor by RP-HPLC shows that the hyperglycaemic activity is confined to two regions. A myoactive factor appears to elute just after the second hyperglycaemic material, whereas a cardio-inhibitory factor has more hydrophobic properties. Synthetic neuropeptides adipokinetic hormone I and red pigment concentrating hormone injected as a dose of 8 pmol produce a weak hyperglycaemic effect, whereas myoactive factors I and II and hypertrehalosaemic factor II cause no hyperglycaemia. No adipokinetic response was shown upon injection of any of the neuropeptides tested. A very small stimulation of the adult heart beat frequency was observed upon administration of 20 pmol adipokinetic hormone I and myoactive factor I; all other peptides were without effect. In conclusion, the bioassays with Tenebrio appear to show some species-specificity and the hyperglycaemic/myoactive factors present in the corpus cardiacum-allatum neurosecretory complex may possess distinctive properties compared with adipokinetic hormone I, red pigment concentrating hormone, myoactive factors I and II, and hypertrehalosaemic factor II.

Studies made on trehalase activity in some tissues of Tenebrio molitor larvae have shown that enzyme activity is highest in the intestine, with a low level of activity in the fat body and muscles and traces of activity in the haemolymph. Higher intestinal trehalase activity was found in larvae fed on flaked oats with the addition of brewers yeast and fresh carrot (diet A) than in larvae fed the same diet without carrot (diet B). Starvation of larvae previously reared on diet A caused a decrease in trehalase activity in all the examined tissues except the haemolymph where traces remained. Refeeding starved larvae on diet A caused an increase in intestinal trehalase activity after as little as 2 hr.

1. 1. Insulin-like peptide from heads and midguts of Tenebrio molitor larvae was partially purified by acid/ethanol extraction and gel filtration. 2. 2. The presence of insulin-like hormone was confirmed by bioassay and radioimmunoassay. 3. 3. A hyperglycemic factor was probably present in the obtained preparations.