Novel Protocol for the Chemical Synthesis of Crustacean Hyperglycemic Hormone Analogues — An Efficient Experimental Tool for Studying Their Functions

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool

The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10(3)Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001).The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals

The main actors involved in parasitization of Heliothis virescens larva

At the moment of parasitization by another insect, the host Heliothis larva is able to defend itself by the activation of humoral and cellular defenses characterized by unusual reactions of hemocytes in response to external stimuli. Here, we have combined light and electron microscopy, staining reactions, and immunocytochemical characterization to analyze the activation and deactivation of one of the most important immune responses involved in invertebrates defense, i.e., melanin production and deposition. The insect host/parasitoid system is a good model to study these events. The activated granulocytes of the host insect are a major repository of amyloid fibrils forming a lattice in the cell. Subsequently, the exocytosed amyloid lattice constitutes the template for melanin deposition in the hemocel. Furthermore, cross-talk between immune and neuroendocrine systems mediated by hormones, cytokines, and neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropin hormone, alpha melanocyte-stimulating hormone, and neutral endopeptidase occurs. Thus, parasitization promotes massive morphological and physiological modifications in the host insect hemocytes and mimics general stress conditions in which phenomena such as amyloid fibril formation, melanin polymerization, pro-inflammatory cytokine production, and activation of the adrenocorticotropin hormone system occur. These events observed in invertebrates are also reported in the literature for vertebrates, suggesting that this network of mechanisms and responses is maintained throughout evolution

Circulating hemocytes from larvae and adults of Carabus (Chaetocarabus) lefebvrei Dejean 1826 (Coleoptera, Carabidae):Cell types and their role in phagocytosis after in vivo artificial non-self-challenge.

Carabus lefebvrei Dejean 1826 is an helicophagous Italian endemic ground beetle that lives in central and south Apennines mountain forests, from lower altitudes to about 1500 m. In ground beetles, no morphofunctional data about immune system is available, even though they are well known both taxonomically and ecologically and they have been often used as indicators of the habitat quality due to their specificity to certain habitat types. In the current investigation the cellular population in the hemolymph of adult and third instar larvae of C. lefebvrei has been characterized by means of light and electron microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.9 mm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found both in larvae and in adults: prohemocytes, granulocytes, oenocytoids and plasmatocytes. After in vivo artificial non-selfchallenge treatments, C. lefebvrei showed a non-specific immune response involving phagocytosis performed by plasmatocytes, both in adults and in larvae and by oenocitoids in larvae. In untreated animals, the hemocyte type presenting a firm phagocytic activity, the plasmatocytes, presented a percentage significantly higher in larvae than in adults, and after latex beads injections in larvae there was a tendency of significant difference in plasmatocyte percentage compared to controls injected with phosphate saline buffer. We think that these differences could be correlated with the peculiar morphology (less chitinization) and ecology of larval stages that are more sensitive to pathogens than adults

Functional aspects of cHH C-terminal amidation in crayfish species

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition

Circadian modulation of crustacean hyperglycemic hormone in crayfish eyestalk and retina.

Previous studies suggested the retina could be a putative locus of daily crustacean hyperglycemic hormone (CHH) secretion, as it possesses its own metabolic machinery and is independent of the well-known CHH eyestalk locus responsible for the circadian secretion of this peptide. However, it has been proposed that hemolymph glucose and lactate concentrations play a dual role in the regulation of CHH in crayfish. To elucidate the temporal relationship between these two different CHH production loci and to examine their relationship with glucose regulation, we investigated the expression of CHH daily and circadian rhythms in the eyestalk and retina of crayfish using biochemical methods and time series analysis. We wanted to determine whether (1) putative retina and eyestalk CHH rhythmic expressions are correlated and if the oscillations of the two metabolic products of lactate and glucose in the blood due to CHH action on the target tissue correlate, and (2) retina CHH (RCHH) and the possible retinal substrate glycogen and its product glucose are temporally correlated. We found a negative correlation between daily and circadian changes of relative CHH abundance in the retina and eyestalk. This correlation and the cross-correlation values found between eyestalk CHH and hemolymph and glucose confirm that CHH produced by the X-organ sinus gland complex is under the previously proposed dual feedback control system over the 24 h time period. However, the presence of both glycogen and glucose in the retina, the cross-correlation values found between these parameters and hemolymph lactate and glucose, as well as RCHH and hemolymph and retina metabolic markers suggest RCHH is not under the same temporal metabolic control as eyestalk CHH. Nonetheless, their expression may be linked to common rhythms-generating processes

Brain-derived neurotrophic factor mRNA and protein are targeted to discrete dendritic laminas by events that trigger epileptogenesis

Dendritic targeting of mRNA and local protein synthesis are mechanisms that enable neurons to deliver proteins to specific postsynaptic sites. Here, we demonstrate that epileptogenic stimuli induce a dramatic accumulation of BDNF mRNA and protein in the dendrites of hippocampal neurons in vivo. BDNF mRNA and protein accumulate in dendrites in all hippocampal subfields after pilocarpine seizures and in selected subfields after other epileptogenic stimuli (kainate and kindling). BDNF accumulates selectively in discrete dendritic laminas, suggesting targeting to synapses that are active during seizures. Dendritic targeting of BDNF mRNA occurs during the time when the cellular changes that underlie epilepsy are occurring and is not seen after intense stimuli that are non-epileptogenic, including electroconvulsive seizures and high-frequency stimulation. MK801, an NMDA receptor antagonist that can prevent epileptogenesis but not acute seizures, prevents the dendritic accumulation of BDNF mRNA, indicating that dendritic targeting is mediated via NMDA receptor activation. Together, these results suggest that dendritic accumulation of BDNF mRNA and protein plays a critical role in the cellular changes leading to epilepsy