Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge.

The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an important aquacultured decapod species as well as an invasive species in some European countries. In the current investigation we characterized the different classes of circulating blood cells in A. leptodactylus by means of light and electron microscopy analysis and we explored their reaction to different latex beads particles in vivo by total and differential cell counts at 0.5, 1, 2 and 4h after injections. We identified hemocytes by granule size morphometry as hyaline hemocytes with no or rare tiny granules, small granule hemocytes, unimodal medium diameter granule hemocytes and both small and large granule containing hemocytes. The latter granular hemocytes showed the strongest phenoloxidase l-DOPA reactivity both in granules and cytoplasm. A. leptodactylus respond to foreign particles with strong cellular immune responses. All treatments elicited a total hemocyte increase with a conspicuous recruitment of large granule containing hemocytes. All hemocyte types mounted some phagocytic response but the small granule hemocytes were the only ones involved in phagocytic response to all foreign particles with the highest percentages. These results (1) depict the variability in decapod hemocyte functional morphology; (2) identify the small granule hemocyte as the major phagocytic cell; (3) suggest that the rather rapid recruitment of large granule hemocyte in all treatments plays a relevant role by this hemocyte type in defense against foreign particles, probably in nodule formation.

Role of biogenic amines and cHH in the crustacean hyperglycemic stress response.

In this study, we investigated (using bioassays and ELISA) the variation of cHH (crustacean hyperglycemic hormone) level in the eyestalks and hemolymph of Palaemon elegans (Rathke) (Decapoda, Caridea) following injection of serotonin (5-HT) and dopamine (DA) and correlated cHH profile with the variation in amount and time course of glycemia. 5-HT induced in P. elegans a rapid and massive release of cHH from the eyestalk into the hemolymph followed by hyperglycemia. On the contrary, DA did not significantly affect cHH release and hyperglycemia. In addition, we measured the level and variation of 5-HT in the eyestalk and hemolymph of P. elegans following copper contamination. The release of 5-HT from the eyestalk is very rapid and dose dependent. In the hemolymph, a peak of 5-HT occurs after 30 min, and again the circulating concentration of 5-HT is dose dependent on copper exposure. After 1 h, the level of 5-HT slowly decreases to basal level. The release of 5-HT from the eyestalk into the hemolymph after copper exposure precedes the release of cHH, confirming its role as a neurotransmitter acting on cHH neuroendocrine cells. The fact that copper induced a rapid and massive release of 5-HT from the eyestalk can explain its demonstrated role in inducing the release of cHH and the consequent hyperglycemia in intact but not eyestalkless animals.

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins.

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.