title: Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.
abstract: The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly - regardless the time-intervals - at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight "day" effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the "day" effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails' immunocompetence.
authors: Irstea, UR MALY, Centre de Lyon-Villeurbanne, 5 rue de la Doua, BP 32108, 69616 Villeurbanne Cedex, France.
authors: Paul
authors: P
authors: Boisseaux
authors: Université de Lyon, Université Lyon 1, CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive, 43 Bd du 11 novembre 1918, 69622 Villeurbanne, France; Université de Lyon, VetAgro Sup Campus Vétérinaire de Lyon, 69280 Marcy l'Etoile, France.
authors: Marie-Laure
authors: ML
authors: Delignette-Muller
authors: Irstea, UR MALY, Centre de Lyon-Villeurbanne, 5 rue de la Doua, BP 32108, 69616 Villeurbanne Cedex, France.
authors: Khédidja
authors: K
authors: Abbaci
authors: LIttoral ENvironnement et Sociétés (LIENSs) - UMR 7266, Avenue Michel Crépeau, 17 042 La Rochelle, France.
authors: Hélène
authors: H
authors: Thomas
authors: Irstea, UR MALY, Centre de Lyon-Villeurbanne, 5 rue de la Doua, BP 32108, 69616 Villeurbanne Cedex, France. Electronic address: jeanne.garric@irstea.fr.
authors: Jeanne
authors: J
authors: Garric
mesh_terms: D000818:Animals; D006434:Hemocytes / Q000648:ultrastructure*; D006458:Hemolymph; D008195:Lymnaea / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D013048:Specimen Handling
keywords: Electron microscopy; Flow cytometry; Frequency of collection; Hemocyte density; Hemocyte viability; Linear mixed-effect models; Repeated collections; Snail survival