Ph. Roch

Scanning electron-microscopic study of the involvement of coelomic cells in earthworm antibacterial defense

SummaryThe first steps of an antibacterial reaction in the earthworm Eisenia fetida andrei were investigated. The main cellular mediators of this activity are the chloragocytes, a class of free coelomocytes existing only in annelids. Our observations using scanning electron microscopy have shown that chloragocytes were able to agglutinate and perhaps to destroy pathogenic bacteria such as Bacillus megaterium in the same way that they agglutinate and lyse vertebrate erythrocytes. Bacteria known to be non-pathogenic for the worm, such as Acinetobacter, were not agglutinated but slowly eliminated by segregation into brown bodies. Chloragocytes maintained in vitro, lost their chloragosomes and exhibited stronger agglutination activity against pathogenic bacteria than chloragocytes in situ. From this increased efficiency of chloragocytes in vitro, we infer that, in normal living conditions, chloragocytes probably intervene in antibacterial defense mainly after their extrusion from the coelomic cavity and their spreading and degranulation at the surface of the integument.

Protein analysis of earthworm coelomic fluid IV. Evidence, activity induction and purification of Eisenia fetida andrei lysozyme (Annelidae)

Abstract 1. 1. Lysozyme activity was found in the coelomic fluid of the earthworm Eisenia fetida andrei . 2. 2. Background activity was 20-fold enhanced by one injection of either Gram+, Gram−, worm pathogen or non-pathogen bacteria, SRBC. 3. 3. Maximum induced activity occurred 4–5 hr after injection. 4. 4. Purification has been achieved by three steps fast protein liquid chromatography (FPLC): gel filtration; chromatofocusing; and reversed phase chromatography (RPC). Only one active molecule was isolated: MW mol. wt 20,000, pI > 9.0. 5. 5. Comparing numbers of cystein residues, E.f. andrei lysozyme is close to vertebrate type c lysozyme, but its amino acid composition seems to be related to Asterias rubens lysozyme (Echinoderm).

Purification of three serine proteases from the coelomic cells of earthworms (Eisenia fetida)

Abstract 1. 1. Proteinase activity is evidenced in coelomic cell lysate of Eisenia fetida andrei. 2. 2. Tested on gelatin plates, the proteolysis develops rapidly during the first hr of incubation at 37°C. 3. 3. The proteolysis is pH-dependent with two optima for pH 7.0 and 10.0. 4. 4. The proteolysis is dose-dependent. 5. 5. The proteolysis is not influenced by ionic strength. 6. 6. The proteolysis is thermo-sensitive but not totally suppressed by 15 min at 100°C. 7. 7. Three serine proteases, A2 (Mr 42 kDa), B2 (Mr 46 kDa) and C2 (Mr 48 kDa), were purified by affinity chromatography on benzamidine-Sepharose. 8. 8. According to various inhibition and activity tests, A2 is of trypsin-like type, Ca2+-independent but Mg2+- and Mn2+-dependent and C2 is of chymotrypsin-like type.

Evidence and characterization of lysozyme in six species of freshwater crayfishes from astacidae and cambaridae families

Abstract 1. 1. A lysozyme activity was found in four Astacidae and two Cambaridae species. 2. 2. Tested on Micrococcus luteus cell wall, the reaction was stable when heated in acidic buffer but heat labile in an alkaline one. 3. 3. The activity is present in serum, hemocyte lysate and plasma. 4. 4. In serum, the lysis occurred within the first 15 min; the less active was the plasma. 5. 5. In the six species, the kinetics followed the same pattern. 6. 6. Different animals of the same species exhibited a great variability in lysozyme activity: 32–53.8% of lysis after 1 hr in serum, 7–31.7% in plasma, and 20–50.3% in hemocyte lysate. 7. 7. Different stresses weakening the animals lowered the response to less than 25% of lysis after 1 hr instead of 60–86% in serum or 38–72% in hemocyte lysate. 8. 8. The lysozyme molecules seemed to be located in hemocytes and released during experimental cell lysate or in vitro coagulation. 9. 9. Plasma reaction could represent the background of the hemolymph activity occurring in natural conditions.

Amino acid compositions and relationships of five earthworm defense proteins

Abstract 1. 1. Amino acid composition of five earthworm proteins involved in several defense processes was established. 2. 2. The SΔQ statistical test of Marchalonis and Weltman (1971) gave various degrees of similarity within these proteins called α, β, γ, δ and ϵ. 3. 3. Close similarities of proteins β, γ and δ confirm our previous physiological and genetical studies on their common allelic origin. 4. 4. Compositional similarities were also suspected with Asteria rubens lysozyme, three invertebrate agglutinines, human fibrinogen, mouse B2-M, H2 and complement C3.

EVIDENCE OF A MLR-LIKE REACTION IN AN INVERTEBRATE, THE EARTHWORM EISENIA FETIDA

ABSTRACT The incorporation of 3H-TdR by leucocytes arising from 2 Eisenia fetida and mixed together, in vitro, in a ratio of 50/50 was studied. A spontaneous increase of this incorporation was noted in about 8 per cent of the tested combinations. Combinations having shown a positive response at the time t, also gave a positive response at the time t + 4 months. The nature of stimulated leucocytes was studied by autoradiography: incorporating cells were lymphocyte-like cells (Φ = 10 to 12 μm). Such a type of cells was also found to be involved in graft rejections, cytotoxic reactions and mitogenic stimulations.