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First published online March 17, 2006
Journal of Experimental Biology 209, 1326-1335 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02118
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Interaction between non-specific electrostatic forces and humoral factors in haemocyte attachment and encapsulation in the edible cockle, Cerastoderma edule

Emma C. Wootton*, Elisabeth A. Dyrynda{dagger} and Norman A. Ratcliffe

School of Environment and Society, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK


Figure 1
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  		Fig. 1. Encapsulation of positively charged, DEAE Sepharose beads by haemocytes in the presence of plasma. (A) Onset of encapsulation (10 min incubation), characterized by attachment of individual haemocytes to beads. (B) End point of encapsulation (2 h incubation), characterized by formation of bead/haemocyte aggregates with large numbers of haemocytes attaching to the beads. Phase-contrast microscopy. Scale bars, 80 mm.

 

Figure 2
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  		Fig. 2. Metacercarial cysts of Himasthla sp. in the foot of C. edule. (A) Squash preparation showing both un-encapsulated (Un) and encapsulated (En) cysts within the same individual. (B) Histological section stained with Cole's Haematoxylin and Eosin showing a thick haemocytic capsule (HC) surrounding the parasite cyst (P). Scale bars, 200 µm.

 

Figure 3
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  		Fig. 3. In vivo and in vitro cell attachment and encapsulation experiments. Times were recorded for the time taken for onset (light grey) and completion (dark grey) of cell attachment/encapsulation. The rate of encapsulation (both onset and completion) was significantly different between (but not within) targets of different surface charges (P<0.001). (A) In vivo response. (B) In vitro response, in which times were not recorded for completion of cell attachment/encapsulation of neutral targets (N) as no further progression of encapsulation was observed after initial attachment of individual haemocytes. DEAE, DEAE Sepharose; QAE, QAE Sephadex; Nylon, Nylon thread; CM, CM Sepharose; Seph, Sepharose; Toyo, Toyopearl; +, positive; –, negative; N, neutral; 1, haemocytes in the presence of plasma; 2, haemocytes in the absence of plasma; x, no encapsulation. N=10 for each target. Samples with different symbols are significantly different.

 

Figure 4
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  		Fig. 4. Differences in haemocyte morphology observed in vitro, in the presence and absence of plasma. (A) Haemocytes with plasma, spread on uncoated 24-well culture plate (1 h incubation); scale bar, 10 µm. (B) Rounded haemocytes without plasma, attached to uncoated 24-well culture plate (1 h incubation); scale bar, 10 µm. (C) Haemocytes with plasma, spread on surface of DEAE Sepharose beads (1 h incubation); scale bar, 80 µm. (D) Rounded haemocytes without plasma, attached to surface of DEAE Sepharose beads (3 h incubation); scale bar, 80 µm.

 

Figure 5
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  		Fig. 5. Identification of negatively charged surfaces using FITC-poly-L-lysine. (A) Negatively charged CM Sepharose beads; scale bar, 80 µm. (B) Negatively charged viable haemocytes (without plasma); scale bar, 10 µm. (C) Himasthla sp. cyst under phase contrast; scale bar, 100 µm. (D) Identical cyst showing negatively charged cyst wall; scale bar, 100 µm.

 

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© The Company of Biologists Ltd 2006