Separation and behavior in vitro of hemocytes from the moth, Pseudoplusia includens

Abstract

Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera:Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.

Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens

“…For in vitro experiments, haemocytes were collected from unparasitized P. includens larvae and were purified on Percoll cushions (Pech et al, 1994). Briefly, larvae were surface sterilized with 70 % ethanol and bled from a cut proleg directly into ice-cold anticoagulant buffer (0.098 M-NaOH, 0-186 M-NaCI, 0.017 M-EDTA and 0-041 M-citric acid pH 4.5).…”

“…After rinsing in Ex-cel1400 medium, haemocytes were pelleted by centrifugation at 250 g. Plasmatocytes and granular cells were then purified on 47 and 63 % Percoll cushions, respectively. Resulting granular cell and plasmatocyte populations were 99 % and 95 % pure as identified by established morphological criteria (Pech et al, 1994). To inoculate cells in vitro, haemocytes were cultured (l x l0 s cells/well) in 70 gl of Ex-cell 400 medium containing 0.50 wasp equivalents of calyx fluid or 1.0 wasp equivalent of MdPDV plus venom.…”

Specific Residues in Plasmatocyte-spreading Peptide Are Required for Receptor Binding and Functional Antagonism of Insect Immune Cells

“…The three-dimensional structure of PSP consists of a disordered N terminus (residues 1-6) and a well defined core (residues 7-22) stabilized by a disulfide bond between Cys 7 and Cys 19 , hydrophobic interactions, and a short ␤-hairpin turn (11). Comparison with other proteins reveals that the core region of PSP adopts a very similar structure to the C-terminal subdomain of human epidermal growth factor and the anticoagulant protein thrombomodulin.…”

“…Using alanine replacement and deletion mutants, we also have identified residues in both the structured and unstructured regions of PSP that are essential for plasmatocyte-spreading activity (16,17). These include Cys 7 and Cys 19 , which form the disulfide bond required for the proper threedimensional structure of PSP, and the charged residue Arg 13 within the ␤-hairpin turn. Deletion mutants in the unstructured N terminus also eliminate all biological activity.…”

“…The percentage of plasmatocytes that spread in an assay was scored 30 min later by counting 100 cells from a randomly selected field of view. Plasmatocytes were scored as spread if they assumed a flattened morphology and were Ն35 m along their longest axis (6,19). Unspread plasmatocytes remained nonadhesive and spheroidal in shape.…”