All in vitro microaggregates displayed

clustered granular cells with plasmatocytes rosetting the edges of the cluster ( Fig. 2B). The leading, fan-shaped edges of the plasmatocytes were directed away from the cell clusters like those in hemocyte clusters of the lepidopteran Ephestia kuhniella suggesting exomigration of the plasmatocytes [23]. To determine if the CTX effect reflected the influence of its individual components, hemocytes were treated with corresponding stoichiometric levels of individual CTB (levels being selleck kinase inhibitor 0, 6, 30, 150, 300, and 600 nM since CTX has five CTB subunits) or CTA. Total individually attached hemocytes from 0 to 30 nM CTB were statistically similar (p<0.05), whereas those treated with higher CTB concentrations (60–600 nM) decreased linearly (r2=−0.94, p<0.05; Fig. 2C). Both attached granular cells and plasmatocytes displayed decreasing attachment levels with increasing CTB concentration BTK inhibitor (granular cells, r2=−0.89; p<0.05; plasmatocytes, r2=−0.75; p>0.05; Fig. 2C). Total aggregated hemocytes increased 82% (52.3–59.5) above control levels at 30 nM CTB, followed by a small (p>0.05) decrease at 60 nM CTB, and increased to a maximum plateau [147% (24.5–32.8) above control levels] by 150 nM CTB ( Fig. 2C). This implies that individual CTB may enter cells or perturb the hemocyte cell membrane generating these effects. Increasing CTA concentrations had no effect on

total individually attached hemocytes, granular cells and plasmatocyte attachment, or total aggregated hemocytes ( Fig. 2D) suggesting it about either did not bind to the hemocytes or bound but did not enter the cells. CTX at high concentrations (12–120 nM) produced adhering total hemocyte counts statistically similar (p>0.05) to higher concentrations of CTB (30–300 nM) indicating CTB may explain higher CTX results. For granular cells, CTB did not change counts until 30 nM after which the concentration of adhering granular cells declined, and CTX effects from 12 to 120 nM were similar to CTB (60–600 nM). Plasmatocyte adhesion at the higher

levels of CTX (6–120 nM) and CTB (30–600 nM) were identical (p>0.05). CTX elevated the total number of cells that formed in vitro microaggregates, but CTB had no effect until 30 nM, elevating total aggregated hemocytes to the CTX values (6–120 nM). Collectively the data established a modulatory role for CTX on the hemocyte that is linked, in part, to CTB. However, it is not known if the CTB effects are directly on each hemocyte type or indirect with the granular cells modifying the plasmatocytes as in M. sexta, wherein granular cells-released proteins stimulate plasmatocyte activity [50]. The sum total of adhering individual hemocytes and total aggregated hemocytes was statistically the same (p>0.05) with increasing CTX treatment except for a decline at 1.2 nM CTX ( Fig. 2E), which may reflect reduced adhesion, hemocyte detachment or lysis at this concentration.