Human granulocyte adhesion to glass is inhibited by increasing intracellular cAMP concentrations [12]. In bivalve mollusks, hemocytes with elevated cAMP do not attach to foreign surfaces [16]. LPS-stimulated amoebocyte exocytosis and spreading is inhibited by intracellular cAMP-elevating drugs in non-insectan arthropods [7]. In lepidopteran insects, hemocyte adhesion to glass and bacteria and phagocytosis of bacteria are inhibited by increased intracellular cAMP and cAMP-activated protein kinase A (PKA; [11], [45] and [34]). Eicosanoid-stimulated G proteins are involved in lepidopteran hemocyte–hemocyte interactions including bacterial-induced microaggregations

by activating adenylate Ion Channel Ligand Library cost cyclase [47] and [68]. Cholera toxin (CTX) is used in insects to examine a myriad of cellular activities

including gene expression [10], and cAMP-mediated signaling in fat body tissue [75] and Ca2+-channels [57]. The link of cAMP to insect hemocyte–hemocyte interactions including microaggregation and nodulation is not known. However, CTX acts as an adjuvant with vertebrate immune systems [29] and in view of the physiological similarities of lepidopteran hemocyte to innate mammalian immunocytes ON-01910 purchase including human neutrophils [9] and that the B-subunit elicits raft formation on M. sexta hemocytes [50], it is likely that CTX and its moieties, possibly through cAMP mediation, would influence insect hemocyte–hemocyte interactions including microaggregation

and nodulation of G. mellonella. This Meloxicam communication shows that cholera toxin and its B-subunit at levels less than those traditionally used in insect studies has a concentration-induced bimodal effect on in vitro hemocyte microaggregation and in vivo bacterial removal and nodule formation. These responses may be independent of intracellular cAMP. RGD peptide inhibition of cholera toxin-induced microaggregation suggests integrin mediation. G. mellonella larvae were raised at an ambient incubator temperature of 28 °C (producing a dietary temperature of 37 °C due to insect metabolism) under constant light conditions on a multigrain diet supplemented with glycerol and vitamins [27]. Fifth instar larvae weighing 250±10 mg were used for all experiments. All hemocyte experiments, in vitro and in vivo, were conducted at 37 °C reflecting temperatures in G. mellonella-occupied honeybee colonies [66] and in pure G. mellonella colonies [14]. Stock cultures of Gram-positive Bacillus subtilis (Boreal Biological, Mississauga, Ontario, Canada) were grown in darkness on Luria agar (1 g NaCl, 1 g yeast extract, 2 g bactotryptone, 3 g agar; 200 ml water) at 25 °C and subcultured every fortnight. For experimental purposes, B. subtilis was added to Luria broth (5 ml) in scintillation vials (20 ml) and incubated overnight on a horizontal gyratory shaker (25 °C, 200 rpm).