Morphology and Cytochemistry of Hemocytes and Analysis of Hemolymph from Melanoplus sanguinipes (Orthoptera: Acrididae)

Abstract

The hemolymph and the hemocytes from the migratory grasshopper, Melanoplus sanguintpes (F.), were characterized. Ten-d-old male grasshoppers had similar hemolymph osmolality (375 m3sm/kg) and pH (7.6) compared with female grasshoppers. However, females had twice the concentration of hemolymph protein and carbohydrate compared with males. Furthermore, SDS-PAGE separation of hemolymph proteins showed sexual differences in the number and quantity based on the intensity of staining of protein species. Various buffers were evaluated for effectiveness in maintaining hemocyte stability. In some buffers, the hemocytes lysed, clumped together, or were osmotically unstable, A physiologicallyisotonic saline buffer, which maintained hemocyte integrity for up to 30 min, was formulated. This buffer consisted of 1.5 m M K2HPO4, 8.0 m M NaH2PO4, 0.9 m M CaCl2, 2.7 m M KCl, 0.5 m M MgCl2, and 0.3 m M NaCl. Two major types of hemocytes were observed in M. sanguinipes : granulocytes comprised ≈95% of the total hemocyte population and plasmatocytes constituted the remainder. Scanning electron and phase-contrast microscopy revealed that granulocytes were round in shape and had a central nucleus and a granular cytoplasm. Plasmatocytes had a larger nucleus than that of granulocytes and the cells were spindle-shaped. The two types of hemocytes could not be distinguished from one another when the hemocytes were exposed to enzymatic or nonenzymatic cytochemical stains. The granulocytes of grasshoppers that had been previously injected with conidia of the entomopathogenic fungus Beauveria bassiana were observed to lyse and exude mucopolysaccharide strands to which conidia, melanized particles, and other granulocytes adhere.