Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Abstract

Primary cultures of hemocytes from the Caribbean spiny lobster Panulirus argus were developed for studies on the in vitro propagation of Panulirus argus Virus 1 (PaV1). A modified Leibovitz L-15 medium supported the best survival of hemocytes in in vitro primary cultures. However, degradation of the cultures occurred rapidly in the presence of granulocytes. A Percoll step gradient was used to separate hemocytes into three subpopulations enriched in hyalinocytes, semigranulocytes, and granulocytes, respectively. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability (∼80%) after 18 days incubation compared with granulocytes, which degraded over 2–3 days. Susceptibility of the cell types was investigated in challenge studies with PaV1. Hyalinocytes and semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were observed as early as 12 h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 24 h of infection. The presence of virus within cells was further confirmed by in situ hybridization using a specific DNA probe. The probe gave a unique staining pattern to cells infected with PaV1 24-h post-inoculation. Cells in the control treatment were intact and negative to hybridization. This assay was further applied to the quantification of infectious virus in hemolymph using a 50% tissue culture infectious dose assay (TCID 50) based on CPE. These tools will now allow the quantification of PaV1 using established culture-based methods.