Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro

Abstract

Hemocytes from the red swap crayfish Procambarus clarkii were classified into three subpopulations: hyalinocytes (H), semigranulocytes (SG) and granulocytes (G) by morphologic observation and flow cytometry (FCM). The primary hemocyte culture was then established for studies on the in vitro propagation of Spiroplasma eriocheiris isolated from naturally infected P. clarkii. Grace's insect medium supplemented with 15% fetal bovine serum (FBS), along with 100Uml−1 penicillin, 100μgml−1 streptomycin, with a final pH of 7.2–7.4, incubated at 28°C, supported the best survival in primary cultures. However, the granulocytes dehisced rapidly in culture medium, potentially impacting the survival of the other cell types. A two-step density gradient centrifugation with Percoll was developed to separate the hemocytes. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability (>75%) after 16days of incubation compared with granulocytes, which degraded over 2–6days. After a challenge with S. eriocheiris, cytopathic effects (CPE) of the cultured hemocytes were observed as early as 48h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates in inoculated cultures. Cell lysis was noticeable within 60h after challenging. A quantitative real-time RT-PCR was conducted to detect the immune responses during the challenging process at 2h, 4h, 8h, 10h, 24h, 48h and 60h, respectively. Clear time-dependent expression patterns of the peroxinectin gene (referred as Pcpxin), recently isolated from the crayfish hemocytes, and heat shock protein 70 (HSP70) gene were observed after S. eriocheiris challenge. The results above should be helpful in promoting research with S. eriocheiris, including elucidation of pathogenesis, host pathogen interaction and the defense mechanisms, and ultimately lead to prevention of this crustacean disease.