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Diseases of Aquatic Organisms

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DAO 55:3-10 (2003)  -  doi:10.3354/dao055003

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus

Kanokpan Wongprasert1, Kornnika Khanobdee2, Supatra Somapa Glunukarn1, Prasert Meeratana3, Boonsirm Withyachumnarnkul1,4,*

1Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
2Neurobehavioral Biology Center, Institute of Science and Technology for Research and Development, Mahidol University at Salaya, Nakhornpatom, Thailand
3Department of Medical Science, Faculty of Science, Burapha University, Chonburi, Thailand
4Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
*Corresponding author. Email:

ABSTRACT: This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4,6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.

KEY WORDS: Apoptosis · WSSV · TUNEL · DAPI · Penaeus monodon · White-spot disease · Caspase-3

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