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

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.     

Dysfunction of heterotrimeric kinesin-2 in rod photoreceptor cells and the role of opsin mislocalization in rapid cell death

Due to extensive elaboration of the photoreceptor cilium to form the outer segment, axonemal transport (IFT) in photoreceptors is extraordinarily busy, and retinal degeneration is a component of many ciliopathies. Functional loss of heterotrimeric kinesin-2, a major anterograde IFT motor, causes mislocalized opsin, followed by rapid cell death. Here, we have analyzed the nature of protein mislocalization and the requirements for the death of kinesin-2-mutant rod photoreceptors. Quantitative immuno EM showed that opsin accumulates initially within the inner segment, and then in the plasma membrane. The light-activated movement of arrestin to the outer segment is also impaired, but this defect likely results secondarily from binding to mislocalized opsin. Unlike some other retinal degenerations, neither opsin–arrestin complexes nor photoactivation were necessary for cell loss. In contrast, reduced rod opsin expression provided enhanced rod and cone photoreceptor survival and function, as measured by photoreceptor cell counts, apoptosis assays, and ERG analysis. The cell death incurred by loss of kinesin-2 function was almost completely negated by Rho^−/−. Our results indicate that mislocalization of opsin is a major cause of photoreceptor cell death from kinesin-2 dysfunction and demonstrate the importance of accumulating mislocalized protein per se, rather than specific signaling properties of opsin, stemming from photoactivation or arrestin binding.     

Evidence for apoptosis correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus

Histological, cytochemical and ultrastructural changes in giant black tiger shrimp Penaeus monodon were investigated at various time intervals after injection with yellow head virus (YHV). Hemocytes, lymphoid organs (LO) and gills were the main focus of the study. After injection with YHV, onset of mortality varied from 36 h onward. By normal hematoxylin and eosin staining, the 3 tissues showed clear and increasing prevalence of nuclear condensation, pyknosis and karyorrhexis from approximately 36 h post-injection (p.i.) until death, although pathology was evident in the LO as early as 12 h p.i. in some shrimp. By nuclear DNA staining with 4',6-diamidino-2-phenylindole (DAPI) and by specific labeling of 3'-OH ends of nuclear DNA using a technique called terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells of the 3 tissues showed evidence of chromatin condensation and DNA fragmentation, respectively. Both are generally considered to be characteristic of apoptosis. In addition to TUNEL labeling, evidence for DNA fragmentation was supported by the appearance of approximately 200 base pair DNA ladders at approximately 48 h p.i. in hemocytes of YHV-infected but not uninfected shrimp. Transmission electron microscopy (TEM) of LO tissue revealed features of apoptosis in tissues of YHV-infected shrimp only. These included marginated, condensed and fragmented chromatin without concurrent cytoplasmic damage. Histological, cytochemical, ultrastructural and biochemical data were consistent with the hypothesis that widespread and progressive apoptosis occurred in susceptible shrimp infected with YHV. Although no specific tests were carried out to determine whether this purported apoptosis was the cause of mortality, moribund shrimp had extensive deterioration of vital tissues such as the hemolymph, gills, heart and LO, suggesting that many essential bodily functions had been severely compromised. This probably resulted in the gross signs of lethargy and weakness seen, and it is reasonable to suggest that further, progressive deterioration could have led to the collapse of vital functions followed by death.