Flow cytometry assay shows the percentage of normal cells was high in the control, clearly denoting that the hemocytes are in suitable stage to analyze the apoptosis event (Fig. 1B) after sample preparation. Wongpraset et al [22] described that around 3% of apoptosis occur in haematopoietic tissue at 36h post-infection and as low as at 24h and as high as 20% at 60h in hemocytes of P. monodon against WSSV. In the present study, after 48hrs of WSSV infection in WSSV group early apoptosis was observed 7.6 times higher than the late apoptosis; necrosis was observed at 7.88 times higher than early apoptosis (Fig. 1F & 2). This is because, as viral infection increases rapidly more and more tissues were subjected to necrosis stage.
Early apoptosis was observed to be lower in combined dsRNA (Pmrelish/Rab7/IAP) injected group after WSSV challenge than Rab 7 specific and Relish specific dsRNA individually injected group (Fig. 1C, 1D 1E & 2) which was much higher. Khanobdee et al [23] reported that continuous events of apoptosis occurred leading to mortality of P. monodon against YHV infection. Flegel and Paharawipas [24] suggested that apoptosis might be involved in shrimp mortality at that time of viral infection.
Caspase-3 knock down studies have indicated that shrimp mortality was reduced when challenged with low dose of WSSV [25]. This shows that silencing genes involving in apoptosis failed to control viral replication if the viral copy number are very high paving way for necrosis. WSSV possesses three anti-apoptosis proteins such as wssv449, wssv143 and wssv322, which could inhibit caspase-mediated apoptosis for its replication [26]. In the present study necrosis was found to be very high in combined dsRNA administered group than individual dsRNA adminsitered group, while only WSSV challenged group showed very less necrotic cells (Fig. 1C, 1D, 1E 1F & 2). Necrotic cells might increase because of two reasons; either the administered WSSV copy is higher or the combinely knocked out genes play a critical role in shrimp during infection periods. We found that, the percentage of apoptosis was lesser in Rab7 dsRNA group than only WSSV group, at the same time combined dsRNA group suffered high necrosis than apoptosis. Rab7 was involved not only in endosome maturation and transportation to lysosomes but also in several cellular physiological processes including apoptosis [27] while deprivation of growth factor, Rab7 activation on lysosomal membranes cause’s apoptosis [28].
Relish-dsRNA group showed a very high early apoptosis event as equal to early apoptosis event in only WSSV group. This shows silencing of relish gene has no effect on control of viral infection (Fig. 1D, 1F & 2). PmStat gene expression found up regulated by ie1 promoter of WSSV with the help of STAT binding motif in Penaeus monodon [29]. Viruses like WSSV in crustacean and HBV in human can counteract the host cellular progress such as STAT pathway for its own replication and progressive apoptosis leading to necrosis. IAP was required for cell viability in both shrimp and Drosophila and the mechanism to regulate apoptosis is similar to drosophila [30]. In our study too, dsIAP injected shrimps showed mortality within 48hrs (Fig. 7) which is similar to earlier study [31].
Shrimp stat gene expression found to be increased in response to WSSV infection [29]. An attempt was made in this study to know the status of stat gene expression when Relish/Rab7/IAP genes were silenced. Stat gene expression was upregulated only in gills and to a much lesser extent in pleopod and muscle of Rab7 silenced group (Fig. 4). But in case of Relish and IAP silenced group, stat gene expression was observed to be significantly up-regulated in pleopods and muscles (Figs. 5 & 6). Similar kind of results were observed in stat gene up-regulation in P.monodon when IAP gene was silenced [32]. Phylogenetic analysis revealed that shrimp stat was closely related to ancient stat family of all insects [33]. Silencing of P. monodon stat gene showed decreased level of WSSV ie1 gene expression and copy numbers in significant range, emphasising that WSSV needs host stat genes for its replication [34]. However stat gene is also involved in anti-viral immune response in drosophila against drosophila C virus [35]. Similarly, in Aedes albopictus mosquito cell line the stat gene showed antiviral immune response [36].
In our study, stat gene is not much up-regulated in case of Rab7 silencing but considerably up-regulated in Relish and IAP gene silencing which shows that Rab7 gene has a control over stat gene expression whereas relish and IAP do not. Hence Rab7 silencing leads to reduced mortality which indirectly reduced expression of stat genes that are required for virus replication. Flow cytometry analysis indicated reduction in the percentage of early apoptotic cells in Rab7 knocked down animals compared to only WSSV infection and Relish-dsRNA administered group, suggesting that less number of Rab 7 receptor and low expression of stat gene leading to reduced early apoptosis.
Crustins belong to a family of multi-domain cationic Anti-Microbial Proteins (AMPs) found in hemocytes of numerous crustaceans. There are a number of studies reporting the importance of crustins against bacterial infections [37, 38]. However, crustin’s anti-viral function in shrimps remains unclear even though some studies showed that crustins are involved in anti-viral mechanism [39, 40]. In this study, Rab 7 gene silencing was found to upregulate crustin gene in gills and muscle and on contrary silencing of Relish and IAP gene, had no effect on crustin gene regulation (Figs. 4, 5 & 6). Similarly in FACS analysis, percentage of early apoptotic cells was less when compared to only WSSV and Relish-dsRNA administered group, suggesting that less number of Rab 7 receptor and high expression of crustins leading to less early apoptosis and crustin may act as anti-viral. Earlier studies reported that Pmcrustin5 might be upregulated during the time of stress such as heat and hyperosmotic salinity [41].
In our study, Relish gene-silencing leads to significant upregulation of stat gene and Rab7 gene expression (Fig. 5). Consequently stat gene product would be available for WSSV replication and expressed rab7 receptor for attachment into shrimp cells paving way for more WSSV infection similar to increased susceptibility to YHV infection [42]. FACS analysis too showed high percentage of early apoptosis event for relish-dsRNA silenced group than combined dsRNA and Rab7-dsRNA silenced group. Pmrelish gene and crustin gene found to be upregulated in Rab7 silenced group, supporting that Pmrelish is important in antiviral immunity. Pmrelish also involved in regulation of penaeidin3 and penaeidin5 against YHV infection [42].
Hemocyte count is directly proportional to extent of apoptosis event occurring inside a cell during an infection, as noticed in decrease in total hemocyte count after WSSV infection in P. monodon [22] and in P. japonicus [43]. In our present study, the group administered with IAP-dsRNA suffered mortality in 48hrs (Fig. 7) of dsRNA injection suggesting they are important in maintaining the cell viablility [30]. Also significant Stat gene up-regulation (Fig. 6) might have made the shrimps more prone to infection. IAP1 gene expression was up regulated in knockdown of IMD genes in shrimp against V. anguillarum and M. lysodeikticus infection suggesting that IAP1 may interact with IMD pathway [44].