More directly involves with antioxidant and immune response in shrimp, we observed different transcription level of SOD, and proPO1, particularly in hepatopancreas, although in the case of proPO1only in this tissue in shrimp kept in BFT. In L. vannamei, differences in SOD transcription or in the activity of its corresponding enzymatic product have been reported. Hsieh, Ruan, Li, Hsieh, Hu & Kuo (2008) demonstrated that SOD activity decreased after 12 h p.i. with Vibrio alginolyticus. On the other hand, Chiu, Guu, Liu, Pan & Cheng (2007) observed an opposite result also upon bacterial challenge, when shrimp were fed with diet containing Lactobacillus plantarum. Moser (2011) showed that, after exposure to permethrin, transcription of SOD in L. vannamei gills was inhibited after 6 and 12 h after exposure, but increased after 48 h and 72 h of exposure to the pesticide. In addition to these studies, our findings can be taken as indicative of SOD response being a defense response elicited upon viral challenge and corroborating this cellular response as a general indicator of stress (Smith et al. 2014). Moreover, our results could be pointing out that shrimp phagocytic cellular process could be more active at 48 h p.i., reflecting in a more intense production of ROS.
Associated with host defense response against infection are proPO1 and H1. Prophenoloxidase activating system recognizes bacteria and fungi (Smith et al. 2014), and is one of the most important immune response in crustacean. Different to our results, Yeh, Chen, Hsieh, Cheng & Liu (2009) showed that L. vannamei infected with WSSV or with WSSV in a co-infection with IHHNV (Infectious Hypodermal and Hematopoietic Necrosis Virus) had a decrease in the transcription level of proPO. Moreover, authors suggested that one of the causes of mortality in shrimp was the WSSV infection itself, plus the suppression in some immune-related gene expression, such as proPO.
Indeed, low or absent gene transcription and / or proPO system activity may be critical for effective immune defense, resulting in more susceptible animals to secondary infections and facilitating death by viral infection. Our results indicate that, in most tissues there was no difference in proPO transcript levels, which can be understood as an overall fragile immunological condition in both systems. This evidence can also be corroborated by the absence of difference in H1 transcripts, since histones are involved with the extracellular traps, essential for bacteria clearance (Ng et al. 2013).
It is noteworthy pointing out the exception related to the high transcription level of proPO1 in hepatopancreas of infected animals kept in BFT. This group also presented a high transcription of SOD. Although it was not effective to result in greater survival of shrimp, an increase in proPO occurred in this group. As this route results in toxic molecules, e.g. ROS (Cerenius & Söderhäll 2004), some cellular strategies to reduce excessive ROIs was necessary, such as modulation of SOD transcription.
Seeking to further explore the responses against WSSV infection and the related phagocytosis, we also addressed the transcription profile of Ras-related nuclear gene, the Ran gene. This gene is described as being associated to shrimp antiviral immunity (Han & Zhang 2007). Han & Zhang (2007) showed that its transcription was detected in different tissues, including hepatopancreas, haemolymph, and gills of Penaeus japonicus infected with WSSV; an up regulation was seen at 4 h p.i. Similarly, Qiao, Xu, Wang, Jang, Qi, Zhang & Kim (2015), although they observed some fluctuation in the transcript levels of this same gene along the infection of L.vannamei with WSSV, co-infected with Vibrio anguillarum, an up regulation was seen at 48 h p.i. Interestingly, the authors concluded that transcriptional suppression can be directly related to high mortalities. In the present study, even with an up regulation observed in both gills and hepatopancreas in shrimp kept in BFT, and probably a consequent increase in the phagocytosis process, this scenario was not enough to prevent high mortalities.