Survival of L. vannamei
Up to 24 h p.i., no death was recorded in any of the tanks, although some clinical signs could be clearly observed, such as lethargy, reddish body, prostration, and low feed consumption. After 24 h, clinical signals were intensified and animals from both systems started to die.
Shrimp survival rate in the two different systems was significantly different, i.e., a higher number of shrimp died in biofloc. Moreover, deaths took place more rapid biofloc in comparison to clear seawater. At 72 h, a small number of animals with subclinical infection (asymptomatic shrimp) was observed. Cumulative mortality was higher at 48 h p.i. in comparison to 72 h p.i.
Furthermore, only animal survived to WSSV challenge in biofloc for 72 h p.i. Interestingly, it showed no detectable clinical signs. Data of survival (%) of L. vannamei are presented in Figure 2. It is noteworthy that no shrimp has died along the same period (72h) in both control groups and no difference was observed in shrimp final body weight (10.36 g ± 0.18 in biofloc; 10.98 g ± 1.28 in clear seawater).
Viral quantification by RT-qPCR
Quantification of WSSV in pleopod samples was higher in animals kept in biofloc system, at 48 h p.i. On the other hand, it is noteworthy that, in the only infected individual that survived in the biofloc up to 72 h p.i., viral load showed to be comparatively lower. This finding paralleled with the observation that this individual was one of the few survivors with no visible clinical signs. Moreover, no difference in viral load was seen between 48 h and 72 h p.i. in shrimp kept in clear seawater (Table 2).
Target gene transcripts
Comparing pre and post-infection gene transcription profile, a significant difference was observed when comparing infected and non-infected shrimp.
?-tubulin, Calreticulin, and proPO1 were differently expressed only in hepatopancreas. On the other hand, EF-1?, QM, SEC 61, Ran, SOD, and Ubiquitin were differently expressed in different tissues.
Transcript levels in H1and GAPDH showed no difference between infected and non-infected animals. Considering the latter, our results corroborated the common use of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) as internal control or reference gene (Dhar, Bowers, Licon, Veazey & Read 2009), since it showed no difference in transcription level, independently either of the assessed tissue or the system shrimp were kept.
Differences in gene transcription level between WSSV challenged and not challenged shrimp were much greater in the case of genes that showed to be up regulated. Moreover, higher differences in transcription levels were more evident in hepatopancreas of shrimp kept in biofloc. Overall, hemocytes presented no observable transcription level changes of the selected target genes (Table 3).
Comparing transcription profile among systems, differences were observed only after viral infection, i.e., differential transcription between CSW versus BFT raised animals were only post infection. After WSSV infection, gills from CSW showed more differences in transcript levels, and always up regulated. Calreticulin, GAPDH and H1 genes presented the same level of transcription in all infected animals, even comparing animals kept in two different experimental systems. QM, proPO1, and SEC 61 genes were highlighted, with high up regulation (? 50 x) after viral infection, in their respective system and tissue.
WSSV infection interfered similarly between systems (BFT vs. CSW) as well as among systems (pre infection vs. post infection), particularly in gills. However, in hemocytes, this similarity was not observed. Figure X shows levels of transcripts with significant difference, pointing to tissue and system.