To better understand the molecular responses elicited by WSSV infection in juvenile Pacific white shrimp, Litopenaeus vannamei, as well as, a possible effect of the cultivation conditions on such responses, we addressed the transcription of target genes in experimentally infected shrimp kept in biofloc (BFT) and in clear seawater (CSW).           It is essential to keep in mind that outbreaks of diseases in shrimp farming do not occur solely because of the presence of a given pathogen; usually the cause is multifactorial. Onset, susceptibility and evolution course of diseases are influenced by a multitude of factors, such as environment conditions, nutrition, genetics and animal physiology (Cuéllar-Anjel, Corteel, Galli, Alday-Sanz & Hasson 2014).

            Recent studies have suggested that BFT can enhance animal immunity, and, further, that shrimp growing in this system can be related to a higher survival, growth and expression of immune-related genes (Ekasari, Azhar, Surawidjaja, Nuryati, Schryver & Bossier 2014; Kim, Pang, Seo, Cho, Samocha & Jang 2014).

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Despite these reports, we observed higher cumulative mortality among animals kept in BFT in comparison with those kept in CSW, after WSSV challenge. Our findings may be partially attributed to the conditions of the viral challenge we established, e.g., use of a medium level viral load (Souza 2008), aiming to establish an acute infection (72 – 96 h), as well as WSSV virulence. The mentioned studies either did not include any pathogen challenge, or the challenge of shrimp was performed with a different virus, e.g., infection myonecrosis virus (IMNV).

On the other hand, it should be taken into account that in a rich environment, such as biofloc, which displays a very abundant number of diverse bacteria and other microorganisms, it would be possible to consider that defense responses, including those related to shrimp immune system, are being held on a constant stimulated level, maybe even close to a threshold limit. Such situation could either facilitate the triggering of a more intense and/or integrated response or lead to a certain limitation or wear out of the defense system upon the challenge of coping with a virulent pathogen under such scenario.

            Therefore, one could speculate that upon a greater immune challenge represented by highly virulent pathogen, such as WSSV, the immune system can no longer be so effective in protecting the host against the infection, and high mortalities succeed.  On the other hand, one could consider that shrimp kept in CSW are not facing such constant stimulation of the defense responses and, consequently, this could partially explain our observation regarding the higher survival in this experimental group. This hypothesis is also supported by the lower viral load observed in shrimp kept in CSW at 48 h p.i., as well as, the higher viral replication observed in shrimp from BFT at 48 h p.i.. In other words, when facing a virulent pathogen, shrimp from BFT seemed not able to deal with the challenge, failing or lowering the level of an adequate defense response.

            Despite the postulate that geographic isolates of WSSV do not mutate frequently, due to the DNA nature of its genome (Cuéllar-Anjel et al. 2014), differences in virulence among such isolates have been already reported.  Rahman, Corteel, Escobedo-Bonilla, Wille, Alday-Sanz, Pensaert, Sorgeloos & Naumynck (2008) showed that three different geographical WSSV isolates, two from Thailand and one from Vietnam, differed in terms of virulence when infecting juvenile L. vannamei. The most virulent isolates were those from Thailand, since mortality began at 36 h p.i., whereas for the isolate from Vietnam mortality was recorded at 36 h-60 h p.i..  Based on our data concerning the first recorded mortality at 24 h p.i., and also the cumulative mortality at 48 h p.i., one can consider our isolate as highly virulent. Therefore, differences in WSSV virulent behavior may be related to subtle variations in the viral genome, as we have previously shown in a comparative analysis of target genomic  regions among the Santa Catarina (SC-WSSV) and other WSSV geographical isolates (Müller, Andrade, Tang-Nelson, Marques & Lightner 2010).

            Based on our results relative to viral load quantification, infection behavior along time, a distinct individual pattern of response to infection was seen.  This finding was evidenced by the sole shrimp survivor in BFT at 72 h p.i.. Interestingly, this shrimp did not present any visible clinical signs. Viral infections in crustaceans can be characterized by being persistent and by showing extremely low viral load after surviving the acute phase (Cuéllar-Anjel et al. 2014). In our study, the only survivor shrimp at 72 h p.i. in BFT could be considered as an asymptomatic carrier, since its viral load increased along the 72h period of infection. Nonetheless, this viral load was the lowest one when comparing the viral load range among other shrimp, independently of the systems animals were kept in, CSW or BFT. Therefore, this sole shrimp could be considered as being less susceptible to the experimental WSSV infection. Although we cannot affirm that the 72 h p.i. survivor shrimp from BFT would further behave as a persistent infected host, as well as, an asymptomatic carrier, we could speculate that it would be much likely. This observation can point out to the potential importance of not only addressing host defense response toward pathogens, but also host genomic molecular markers and genetic diversity, as well.

            As expected, a lower level of total viral particles was detected in water samples in comparison to shrimp tissue. Since viruses can remain infective in water for a long time, production water being returned to the environment without treatment to eliminate these particles can contribute to the outbreak of viral diseases. It is interesting to note the detection of viral particles in the biofloc sediment. This finding emphasizes that virus may remain in the sediment, which represents a further source of infection for successive production cycles. In terms of virological safety, waterborne transmission means a threat to animal health, especially for animals in the molting phase, when they become even more susceptible to be infected by this route (Corteel, Dantas-Lima, Wille, Alday-Sanz, Pensaert, Sorgeloos & Nauwynck 2009).

            The identification of potentially WSSV responsive genes involved with stress and immune response in L. vannamei, after viral infection and under different cultivation conditions, was addressed here in order to contribute to a more comprehensive biochemical and molecular panel of the host defense strategies against viral infection.

            Considering the establishment and persistence of WSSV infection, we addressed different tissues to compare the transcription levels of the selected target genes.  Interestingly, we observe few significant differences in the transcription profile of the selected genes in hemocytes between WSSV challenged shrimp and not infected animals. On the other hand, several significant differences in gene transcription were seen in gills and hepatopancreas. Gills are expected to be more exposed and in constant contact with the surrounding water, whereas hepatopancreas play a definite roll in the natural infection through oral route.

A fact to take into consideration is the time of infection we performed in the present study. Duan, Liu, Li, Wang, Li &  Chen (2014) showed that the expression profile of calreticulin, analyzed in hemocytes and hepatopancreas of the white prawn Exopalaemon carinicauda, was up regulated in WSSV and Vibrio anguillarum challenged shrimp at 6 h and 12 h p.i. In case of the present study, genetic expression could be changed prior to mortality.

            WSSV is an enveloped virus, with a double-stranded DNA genome and can replicate in tissues of ecto- and mesodermal origin of a wide host range, and probably also have a very stable genome (Cuéllar-Anjel et al. 2014). Arts, Taverne-Thiele, Savelkoul & Rombout ( 2007) demonstrated that in P. monodon, after 48 and 72 h p.i. through immersion, a significant number of gill cell nuclei are infected with WSSV, and at the same time more hemocytes were observed in the gill of infected animals.

            Many of our results concerning gene transcription may be understood as a direct consequence of viral action against the host cell. Several cellular pathologies caused by virus-cell interactions are known. Some examples among such interactions, include interfering on transcription mechanism, processing and mRNA transport; inhibition of mRNA translation; and inducing the cell to enter in apoptosis (Murphy, Gibbs, Horzinek & Studdert 1999).

            Programmed cell-death is a response against a pathogenic infection, and apoptosis is one of cellular processes responsible for the shrimp death (Flegel, 2007) during WSSV infection, since the virus is capable to trigger the cascade of reactions that lead to apoptosis of the host cell. The undertaking of this strategy can be suggested as well in the present study, since WSSV infection elicited an increase in the transcription level of QM, in all of the analyzed tissues, as well as, in EF-1? transcription level in gills in CSW.

            During an active infection, viral replication produces new viral particles that need to leave infected cell. SEC 61 showed an increase in the number of transcripts in gills of shrimp from CSW and also in hepatopancreas from both system, BFT and CSW. Possibly this enhance was due to the increased quantity of viral protein in the cell, using the channel as a transport gate towards virus particle assembly. Likewise, ? tubulin displayed the same profile in hepatopancreas.

            According to Vidya, Gireesh-Babu & Prasad (2013), WSSV modifies the host ubiquitination and redirects the host immune proteins in order to improve the progressing of viral diseases. Viruses can interfere with cellular mechanisms, such as protein modification, place and maturation (Murphy et al. 1999). In our study, we observed an up regulation of ubiquitin in all tissues of shrimp kept in BFT, indicating that, at least in the conditions provided by this system, host ubiquitin gene transcription is modulated/affected by WSSV infection.

            Duan et al. (2014) observed difference in the transcription level of calreticulin at 6 h and 12 h p.i. Additionally, Gao, Tian, Huang, Yao, Xu & Guo (2016), in a study of hypo-osmotic stress in L. vannamei, found an increase in this gene expression in hepatopancreas and hemocytes, at 6 and 12 h p.i. respectively, followed by a corresponding decrease at 12 h and 24 h, but not in intestines and gills. In our study, calreticulin transcription level only showed difference in hepatopancreas of BFT, at 48 h p.i. 

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