4.1. Viral challenge and WSSV viral load
Many studies have investigated the presence of viral agents in the aquatic environment, as well as in cultivation systems, where crustaceans and other aquatic organisms may act as vectors (Flegel, 2006, Mijangos-Alquisires et al., 2006). However, relative few studies have considered the possibility of infection of wild animals generated by aquaculture activity itself. In this context, crabs Neohelice granulata collected in the vicinity of shrimp ponds were analyzed for the presence of WSSV and, in case of a positive result, had their viral load determined by quantitative real time PCR, as well. The viral load range fell between 102 to 103 copies.µl− 1. According to Shekhar et al. (2006), Souza (2008), and Walker et al. (2011), the degree of the viral infection severity can be predictable according to the viral load measured in the animals. So, based on the criteria pointed out in their work, wild crabs naturally infected by WSSV showed a light WSSV infection. On the other hand, qPCR results showed that naturally infected shrimp displayed 106 copies.µl− 1, corresponding to a severe WSSV infection.
Regarding the WSSV experimental infection, results showed that the adopted protocol was equally efficient for both crustacean species.
Animals challenged with the virus, showed positive reactions, when analyzed by nested PCR, indicating that the virus was present and, as a consequence, a potential infectious process was established. Moreover, qPCR analysis in previously nested PCR WS-positive individuals has revealed that WSSV was capable of replication within the tissues of shrimp, as expected, as well as in crabs. This is the first report of experimental infection in N. granulata crabs. Furthermore, to date, no work has shown the proliferation of the virus, based on the quantification of viral load through the time course of infection. An effective injection of WSSV into blue crab Callinectes sapidus has been reported by Blaylock et al. (2019) and in the mud crab Scylla serrate by Liu et al. (2011), both as examples of commercial species of interest for aquaculture. N. granulata investigated in the present study occurs widely throughout the state of Santa Catarina and is abundant in nurseries and cultivation pond areas, being part of the fauna accompanying and surrounding the farms (Marques et al., 2011). N. granulata has no commercial interest, unlike the blue crab. However, it can be considered as an emergent animal model for biochemical, physiological and ecological research (Spivak, 2010), besides comparative studies related to pathogenic infections in crustacea.
The qPCR results in gills of WSSV-challenged animals confirmed that the number of WSSV viral copies increased over time. Surprisingly, no clinical signs were seen in crabs along the progress of infection. Nonetheless, based on viral load results we can infer that the N. granulata crabs were effectively infected through laboratory viral challenge. Therefore, this crustacean specie serves as asymptomatic carriers of WSSV, and probably other viruses of high-prevalence, without developing the disease, which represents a potential source of reinfection of farmed shrimp and maintenance and spread of the pathogenic agent in the environment.
Viral particles present in water, sediment and vectors may represent important reservoirs and source of contamination, reintroducing diseases and making future crops unfeasible. According to OIE (2016) all crustacean species are potential hosts for WSSV. In addition, insects (Lo et al., 1996a), rotifers (Yan et al., 2004) and polychaetes (Vijayan et al., 2005) also function as vectors of the disease. These organisms can act as important vectors in the transmission of WSSV to shrimp, both in crops and in the wild. Thus, the presence of crabs in shrimp farms should be strongly avoided to disrupt this flow between infected and uninfected animals (Marques et al., 2011). An alternative to prevent the entry of larvae and juveniles of these vectors into nurseries is the use of 150–200 micron mesh (Balasubramanian et al., 2018). Chlorine water treatment also appears to be effective in eliminating vectors, such as crab larvae and copepods (Fegan and Clifford, 2001). Additionally, the use of systems without water exchange is a measure that reduces the risk of disease introduction (Pruder, 2004; Hasan et al., 2020).
However, Fegan and Clifford (2001) and OIE (2016) state that although water may act as an important vehicle for the spread of viruses in the aquatic environment, a high viral load is required for the animal to become infected and ill. Water transmission of WSSV has been demonstrated in different experiments. However, most of them were done with high viral titrations or unnatural proximity of infected to uninfected animals. In our study, crabs were collected inside and in the vicinity of the shrimp farms and their viral loads were measured by qPCR. Although data in the literature demonstrate high mortality as a consequence of WSSV infection, it appears to be dependent on both free virus concentration in water and the health status of exposed animals. Along with the fact that the virus does not remain viable outside a host for more than a few days, it indicates that the risk of water transmitting WSSV is considered lower than previously believed, except when water is discharged with a high viral load during a period of outbreak (Fegan and Clifford, 2001). It is not clear whether crabs were infected by WSSV-positive farmed shrimp or, on the contrary, were responsible to spread the virus to shrimps. However, once crabs are asymptomatic vector of WSSV, an understanding of the reasons for differences in mortality from equally heavy viral loads may allow us to develop strategies for limiting mortality from viral pathogens in shrimp aquaculture (Kanchanaphum et al., 1998; Marques et al., 2011; Bateman and Stentiford, 2017).
So far, evidences regarding whether N. granulata crabs show some kind of resistance to WSSV due to the ability of either clear out the virus, inactivate it, or even slow down its multiplication is not yet possible to say. We observed that equal viral loads promote differences in some molecular responses between shrimp and crabs, as seen in the transcript level of some target genes. So apparently crabs may modulate some molecular pathways or adaptive strategies to avoid the drastic effects of WSSV infection, while shrimp succumb faster to this viral pathogen. Comparatively, viral loads, in both species, were different along the course of infection. WSSV replicated more rapidly in shrimp when compared with crabs. Moreover, our results show that in general terms crabs were less susceptible to WSSV.
4.2. Differential expression in N. granulata and L. vannamei related to the same viral load
Our gene transcript findings showed statistical differences in crabs and shrimp at the same viral load, pointing out similar potential effects of WSSV infection in molecular processes in both species, such as apoptosis, ions storage, and involvement of chaperone proteins. Along the progress of infection, a wide range of molecular interactions occur between WSSV and its host cells. These molecular interactions play an important role in determining host susceptibility to the pathogen.
Verbruggen et al. (2016) have listed a variety of molecular mechanisms that respond and even prevent WS host infectivity. These mechanisms involve several pathways, including some of genes we targeted in the present study. Genes associated with chaperone and apoptosis pathways, such as CALC, IAP, HSP 70, H2a and UBQ, and others associated with phenoloxidase activation, such as PHEN, QM and FERR were cleared induced in both, shrimp and crabs. Upon reaching the threshold of 106 viral copies, all the shrimp died while the crabs remained alive, showing no visible signs of disease. Despite the antiviral responses caused by the shrimp cells, this molecular defense was not enough to keep the host alive.
Apoptosis is considered an important cellular defense mechanism that inhibits viral multiplication and eliminates infected cells in multicellular organisms (Everett and McFadden, 1999). Leu et al. (2013) propose a model for apoptotic interaction between WSSV and shrimp through the activation of signaling pathways that lead to (1) the expression of pro-apoptosis proteins, like caspase modulators; and (2) mitochondrial changes. In a few cases where viruses intentionally induce apoptosis to release progeny virus, the inhibitor of apoptosis proteins (IAP) plays an important role in both apoptosis and innate immunity. The IAP proteins are considered as strict regulators of caspase/apoptosis activity and are influenced by viral infections. Kulkarni et al. (2014) state that in Penaeus monodon when IAP expression was present, caspases level decrease. Comparative analyses in our study, between WSSV-challenged groups, showed a decrease of CASP-3 levels in WSSV-infected shrimp, and a decrease of CASP-2 and CASP-3 levels in WSSV-infected crabs. To manipulate host apoptosis, WSSV modulates the expression of apoptosis-related genes, such as caspase and fortilin, as described to shrimp Marsupenaeus japonicus, Penaeus monodon and to crab Eriocheir sinensis and Cherax quadricarinatus, to actively promote apoptosis to spread virus progeny to neighboring cells (Wang et al., 2013; Qu et al., 2018; Li et al., 2019).
Further, HSP60 is considered a pro-apoptotic protein, whereas HSP27, HSP70, and HSP90 proteins are predominantly anti-apoptotic (Murthy and Ravishankar, 2016). In L. vannamei, HSP family was consistently or specifically expressed in response to thermal or pH stress, heavy metal exposure (Qian et al., 2012), besides viral infection stress. According to Valentim-Neto et al. (2014a), shrimp positive to Hypodermal and Hematopoietic Necrosis Virus (IHHNV) or WSSV with changes in HSP 70 expression levels had a higher rate of survival during the period of cultivation. On the other hand, we observed that in crab and shrimp with severe WSSV infection, transcripts of HSP 70 showed to be up-regulated, as expected, since a role in shrimp antiviral response has been attributed to this gene (Moser and Valentim-Neto, 2020). Perhaps viral infections, such as that caused by WSSV, may cause similar tissue and protein damages as those attributed to heat shock stress. On the other hand, the up-regulation of HSP 70 genes may act to repair protein damages and may play an additional role as signaling molecules to modulate the innate immune response in host shrimp (Janewanthanakul et al., 2019). We also observed a decrease in HSP60 transcript at 24 to 48 hpi, when the WSSV load raised from 101 to 106 copies.µl− 1. In crabs, WSSV-infection increased slower between 96 to 120 hpi. Sun et al. (2013) also report that HSP 60 expression inhibited in gills of WSSV-infected L. vannamei. Zhou et al. (2010) reported a significant HSP 60 expression in L.vannamei gills after Vibrio alginolyticus challenge followed by a decrease to normal levels after 24 h.
The transcript levels of ubiquitin (UBQ) were up-regulated in WSSV infected shrimp and crabs at the same viral load. Our results also showed that levels of ubiquitin transcripts varied depending on the WSSV post-infection time interval, as well as the viral load, both in cabs and shrimp. Vidya et al. (2013) suggested that one of the viral strategies to keep progressive infection involves modifying host ubiquitination. Protein degradation pathway is the most well-studied aspect of the ubiquitin-proteasome system; protein ubiquitination is also responsible for regulating cell signaling by controlling the endocytosis. In addition to the protein degradation pathway, ubiquitin also participates in other cell functions, such as activation of immune cells (Ben-Neriah, 2002) and apoptosis (Leu et al., 2013). As ubiquitin is a protein that acts in several processes the data found in the current literature refer to both a viral pro-infection function induced by WSSV, as well as a possible anti-viral function produced by the host itself. An induction of UBQ has also been seen in other studies of shrimp infected with WSSV (He et al., 2009; Wang et al., 2006 a). Levels of ubiquitin were up-regulated in WSSV-challenged L.vannamei after 24 hpi in comparison to non-infected shrimp, whereas after 72 hpi the same protein was present only in infected shrimp (Valentim-Neto et al., 2014a). Viral load was also monitored in the same study and ranged from 8.38x101 (24 hpi) to 7,79x105 copies (72 hpi). Further, Chen et al. (2016) report that in shrimp P. monodon injected with two viral proteins with the purpose of gaining resistance against WSSV, the list of up-regulated protein spots found exclusively in WSSV-vaccinated shrimp included ubiquitin, calreticulin and HSP 70. All together, these findings show the important role of ubiquitin along the WSSV infection, as well as the involvement of stress proteins and proteins related to calcium metabolism, calreticulin (Chen et al., 2016) and calcitonin-like (Valentim-Neto et al., 2014a).
The histone H2ɑ has been recently implicated in the apoptosis process and identified among the participant proteins in WSSV infection studies (Encinas et al., 2019; Wang et al., 2008). The histone H2ɑ is responsible for the packaging and compactation of nuclear DNA and plays an important role during the regulation of genes at the nuclear level. In addition, H2ɑ displays a high diversity of variants that are involved in apoptosis DNA repair, gene regulation, and genome integrity (González-Romero et al., 2012). Wang et al. (2008) showed that ICP11, a WSSV protein, bound with histone proteins in the cytosol of WSSV-infected hemocytes and HeLa cells, prevent them to participate in nucleosome assembly, inducing incidental apoptosis. Our results show that H2ɑ gene is up-regulated in crabs and shrimp at the same viral load. But, interestingly enough, we observed a significant higher transcription before viral load achieve such level (106 copies.µl− 1), that is, at 24 hpi in shrimp and at 96 hpi in crabs. These results corroborated with Encinas et al. (2019) that reported that maximum levels of expression of this protein were reached at 12h post-WSSV-infection in L. vannamei, declining sharply after that. Feng et al. (2014a) observed that H2ɑ transcript levels in WSSV-challenged Fenneropenaeus chinensis displayed peaks of expression at 6 and 36 hpi. These results indicate an early response of histone protein against WSS|V, as long as the viral infection is considered mild or moderate.
Under severe WSSV-infection our study identified the up-regulation of QM in crabs, as in shrimp further implicating the role of the product of this gene in defense reactions in crustaceans. Xu et al. (2008) demonstrated that the M. japonicus QM protein could regulate the phenoloxidase activity by interaction with hemocyanin, suggesting the involvement of QM protein in the prophenoloxidase (proPO) activation system in shrimp immunity. Liu et al. (2014) report that QM transcripts in L. vannamei were significantly increased after challenge with Vibrio anguillarum. Likewise, an up-regulated expression on phenoloxidase (PHEN) was observed in our study in response to WSSV in both crustacean species. On the other hand, the expression of proPO in the WSSV-infected shrimp was not significant, even considering that proPO was primarily expressed in hemocytes and many tissues infiltrated by hemocytes, like gills, and has been well described as shrimp viral defense according to several reports (Burnett and Burnett, 2015; Wang et al., 2006b; van de Braak et al., 2002). On the contrary, our study detected statistical differences in proPO gene in WSSV-infected crabs. Thus, apparently high viral load induced QM and PHEN gene transcript levels in both challenged crustacean species but was not able to active the whole pro-phenoloxidase system.
Different research results have confirmed the existence of a correlation between iron metabolism and the immune system in crustaceans, once iron is a co-factor of ribonucleotide reductase and is necessary for enzyme activity (Zhang et al., 2014). The depletion of cellular iron can lead to the inhibition of ribonucleotide reductase, preventing virus proliferation (Lin et al., 2015; Verbruggen et al., 2016). Ferritin is a major iron storage protein in living cells and plays an important role in iron homeostasis and also in host's innate immune response to various pathogens (Ye et al., 2015). Our findings revealed up-regulated ferritin gene expression (FERR) in crabs and shrimp, in response to severe WSSV infection. In invertebrates, FERR was found to be up-regulated after pathogens challenge and is considered to be an important element in the innate immune system. The transcripts of FERR in shrimp gills were reported to be up-regulated post WSSV challenge by Ye et al. (2015) once the expression of ferritin was significantly increased 12 h after WSSV injection and was kept in high level untill 96 h after WSSV injection in shrimp. In addition, the up-regulation of ferritin has also been observed in WSSV-infected M. japonicus (Feng et al., 2014b) and in C. quadricarinatus (Chen et al., 2018).
The transcripts of calreticulin (CALR), a highly conserved endoplasmic reticulum luminal resident protein, were up-regulated in shrimp and down-regulated in crab gills at the same viral charge. In crustaceans, calreticulin is known to play important roles in in calcium homeostasis, molting, immune functions, and stress response to viral infection (Luana et al., 2007; Huang et al., 2019). Also, the calcified cuticle proteins (CALC) are related to the cuticle synthesis in crustaceans, with their transcription increased during the molting phase (Kuballa et al., 2007). This protein has never been directly related to WSSV infection, prior to Müller studies (2009), but its induction may be related to the abnormal deposit of calcium salts in the cuticle of the animals, which characterizes WSD. In our experiment, CALC transcript levels were up-regulated in severe WSSV infection, both in crabs and shrimp. It’s well established that shrimp acutely infected with WSSV often show abnormal deposits of calcium within the cuticle (Wang et al., 2006), as well as soft cuticles in advances stages of infection, signs that may be related with the modulation of transcripts encoding to CALR and CALC genes.
Decapod crustaceans (marine and freshwater) are susceptible hosts to the development of the WSD, while non-decapod crustaceans can accumulate high concentrations of viral particles without proof of viral replication in these organisms. However, this is the first report of experimental infection by WSSV and viral proliferation in N. granulata crabs. The set of genes investigated in our study could be used as a complementary early-warning biomarkers to monitor the health status and susceptibility of shrimp and other crustaceans to WSSV, since these responses are directly related to the viral infection. Additionally, the same genes could also be analyzed in early phases or along the time course of the infection to better understand the dynamics of WSSV infection, in shrimp and crabs, and better study the difference in susceptibility to the infection.
A better knowledge of the molecular defense related to host antiviral responses may contribute to understand and explore the mechanisms triggered by WSSV. The absence of mortality or clinical signs, as seen in N. granulata, may provide the virus a better opportunity for replication and efficient continuing transmission. Moreover, the passage of a WSSV strain through different hosts induces genomic variation and alters the pathogenicity of the virus (Waikhom et al., 2006; Müller et al., 2010), which could explain the variation in WSSV-stress responses. A better understanding of the reasons, as well the molecular scenario, for differences in mortality at equally heavy viral infections may allow us to develop strategies for limiting mortality from viral pathogens in shrimp aquaculture.