Susceptibility of Neohelice granulata (Decapoda, Varunidae) to white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) continues to be the most severe viral pathogen to the shrimp industry worldwide. Pacific white shrimp Litopenaeus vannamei is particularly affected by WSSV, and this virus has been detected in a wide range of wild crustaceans, including penaeid and non-penaeid shrimp and crayfish, as well as crabs. Crabs have been considered as a dangerous threat to shrimp farms because they are generally believed to be highly tolerant to WSSV and to remain infected for long periods without signs of disease. The burrowing crab Neohelice (= Chasmagnathus) granulata can be commonly found in the surroundings of the shrimp farms in southern Brazil. Here, we investigated the susceptibility of N. granulata to WSSV infection in comparison to L. vannamei. WSSV infectability and host susceptibility were investigated by viral challenge (intramuscular injection) in both species. Viral load in challenged animals was quantified by qPCR in both hemolymph and gills. Furthermore, the transcript levels of sixteen target genes related to the molecular defense status were assessed. The results revealed that N. granulata experimentally infected by WSSV, as well as those naturally infected, showed lethargy, lack of appetite, and later gross signs of the disease. Moreover, N. granulata showed to be less susceptible to WSSV when compared to L. vannamei. While no death was observed in crabs before a post-challenge interval of 120 h, shrimp death was seen as early as 48 h post-infection. Comparative viral load was also assessed by qPCR in gills of captured wild crabs and farmed shrimp naturally infected by WSSV. Transcript levels of target genes were also investigated after WS challenge between 24 and 48 hpi in shrimp and between 96 and 120 hpi in crab. Differences in gene transcripts were particularly noteworthy with the increase of viral copies at 106 copies µl−1. These results indicated that WSSV infection modulated genes related of important cellular responses, such as apoptosis, chaperone function, and ion storage, in both shrimp and crab.


Introduction
Viruses are extremely abundant in aquatic systems (Suttler 2005;Zhang and Gui 2018) and are considered as the most important pathogens in impacting shrimp farming worldwide. Different life stages of shrimp may be susceptible to viral infections which can cause mortality, slow growth, and deformations (Rahman et al. 2007;OIE 2016). Among the several viruses that have been reported as pathogenic to shrimp, white spot syndrome virus (WSSV) continues to be one of the most severe and associated to huge global economic losses to the shrimp farming. Since the first report of WSSV in Brazil, in the south of Santa Catarina state in January 2005, the disease was soon detected in some farms located in the northern and central regions of the state, as well. Subsequently, the southern region of the state was isolated, and the stocking suspended until September of that year (Seiffert et al. 2005). After the fallowing, new stockings took place, but successive losses were still reported in subsequent years. Since then, WSSV outbreaks have been seen in many areas of Santa Catarina, as well as in other regions of Brazil until now. WSSV caused a drop of more than 90% in the production of shrimp in Santa Catarina (Goularti Filho and Ronçani 2018).
WSSV is a double-stranded rod-shaped DNA virus, which belongs to Nimaviridae family (Mayo 2002), being unique among shrimp viruses due to its large genome size of approximately 290 kbp (van Hulten et al. 2001). Clinical signs of white spot disease (WSD) caused by WSSV include lethargy, reddish discoloration of the animal's body, and emaciation of the carapace, which may display the presence of white spots (Durand et al. 1997). Mortalities on cultured shrimp may reach 100% within 3-10 days after exhibiting the first clinical signs of the disease. Survivor shrimp may carry the virus for life and may also pass the virus to their progeny (Lo et al. 1996a(Lo et al. , 1997. Water discharges from shrimp farms were seen as the source for wild crustacean population infection (Lo et al. 1996a, b) and the consequent high prevalence of WSSV in natural populations (Meng et al. 2009). WSSV propagates quickly throughout the environment and has been found not only in wild penaeid shrimp but also in a wide host range, including crab, lobster, copepod, crayfish, and freshwater crab and prawn, as well (Lo et al. 1996a, b;Maeda et al. 1998;Peng et al. 1998;Wang et al. 1998;Sahul Hameed et al. 2001, 2003Marques et al. 2011;Sánchez-Paz et al. 2015). Mortality among this host range is highly variable according to several reports, suggesting that susceptibility varies between crustacean taxa (Oidtmann and Stentiford 2011). Economic impacts have driven research on WSD in farmed shrimp, especially in Litopenaeus vannamei, and reports in the literature are abundant for this penaeid species. In contrast, the data concerning the effects of the disease in wild noncommercial crustacean populations remain scarce.
A wild crustacean frequently seen in the surroundings of shrimp farms in south of Brazil is the crab Neohelice granulata (Dana, 1851), previously known as Chasmagnathus granulata, before Sakai et al. (2006) revised and reclassified all species formerly attributed to the genera Helice and Chasmagnathus. N. granulata is a burrowing semi-terrestrial crab found in the intertidal zone of estuaries, salt marshes, and mangroves of the Southwestern Atlantic Ocean, ranging from San José Gulf, northern Patagonia, Argentina, throughout Uruguay, to Lagoa Araruama, in Rio de Janeiro, Brazil (Spivak 2010). In southern Santa Catarina, it can be found along the extension of the estuary of the huge Laguna Lagoon Complex, around which most of the shrimp farms are located. This crustacean is abundant in the rearing ponds, digging holes and galleries on the margins (Marques et al. 2011).
N. granulata has been previously reported as a carrier and as a potential host of WSSV, due to natural infection, based on sequence homology data to the viral genome of a nested PCR fragment obtained from specimens collected in Santa Catarina, Brazil (Marques et al. 2011). Later, positive PCR results were also reported for N. granulata collected further south, in Rio Grande do Sul, Brazil (Cavalli et al. 2013).
Crabs have been considered as a particularly dangerous threat to shrimp farms because they are generally believed to be highly tolerant to WSSV and to remain infected for long periods of time without showing signs of the disease, therefore contributing to the maintenance of the viral agent in the environment. Since N. granulata susceptibility to WSSV has not been addressed so far, our aim was to carry an investigation under a comparative perspective to L. vannamei, including the analysis of the transcript levels of target genes related to molecular defense responses.
The identification of potential hosts in penaeid farms, as well as surrounding areas, may contribute to monitoring the presence and behavior of the virus in the environment, as well as to determine its geographic distribution, and provide information about its persistence and pathogenicity (Marques et al. 2011;Moser et al. 2012). These aspects should be considered as a critical topic in sanitary programs and to reinforce management strategies.

Animals
Crabs, Neohelice granulata (n = 115; 5.3 ± 0.6 cm in body length; weight 12.7 ± 1.1 g), were obtained nearby the tanks of a local shrimp farm in Northern of Santa Catarina State (Brazil). Collected crabs were transported to the laboratory and maintained in 30-l fiberglass tanks (n = 4), containing a thin layer of filtered sea water, at 23 ± 1 °C and salinity of 25 g l −1 . Crabs were held under these controlled conditions for 5 days to acclimate prior to viral challenge. During this period, crabs were fed once a day with commercial shrimp feed. Crab identification was carried out according to the key to Brachyura of the Brazilian coast (Melo 1996).
Juvenile intermolt shrimp L. vannamei (n = 110; 8.5 ± 2 cm in body length, weight 10 ± 1 g) were obtained from the same shrimp farm, transported to the laboratory and maintained in 6-l fiberglass tanks (n = 3), with filtered sea water at 23 ± 1 °C and salinity of 20 g l −1 . Shrimp were held under these conditions for 5 days to acclimate prior to viral challenge. During this period, shrimp were fed once a day with commercial shrimp feed.
To investigate the previous presence of infectious hematopoietic necrosis virus hypodermal (IHHNV) and white spot syndrome virus (WSSV), hemolymph samples from shrimp and crab were used for PCR assays. Based on the IHHNV and WSSV screening, only animals that showed to be negative for both viruses were further used for the challenge experiments.
Additionally, to better access the natural WSSV prevalence, a total of other 30 N. granulata crabs (5.8 ± 1 cm in body length; weight 12.5 ± 5 g) and 30 L. vannamei shrimp (14.5 ± 3 cm in body length; weight 20 ± 2 g) was collected in another shrimp farm, located in the southern part of the state. In the case of these other collected specimens, gill samples, from both shrimp and crabs, were used to WSSV screening.

Viral challenge
Crabs and shrimp were challenged with WSSV by injection, using an inoculum corresponding to a Brazilian geographical isolate of the virus, previously characterized (Müller et al. 2010). The inoculum was prepared as follows: WSSV-infected L. vannamei tissues were diluted 1:5 (w/v) with sterile PBS buffer (pH 7.8) and homogenized. The homogenate was clarified by centrifugation, filtered (0.45 µm filter), and used for injection. Prior to injection, the concentration of WSSV inoculum was assessed by qPCR. A standard curve was used for viral quantification, with serial dilutions of recombinant plasmid containing a previously known number of WSSV genome sequence copies per microliter. First crabs (n = 56) were injected with 10 µl purified WSSV (5.3 × 10 4 viral copies per animal) via the base of the walking leg using a sterile 1-ml syringe fitted with a 21G needle. The control group was injected with the same volume (10 µl) of PBS buffer. Subsequently, shrimp (n = 54) were injected with 100 µl purified WSSV (5.5 × 10 4 viral copies per animal) intramuscularly into the third dorsal segment. Likewise, the same volume of PBS buffer was used for injecting the control group. Crab challenge experiments were performed in duplicate, whereas shrimp experiments were performed in triplicate. The difference in the number of experimental replicates between species was due to the availability of animals, since shrimp were obtained directly from the farm, whereas crabs were collected in the field, i.e., in the surroundings of the farm. After challenge, crabs and shrimp were monitored daily for survival, pattern of behavior, mobility, and body coloration, as well as feed consumption.

Sampling and DNA extraction
In the challenge experiments, gill samples were used to monitor viral infection at intervals of every 24-h post-infection (hpi). Samples were collected from eight crabs and nine shrimp at each time. The first sampling was performed prior to infection (0 h) and the last one took place at 72 and 144 hpi, for shrimp and crab, respectively. Accordingly, gills from natural infected farmed shrimp and wild crabs were also used to assess viral infection. DNA extraction from gills was performed according to Marques et al. (2011). The concentration of total DNA of gills was calculated by the optical density (OD) ratio at 260/280 nm using a NanoDrop® 2000 spectrophotometer reader (Thermo Scientific).

WSSV detection and viral load determination
A standard conventional polymerase chain reaction (PCR) was used for initially screen for WSSV natural infection in N. granulata and L. vannamei samples, using primers designed to amplify a 480-bp fragment of the WSSV genome, according to Nunan and Lightner (2011). PCR products were submitted to 2% agar electrophoresis, and qualitative results were documented by digital camera. Viral load determination in these samples, as well as for both WS-challenged N. granulata and WS-challenged L. vannamei, was performed by qPCR. An amount of 100 ng µl −1 DNA was added to a reaction mix prepared according to the protocol of QuantiTect® SYBR® Green PCR-Kit (Qiagen, Hilden). Reaction was performed in a thermocycler (Rotor-Gene Q), with an initial denaturation at 95 °C for 15 min, followed by 39 cycles of 95 °C for 30 s, annealing temperature at 55 °C for 30 s, and extension at 72 °C for 10 s. The set of primers was designed based in the sequence amplified by the nested PCR reaction described by Lo et al. (1996a): WS2F: 5′-TGC CTT GCC GGA AAT TAG TGT GTG -3′ and WS2R: 5′ACA ACA TCC AAC AAT GGT CCC GTG -3′. After amplification, results were analyzed and compared to the standard curves. Standard curves were performed with a serial diluted DNA from a recombinant plasmid containing a previously known number of WSSV genome, belonging to our laboratory collection, as internal positive controls.

Transcript levels of target genes in samples of N. granulata and L. vannamei infected with WSSV
Gills collected from WSSV-challenged animals, and their respective controls were used to investigate differential transcription of target genes. Total RNA from the gill samples was extracted using the TRIzol reagent (Invitrogen), according to the manufacturer's protocol. Total RNA concentration and purity were assessed by spectrophotometer (NanoDrop 2000, Thermo Scientific) through the absorbance at 260 nm and 260 nm/280 nm ratio, respectively. Total RNA from crab and shrimp gills was reversely transcribed using Quantitect Reverse Transcription Kit (Qiagen, Hilden). Diluted cDNA (100 ng µl −1 ) was added to a reaction mix composed of QuantiFast® SYBR® Green PCR kit (Qiagen, Hilden). Amplifications were performed in duplicates for each sample using a Rotor-Gene 6000 real-time PCR thermocycler (Qiagen, Hilden) under the following conditions: 1 cycle at 95 °C for 5 min; 30-40 cycles of amplification at 95 °C for 10 s, 58-60 °C for 30 s. A dissociation curve was carried out to confirm the amplification of a single product. Efficiency of PCR reaction was determined using a standard serial dilution curve of cDNA and negative control. Transcript levels of sixteen target genes were quantified by the method of 2 −CT , using ribosomal 18S-like as a house-keeping normalizing gene (Schmittgen and Livak 2008) and the Rotor-Gene 6000real-time qPCR system software. The analyses were performed using GraphPad Prism 5.0 software (GraphPad Software). The primer designing was based on our previous studies assessing shrimp defense responses through gene transcription and proteomics (Müller 2009;Valentim-Neto et al. 2014a;Valentim-Neto et al. 2015;de Souza Valente et al. 2020;Mattos et al. in preparation) (Table 1).

Statistical analysis
Normal distribution was tested using Shapiro-Wilk test, and homogeneity of variance assumption was tested using D'Agostino test. Grubbs test was performed to exclude outliers. Parametric data were analyzed by one-way y ANOVA followed by Dunnet's post hoc test. Kruskal-Wallis test followed by Dunn's test was performed for non-parametric data. Results are presented as mean ± standard deviation (SD); differences were considered statistically significant for p < 0.05. Data were analyzed using GraphPad Prism. Differences in target gene transcript levels was based on the following comparisons: (1) between WSSV-free and WSSV-infected crab (48 h and 72 h after viral infection); (2) between WSSV-free and WSSV-infected shrimp (24 h and 48 h after viral infection); and (3) between groups (crab and shrimp). The statistical results for this analysis were determined using the t test for independent samples, with a confidence level of 95%.

Table 1
Primers used in the analysis of gene differential expression by RT-qPCRin Neohelice granulata

Results
Viral challenge with injection of WSSV resulted in infection of burrowing crab N. granulata. All WSSV-challenged crab showed PCR positive reactions, evidencing the presence of the virus. WSSV challenge N. granulata survived up to 168 h, with no gross signs of infection. Only on the sixth day (144 h) post-infection, it was possible to observe lethargy and reduction in food consumption when compared either with earlier days or with control group. On the other hand, WSSV-challenged shrimp showed lethargy, reddish body color, and low mobility, clinical signs associated to WSSV, earlier as 48 hpi. Mortalities were recorded at 48 hpi. At 72 hpi, all shrimp were collected. Comparative results show wide variation in responses to WSSV challenge. Although both, N. granulata and L. vannamei, were susceptible to WSSV, L. vannamei showed to be more susceptible than N. granulata. Crab and shrimp survival curves after WSSV challenge can be seen in Fig. 1.

Real-time PCR detection and viral load after WSSV challenge
Gills were used for WSSV detection and determination of viral load, by real-time PCR (qPCR). qPCR showed that WSSV could replicate within the crab tissues, as well as in shrimp (Fig. 2). Through these samples, it was possible to evaluate the progress of WS infection in challenged animals. Results showed WSSV-positive crabs in the challenged group, as expected, while the control group remained negative for the virus during the whole experiment time course. Likewise, all shrimp, sampled after virus injection, were WSSV-positive, whereas the control group remained negative. qPCR analysis revealed significant differences (p < 0.05) in the viral load of WS-positive crabs, according to the time interval following virus infection ( Fig. 2A). After 24 h, an amount of 3.59 copies was detected, which rose up to 2.14 × 10 5 (96 hpi) and further to 4.48 × 10 6 (144 hpi). Accordingly, increases in viral copies in WSSV-challenged shrimp were also seen ( Fig. 2A). Nevertheless, after 24 h, the average number of viral copies in WSSV-challenged shrimp was 8.38 × 10 1 copies µl −1 , whereas after 2 days (48 hpi), the average number of viral copies rose to 2.57 × 10 6 and significantly decreased to 7.79 × 10 5 copies at 72 hpi. qPCR average numbers showed that viral load in gill of shrimp at 48 hpi (2.57 × 10 6 copies µl −1 ) had a similar counterpart in crabs only at 120 hpi (4.39 × 10 6 copies µl −1 ). Also, it was possible to identify viral peaks at some post-WS infection phases.
Comparatively, viral load has increased from 24 to 48 hpi in shrimp, while highest replication in crab was detected from 96 to 120 hpi (Fig. 2B). No viral copies were detected in control crab or control shrimp indicating virus-free animals.

WSSV PCR and qPCR detection on natural infected crustaceans
Thirty N. granulata crabs and thirty L. vannamei shrimp gills were also used to determine the viral load in naturally infected animals. The initial analyses were conducted by conventional PCR and PCR products visualized by electrophoresis in 2% agarose gel. PCR products that showed the presence of a single band equivalent to the expected molecular weight of approximately 480 bp were considered WSSV-positive animals. This band corresponds to the amplification of a specific fragment of the WSSV genome. The samples in which the 480-bp fragment was absent were considered negative for the presence of the virus. Furthermore, the viral load in WSSV positive field animals (crabs and shrimp) was measured by qPCR. In naturally infected field shrimps, viral quantification (10 6 copies µl −1 ) was very close to those found in animals collected at 48 h after experimental infection. The crabs, collected within or adjacent to shrimp farms, exhibited a lower amount of WSSV, ranging between 10 2 and 10 3 copies µl −1 .

Differential gene transcription in WSSV-challenged N. granulata and L. vannamei
Differential transcript levels in N. granulata and L. vannamei challenged with WSSV were assessed in gill samples by RT-qPCR, focusing on sixteen target genes. Comparison was made between two different post-infection time intervals (hpi) and their respective controls (Fig. 3). These data are summarized in Table 2  Relative gene transcription profile to most of the selected genes showed significant statistical differences when compared to the respective controls. In crab, changes in transcript levels of fifteen genes were seen at 96 or 120 hpi, whereas in shrimp, twelve genes showed significant changes at 24 or 48 hpi when compared with non-infected controls. Such genes showed an increase in expression in the range from 1.8-to 24-fold relative to control groups (Fig. 3).
According to qPCR results, experimentally infected shrimp displayed a total of viral particles around 10 6 after 48 h of experimental infection (hpi). Interestingly, this value matches the viral load we found in naturally infected L. vannamei collected in the farm. On the other hand, the same amount of viral particles (10 6 copies µl −1 ) in N. granulata gills was seen only after 120 h of experimental infection (hpi). Thus, samples containing the same amounts of viral particles were chosen to compare the induction or inhibition of some of the target genes. The higher viral load was associated to nine upregulated genes in shrimp (CALC, FERR, CASP-2, PHEN, QM, IAP, UBQ, HSP70, and H2ɑ). This same viral load was associated to fourteen upregulated genes in crab (CALR,CALC,FERR,PROPO,PHEN,QM,IAP,UBQ,HSP60,HSP70,PKC,H2ɑ,. Moreover, when overall expression profile was analyzed and compared between species, out of the sixteen selected target genes, we found eight genes that were modulated by the severe infection. Significant changes were seen on transcript levels of CALR, FERR, PHEN, QM, IAP, UBQ, HSP 70, and H2ɑ in both species at similar viral load (10 6 copies µl −1 ).
No significant changes were seen in the transcription of the genes encoding Prophenoloxidase or ß-Tubulin in shrimp after WS challenge. Moreover, ß-actin transcript levels showed no significant changes in shrimp or crabs.

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. So, in our study, 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 10 2 and 10 3 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 10 6 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 Table 2 Differential expression in Neohelicegranulata and Litopenaeus vannameichallenged with WSSV. Columns represent changes in transcript levels of sixteentarget genes in shrimp at 24 and at 48 hours postinfection (hpi), and in crab at96 and at 120 hpi. Average data represent differences in folds relative torespective control (blue and red bars). Mean statistical differences betweenWS-infected and non-infected animals (controls): (↑) up-regulated or (↓) down-regulated genes in timecourse biochemical, physiological, and ecological researches (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, whether N. granulata crab apparent resistance to WSSV is 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, N. granulata crabs were less susceptible to WSSV.

Differential expression in N. granulata and L. vannamei related to the same viral load
Differences in the relative gene transcription profile seen in infected shrimp and infected crab when compared to the respective controls (non-infected animals) reflect the role of essential host molecular defense processes in both species. At the same viral load, differences in gene transcript findings in crabs and shrimp point out similar potential effects of WSSV infection in molecular responses 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 clearly induced in both shrimp and crabs. Upon reaching the threshold of 10 6 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, caspase 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 and 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).
Furthermore, 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. (2014b), 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 upregulated, 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 upregulation 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 10 1 to 10 6 copies µl −1 . In crabs, WSSV infection increased slower between 96 and 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 upregulated 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. 2006a). Levels of ubiquitin were upregulated 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 8.38 × 10 1 (24 hpi) to 7.79 × 10 5 copies (72 hpi). Furthermore, 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 upregulated 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), andcalcitonin-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-García et al. 2019;Wang et al. 2008). The histone H2ɑ is responsible for the packaging and compaction 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, prevents them to participate in nucleosome assembly, inducing incidental apoptosis. Our results show that H2ɑ gene is upregulated in crabs and shrimp at the same viral load. But, interestingly enough, we observed a significant higher transcription before viral load achieve such level (10 6 copies µl −1 ), that is, at 24 hpi in shrimp and at 96 hpi in crabs. These results corroborated with Encinas-García et al. (2019) that reported that maximum levels of expression of this protein were reached at 12 h 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 upregulation 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 upregulated 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 WSSVinfected 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 upregulated ferritin gene expression (FERR) in crabs and shrimp, in response to severe WSSV infection. In invertebrates, FERR was found to be upregulated 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 upregulated 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 until 96 h after WSSV injection in shrimp. In addition, the upregulation 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 upregulated in shrimp and downregulated in crab gills at the same viral charge. In crustaceans, calreticulin is known to play important roles 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 upregulated in severe WSSV infection, both in crabs and shrimp. It is well established that shrimp acutely infected with WSSV often show abnormal deposits of calcium within the cuticle (Wang et al. 2006a), as well as soft cuticles in advanced 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. Our findings comprise the first report of WSSV experimental infection and viral proliferation in N. granulata crabs and show a noteworthy proliferation pattern, in terms of both time course and viral load. 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 as 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.

Conclusions
In conclusion, our investigation provides evidence that N. granulata is susceptible to WSSV and the virus is capable to replicate into its cells. WSSV challenge was conducted by intramuscular injection and revealed that N. granulata is less susceptive to WSSV than L. vannamei. The WSSV stress caused by viral load increase affected transcript levels of eleven genes in crabs and six genes in shrimp, modulating different cellular mechanisms, such as apoptosis, iron storage and mobilization, and chaperone stress response. This molecular interaction network plays an important role in determining host susceptibility and infectability of the pathogen.
Author contribution MRFM and JRM conceived and designed the research. JRM performed the experiments. MRFM and JRM analyzed the data. MRFM provided funding. Both authors contributed to the manuscript.

Declarations
Ethical statement This manuscript does not involve any study with humans, vertebrate animals, genetically modified animals, cloning, or endangered species.

Competing interests
The authors declare no competing interests.