In this study, we investigate the pattern of expression of four representative members of the polypeptide families that are related to the innate immune response of the Pacific whiteleg shrimp L. vannamei, in the standard natural condition of raring in farms located in the northeast of Brazil. Brazil is becoming one of the main global producers of this shrimp species, and approximately 98% of the national aquaculture industry is concentrated in the northeastern region of the country. This Brazilian region favors shrimp and fish aquaculture due to the particular climate, which is characterized by low temperature fluctuation and low rainfall throughout the year. Despite these excellent environmental conditions for shrimp farming, an anomalous period of excessive rain occurred in the interval from December 2009 to March 2010. Consequently, a virus outbreak began, likely as a consequence of reduced water salinity and osmotic shock that compromised the regional shrimp production in that period. The samples for molecular diagnostic analysis were collected in these circumstances and were inspected for known symptoms of viral and bacterial disease. Based on the distinct pathological signs seen in the shrimp infected with IHHNV and IMNV, the two prevalent viruses in Northeastern Brazil, the samples were classified as asymptomatic, IHHNV- or IMNV-suspected (Table 2). Conventional PCR-based analysis proved that 98% of asymptomatic shrimps were IHHNV carriers, although the gross signals of infection were not manifested when they were sampled. The presence of IMNV in asymptomatic shrimp was not tested in such samples because they were collected in a preservation solution for genomic DNA extraction, and IMNV is an RNA virus. Whether carried IHHNV was in its infective form or silently integrated in the shrimp genome, as previously reported (Tang and Lightner 2006; Teixeira et al. 2010), was not addressed in the present study. However, shrimp samples that were grouped and labeled as IHHNV- and IMNV-suspected were analyzed by RT-PCR to confirm the gross signals of chronic viral disease. Unexpectedly, a relatively high number of shrimp samples were co-infected with both viruses, and the disease outcome was the result of the predominance of one kind of virus that quantitatively proliferates to the detriment of the other (Figures 1 and 2). In fact, the prevalent co-infection of L. vannamei with IHHNV and other virus, like TSV, and even in association with two other viruses (TSV and WSSV), i.e., triple-virus infection, has been found among samples in distinct shrimp farms located in China (Tan et al. 2009). Therefore, IHHNV seems to be a constant co-infecting agent in shrimp that is capable of genome integration events and of being carried by asymptomatic individuals in all phases of shrimp development.
To understand the immune response of L. vannamei chronically co-infected with IHHNV and IMNV in the natural condition of farming, we chose to test four representative classes of polypeptides that are responsible for the first line of defense against pathogenic microorganisms. These molecular markers of the innate immune response include two classes of antimicrobial peptides (crustin and penaeidin), a C-type lectin and a heat shock protein (HSP-70). qPCR was used to evaluate the expression patterns of the transcripts that corresponded to the four polypeptides. The analysis was focused on the gill tissue because this shrimp organ is the first to accumulate pathogenic microorganisms (Burgents et al. 2005), and several studies have reported the differential gene expression related to environmental stress in the gill (Zhou et al. 2010; Wang et al. 2011).
As was observed by means of experimental results with samples that were environmentally exposed and thriving with IHHNV and IMNV co-infection (Figure 3A), the higher the number of IHHNV particles, the higher the level of HSP-70 expression in the shrimp gill, and values reached at least 3 orders of magnitude in comparison with estimates from the basal level of HSP-70 copy number. On the contrary, the proliferation of IMNV in co-infect shrimp neither caused such a proportional increase in the HSP-70 level as that which was observed for IHHNV nor caused a down-regulation of this gene, as statistically tested (Figure 3B). The validation of such an observation is also applicable for samples in which IHHNV or IMNV is the single infecting virus (see Figure 4).
The expression levels of crustin, penaeidin and C-type lectin was assessed in the condition of reciprocal viral co-infection. However, the relationship between IHHNV/IMNV reciprocal co-infection and crustin or penaeidin expression was not observed, which showed a weak positive correlation (Figure 3C,D,E,F,G,H). Crustin and penaeidin, which are primarily produced in high level in hemocytes, were shown to be expressed differently in several shrimp tissues, including the gill. Crustin and penaeidin act mainly as antimicrobial agents against bacteria and fungi (Tassanakajon et al. 2011), despite of the reported data about their role as anti-virals (Amparyup et al. 2008).
The recently cloned CTL-br1 cDNA was evaluated for the purpose of establishing a relationship between its expression in response to the simultaneous presence of IHHNV and IMNV in sub-adult farmed shrimps. Despite some level of induction detected in the gill of sub-chronic IHHNV-infected shrimps (Costa et al. 2011), a correlation was observed in the same tissue of L. vannamei co-infected with both IHHNV and IMNV. In fact, homologous C-type lectin gene products were observed to fluctuate positively in L. vannamei hemocytes in the first 48 h of WSSV infection, which showed a direct anti-viral activity that is displayed by this kind of molecule (Zhao et al. 2009).
To certify that the oscillation in gene expression is virus-specific and not a generalized response, a number of samples were evaluated for the ratio between IHHNV and IMNV copy number and the pattern of expression (Figure 4). When IHHNV is the only virus or when its number predominates in co-infected samples by a difference of twofold, the same pattern of expression for HSP-70 and a positive relationship are observed. Again, no significant relationship was seen when IHHNV is predominant in respect to IHHNV and IMNV co-infected shrimp, although there was a slight increase in samples with the presence of IHHNV alone. The same analysis conducted for IMNV revealed that when IMNV predominates in IHHNV/IMNV double-infected shrimps, or when IMNV is the only infective virus, the level of HSP-70 is still inferior by 3 orders of magnitude when compared to IHHNV in the same circumstances. However, as seen in a number of samples, the levels of crustin, penaeidin, and CTL-br1 are preferentially increased in the shrimp gill when IMNV is present alone. For now, we are unable to speculate this differential pattern of expression, but we can conclude, based on the present data, that the triggering of the innate immune genes by IHHNV and IMNV seemed to be virus-specific and does not overlap even in a double-infected host. The up-regulation of HSP-70 IHHNV might confer a certain degree of immunity in shrimp through activation of the toll-like receptor transduction pathway (Tsan and Gao 2004). Thus, HSP-70 may display a dual role: it exerts immune activation as danger signals, mediates protection against infectious diseases, or exhibits regulatory activities in controlling and preventing disease (Multhoff 2006). In fact, shrimp exposed to sub-lethal doses of virus induce a status of ‘pseudo-vaccination’ of the host, improving the resistance and survival of Parribacus japonicus to high infective doses of WSSV (Wu et al. 2002). However, as observed through our data, with excessive increases of HSP-70 expression, damage to the shrimp tissues may occur, due to pro-inflammatory cytokines that are normally produced, which would facilitate IMNV proliferation. In fact, this explanation is plausible because shrimp carrying IHHNV, or in which the number of IHHNV particles is predominant, develop IHHN disease, but not IMN disease. In contrast, shrimp that develop IMN disease are most likely to be co-infected with both IHHNV and IMNV (Table 2).
In summary, to our knowledge, the present study is the first to correlate the up-regulation of the HSP-70 gene mediated by a virus in naturally double-infected farmed shrimp host. The modulation of HSP expression has implications in the activation of the innate immunity response and may serve as a mean of inducing pseudo-vaccination. However, in double-infected shrimp, the triggered induction of HSP-70 that would contribute to counteract a pathogen assault seems to open a window of immune-modulator imbalance that might be deleterious to the host due to the presence of an opportunistic co-infecting virus. Obviously, more comprehensive studies are required to elucidate the molecular mechanisms that switch on and off in the operation of reciprocal viral proliferation in shrimp, as well as their roles in the molecular arsenal of the host defenses that are necessary to maintain homeostasis. In fact, to better understand the inductive expression of HSP-70 response to IHHNV in white shrimp (L. vannamei) infected with predominant IMNV or only IMNV, we are conducting quantitative expression analysis with samples of shrimps infected in the laboratory under controlled conditions.