Antiviral immunity and genomic studies in the marine shrimp Litopenaeus vannamei
Our studies are focused on identifying the molecular basis for recognition and response to viral infections in the marine shrimp L. vannamei. To better understand the role of critical genes, research has focused on applying a reverse genetics approach using double stranded RNA to silence endogenous shrimp genes. These studies have identified dsRNA as an inducer of antiviral immunity in shrimp, suggesting that recognition of dsRNA as a virus associated molecular pattern is a conserved feature of both vertebrate and invertebrate immune systems.
To gain a better understanding of these antiviral responses, we have generated a cDNA microarray containing approximately 3000 non-redundant expressed sequence tags from L. vannamei. This tool is allowing the identification of the gene expression signatures that characterize viral infections and the induction of antiviral responses. This transcriptomic information should provide a basis on which to construct informed hypotheses regarding the genetics of antiviral immunity in shrimp.
Introduction
Marine shrimp culture has become a major global industry producing an estimated 2 million MT of products. According to World Shrimp Farming 2004, the shrimp aquaculture industry that has developed over the last 30 years now accounts for almost 50% of the shrimp on world markets (Rosenberry, 2004). According to the FAO (2004), crustacean aquaculture produced 2,131,000 tons in 2002 valued at $USD 10.8 billion. This increasing production has resulted in lower prices and has fueled increasing shrimp consumption.
For example, in the US per capita consumption has reached 4 lbs per person per year making shrimp the number one preferred seafood product. To satiate this increasing appetite, US shrimp imports were up 18% in 2003 to a record 1.1 billion lbs (Johnson, 2004). Clearly, cultured shrimp are a major international commodity attracting intense attention and scrutiny. International trade disputes have increased, criticism of environmental sustainability of production practices continues while falling market prices have put more and more pressure on producers.
One of the most important problems continuing to plague the industry is disease control. Shrimp viral pathogens have caused devastating losses in many countries. For example pond-side losses attributed to white spot syndrome virus (WSSV) in 1996 in Thailand alone reached approximately 70,000 metric tons, valued at over half a billion dollars (Flegel and Alday-Sanz, 1998). Rosenberry (2001) estimated that disease due to WSSV “robbed the industry” of approximately 200,000 MT of production in the year 2000, worth more than $1 billion.
Unfortunately, our limited understanding of the shrimp immune system coupled with continuing emergence of new pathogens has limited available alternatives for combating disease. The prophylactic use of antibiotics to maintain shrimp health has come under increasing scrutiny as the application of increasingly sensitive testing has blocked some shrimp imports, particularly into the EU.
Industry disease control efforts have shifted to emphasize biosecurity, pathogen exclusion and improved culture systems. Marginal producers have found it increasingly difficult to compete while farmers willing to apply increasingly advanced culture strategies for water quality management and pathogen exclusion have become the norm. Availability of disease-free stocks has resulted in a shift from farming of wild Black Tiger shrimp to culture of increasingly domesticated white shrimp in much of Asia.
Continued industry evolution towards environmental and financial sustainability in an era of increasing competition will depend upon improving culture systems and achieving a better understanding of the fundamental nature of shrimp defense mechanisms. New tools and strategies resulting from continued targeted scientific research should improve the outlook for more consistent shrimp production.
Shrimp genomics
Our basic understanding of the crustacean response to bacterial and fungal disease is limited. Several different mechanisms of immune defense against microbial pathogens have been identified in crustaceans. A number of reviews have been published summarizing our understanding of humoral and cellular defense mechanisms (Bachere et al., 1995, 2004; Cerenius and Soderhall, 2004; Smith, 1991; Smith and Chisolm, 1992). Unfortunately, almost nothing is known about antiviral defenses in invertebrates. This lack of information coupled with limited scientific resources has hindered research in the area of shrimp viral disease.
A novel approach to understanding host-virus interactions in marine shrimp (Robalino et al., 2003) has been initiated in Charleston, SC, USA. This is part of a multi-institutional initiative that seeks to apply ‘functional genomics’ as a method to investigate the interaction of marine organisms with their environment, including infectious diseases. This approach offers the possibility of rapid gene discovery and the potential to study gene regulation on a large scale, by using gene microarrays and transcript profiling (which provide simultaneous quantitative measurements of the expression of a large suite of genes). These approaches will identify potential immune-function genes that can then be taken into traditional hypothesis-driven research projects and selective breeding programs (Chapman and Browdy, 2003).
The aim of the functional genomics studies on innate antiviral immunity in marine shrimp is to characterize the genes and pathways involved in the response of L. vannamei to WSSV and other viruses. Clearly such a project will require as comprehensive a collection as possible of the genes that are expressed in different tissues of L. vannamei, and in the initial stages of the project it will be beneficial to focus on genes that are potentially involved in the immune response.
How many genes are there in shrimp? The penaeid shrimp genome is about 70% the size of that of the human genome, and while the number of expressed genes in the human is estimated to be somewhere between 30-50,000, it is generally estimated that the number of expressed sequences in invertebrates will be about the same, as these are needed to maintain the basic metabolic and developmental processes of a metazoan. To date, our analysis of 9430 sequences obtained from three tissues of immunological interest (hemocytes, gills, and hepatopancreas) has resulted in the identification of 2735 different and unique genes (termed unigenes), based on searching the GenBank databases maintained by the National Center for Biotechnology Information (NCBI).
Our current shrimp unigene collection represents something less than 10% of the estimated total number of genes. The libraries from which these unigenes were initially collected were not manipulated, and thus could be assumed to give a ‘snapshot’ of the normal physiological expression of genes in these tissues. Thus, by asking how many of the transcripts encode molecules with known functions, there is an opportunity to assess the investment by the shrimp of metabolic energy in distinct physiological systems, including immune function.
This approach revealed, perhaps unsurprisingly, that shrimp hemocytes invest a very large fraction of their metabolic energy in immune defense, transcribing many copies of genes involved in defense against microbial infection, particularly the production of antimicrobial peptides that are active against bacteria and fungi. A surprising and novel discovery was that the shrimp hepatopancreas expresses immune-function genes, including those encoding proteins (e.g. ß-glucan binding protein and lectins) involved in immune recognition and the binding of foreign materials (Gross et al., 2001).
To selectively increase the representation of genes regulated by the immune response, more than half of the genes in our collection have been isolated from gene libraries that have been manipulated to enrich their content of transcripts whose expression is either increased or decreased in animals undergoing specific challenges. Such libraries were constructed from shrimp infected with WSSV and from shrimp whose immune systems had been stimulated with a mixture of heatkilled bacteria (Vibrio parahemolyticus, Aerococcus viridans) and fungal spores (Fusarium oxysporum).
Hyperthermia has been reported to improve the survival of L. vannamei to WSSV, so we also collected sequences from libraries that were enriched for genes up- and down-regulated during hyperthermia and WSSV infection. Our shrimp gene collection, although representing only a fraction of the complete ‘transcriptome’, indicates the presence in shrimp of pathways that have been well-characterized (in other animals) as having an important role in immune function.
Thus, these same pathways are very likely involved in the immune response of shrimp. Our collection contains genes encoding proteins known to play immune roles in other organisms and that had previously been unreported in crustacea (Table 1). Genes of predicted immune functions represent no more than 6% of the current shrimp unigene collection, but the presently unidentified genes (that represent approximately twothirds of the unigene collection) may turn out to be highly important. Given our ignorance of the shrimp immune response, especially regarding the shrimp antiviral immune response, it is likely that this ‘unknown’ group contains genes whose products have important functions in shrimp immunity. The shrimp ESTs that have been collected are publicly available at Marine Genomics, and at the national NCBI database (GenBank).
To understand how the genes we have identified are involved in the immune response and other physiological processes is a challenging project. One approach is to consider how their abundance changes upon environmental stimulation or infection with a disease agent. It is likely that the suite of genes regulated by a specific stimulus will contain the genes that encode the proteins involved in mediating the animal’s reaction to that stimulus. To measure the expression of shrimp genes, a cDNA microarray has been created on which our current shrimp and WSSV gene collections are represented. Our shrimp microarray is based on glass slides, onto which solutions of DNA (as microdrops) for each gene are spotted at high density. Over 100 thousand genes can be spotted to a single glass slide using modern robotic microarray printing techniques.
If we make the reasonable assumption that the physiological status of an organism will be reflected by the levels of expression of specific genes, cDNA microarrays can be used to gain insight into how environmental changes and infection with pathogens can affect shrimp health. The quantification of the relative expression of thousands of genes simultaneously (the ‘transcriptomic signature’) can provide a complex but comprehensive picture of cellular and molecular mechanisms working in concert in situations of stress and disease. The analysis of the transcriptional signature can provide both diagnostic and prognostic information about stress and infection, as well as serve to identify novel genes of potential importance to immune function. The sorts of functional genomics studies that we have embarked on also have the potential to provide new insights into how the shrimp immune response is coordinated and how it is interrelated to other physiological processes in shrimp.
Table 1.Immune effectors and signal transduction pathways regulated during shrimp antiviral response.
Double stranded RNA mediated antiviral immunity in shrimp
Replication of viral RNA genomes and expression of viral RNAs rich in secondary structures often lead to accumulation of double-stranded RNA (dsRNA) in virally infected cells (reviewed by Jacobs and Langland, 1996). In contrast, the occurrence of dsRNA in normal (uninfected) eukaryotic cells is thought to be rare and tightly controlled both temporally and spatially. Thus, it is not surprising that the innate immune system has developed the capacity to interpret the presence of dsRNA as a sign of viral infection.
For instance, in vertebrates the antiviral immune response is strongly stimulated by dsRNA. Exposure of vertebrate cells to dsRNA analogues results in many of the same cellular responses that accompany active viral infections, including apoptosis, suppression of protein synthesis, and production of antiviral cytokines, namely interferons. This situation is very much analogous to the way the immune system recognizes molecular structures that are unique to bacteria, such as lipopolysaccharides and peptidoglycans.
Among invertebrates (e.g. flies and shrimp), recognition of these bacterial-associated molecules is well documented, and some of the antimicrobial responses that result from such recognition have been thoroughly described (reviewed by Hetru et al., 2003). The way invertebrates sense viral infections and the molecular events that constitute the invertebrate antiviral response pose one of the big questions regarding the evolution of the immune system that has remained relatively unexplored.
Invertebrates lack genes homologous to interferons, interferon receptors, and to most of the prominent effectors of the interferon response (e.g. RNAdependent protein kinase). This has been interpreted for a long time as an inability of invertebrates to sense dsRNA as an indicator of viral infections. While it is clear that the cytokines and effectors that mediate a potential antiviral immune response in invertebrates are structurally different from those of the vertebrates, the assumption that invertebrates lack altogether the ability to sense viruses through dsRNA is probably wrong. Our work has demonstrated that the marine shrimp L. vannamei, when exposed to dsRNA, is capable of mounting broad-spectrum antiviral responses (Robalino et al., 2004). These responses were discerned using a sensitive bioassay system capable of resolving statistically significant changes in levels of protection against carefully titrated viral challenges (Prior et al., 2003).
In these studies, we show that: 1) injection of dsRNA prior to viral infection induces significant resistance against at least two unrelated viruses, WSSV and TSV; and 2) dsRNA exerts this antiviral protection through mechanisms that are independent of its sequence and base composition. Although the protection breaks down when shrimp are challenged with higher viral doses, these observations are of great significance in the context of the evolution of antiviral immunity, because they suggest that invertebrates, just like vertebrates, possess general mechanisms of immunity that are active against phylogenetically divergent viruses. The fact that such mechanisms are both broad-spectrum and inducible by dsRNA in a sequence-independent manner, further suggests that invertebrates possess antiviral mechanisms analogous to the interferon response from vertebrates.
Our current work is focused on using the functional genomics tools we have developed to gain insight into the genes responsible for dsRNA-induced antiviral immunity in shrimp. Identification of the profiles of gene expression that result from dsRNA stimulation and viral infections should provide leads into identifying some of the molecular players involved in invertebrate antiviral responses. Phenomena associated with dsRNA mediated gene silencing (RNAi) add further complexity to the function of dsRNAs in vivo.
Cells from many organisms including plants, nematodes, insects and mammals are able to process dsRNA through cellular mechanisms which eventually degrade homologous mRNAs, thus shutting down translation of targeted gene products (Fire et al., 1998; Misquitta and Paterson, 1999; Svoboda et al., 2000). This is thought to be a mechanism to block the production of viral gene products in host cells. Further research in our laboratory is exploring the relationships between the non-specific antiviral response induced by dsRNA and the potential for viral gene silencing by virus specific dsRNA.
Opportunities for the future
The studies described here have important ramifications for improving our fundamental understanding of the shrimp immune system and its genetic control, for developing new opportunities for antiviral chemotherapies for shrimp, and for developing more resistant lines of shrimp stocks.
Using advanced molecular tools and new computational and mathematical tools to analyze the massively paralleled data they generate, we will be able to examine correlations among thousands of genes and the disease resistance of an organism. These analyses should provide for identification of gene-gene interactions, which could be amenable to selection. Traditional hypothesis-based experimentation derived from genetic discoveries will further our understanding of physiological pathways and their genetic control.
For example, our finding of new viral-specific antiviral resistance mechanisms in shrimp has yielded promising research avenues for identification of new cytokines and elucidating mechanisms of signaling pathways. Further research in our laboratory is examining the involvement of RNA interference (RNAi)-like processes in shrimp antiviral immunity by studying the shrimp response to viral sequence-specific dsRNA.
Promising results have been achieved, which have been submitted for publication. A new model of antiviral immunity in shrimp is being developed by which viral dsRNA engages not only innate immune pathways, but also an RNAi-like mechanism, to induce potent antiviral responses in vivo. As one discovery leads to others, effective treatments may be developed which can have very significant repercussions for this growing industry.
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Authors: MARINE GENOMICS GROUP, CHARLESTON, SOUTH CAROLINA, USA
Department of Natural Resources, Medical University of South Carolina, National Oceanographic and Atmospheric Administration, Hollings Marine Laboratory, Charleston, South Carolina, USA
