Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates.In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequencedependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.Double-stranded RNA (dsRNA) is a hallmark of viral infections, and thus, it is not surprising that the immune system has evolved the capacity to recognize dsRNA and respond to it by mounting antiviral responses. In vertebrates, these innate antiviral responses rely in part on the recognition of dsRNA by Toll-like receptor 3 and by RNA-dependent protein kinase (32, 47). The consequences of dsRNA recognition include activation of the interferon system, initiation of apoptosis, and inhibition of cellular protein synthesis. From an evolutionary perspective, innate immune activation by dsRNA has long been thought to be exclusive to vertebrates. This view has been encouraged by the fact that genes encoding homologues of interferons, their receptors, and most of the prominent interferon-regulated genes are absent in fully sequenced invertebrate genomes (1, 7, 10, 11). Nevertheless, it is a reasonable expectation that invertebrates should have an innate immune system capable of recognizing dsRNA as a signature of viral infection. A previous study suggested such a capability by demonstrating that exposure of a marine shrimp to dsRNA induced innate antiviral immunity in a sequence-independent manner (36). The mechanisms underlying this phenomenon as well as its occurrence in other invertebrate taxa remain unknown, but it is clear that the recognition of dsRNA by another pathway, RNA interference (RNAi), is widely distributed among invertebrates and likely an important component of the invertebrate antiviral response.RNAi comprises a set of related cellular processes by which dsRNA molecules direct the suppression of gene expression based on sequence homology between the dsRNA trigger and the target gene. The specific mechanisms u...

Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNAmediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

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Eukaryotic translation initiation factor 4E (eIF4E) is 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m 7 GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described. The unigenes printed on the microarray were derived from the analyses of 7,021 expressed sequence tags obtained from standard cDNA libraries as well as from libraries generated by suppression subtractive hybridization, after challenging shrimp with a variety of immune stimuli. The general utility of the cDNA microarray was demonstrated by interrogating the array with labeled RNA from four different shrimp tissues (gills, hemocytes, hepatopancreas, and muscle) and by analyzing the transcriptomic response of shrimp to a lethal challenge with white spot syndrome virus. Our results indicate that white spot syndrome virus infection upregulates (in the hepatopancreas) genes encoding known and potential antimicrobial effectors, while some genes involved in protection from oxidative stress were found to be downregulated by the virus.

Several shrimp species from the clade Penaeidae are farmed industrially for human consumption, and this farming has turned shrimp into the largest seafood commodity in the world. The species that are in demand for farming are an anomaly within their clade because they grow to much larger sizes than other members of Penaeidae. Here we trace the evolutionary history of the anomalous farmed shrimp using combined data phylogenetic analysis of living and fossil species. We show that exquisitely preserved fossils of †Antrimpos speciosus from the Late Jurassic Solnhofen limestone belong to the same clade as the species that dominate modern farming, dating the origin of this clade to at least 145 mya. This finding contradicts a much younger Late Cretaceous age (ca. 95 mya) previously estimated for this clade using molecular clocks. The species in the farmed shrimp clade defy a widespread tendency, by reaching relatively large body sizes despite their warm water lifestyles. Small body sizes have been shown to be physiologically favored in warm aquatic environments because satisfying oxygen demands is difficult for large organisms breathing in warm water. Our analysis shows that large-bodied, farmed shrimp have more gills than their smaller-bodied shallow-water relatives, suggesting that extra gills may have been key to the clade’s ability to meet oxygen demands at a large size. Our combined data phylogenetic tree also suggests that, during penaeid evolution, the adoption of mangrove forests as habitats for young shrimp occurred multiple times independently.

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