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...

The response of crustaceans to pathogens is believed to depend solely on innate, nonadaptive immune mechanisms, including phagocytosis, encapsulation, clotting, and a variety of soluble antimicrobial activities. Arthropod antimicrobial peptides, while characterized primarily from insects, also have been isolated from crustaceans. Expressed sequence tag analysis of hemocyte complementary DNA libraries from 2 species of shrimp, Litopenaeus vannamei and Litopenaeus setiferus, revealed transcripts with strong sequence similarity to an 11.5-kDa antibacterial peptide (crustin Cm1) found in Carcinus maenas. Crustins were also observed to contain motifs common to proteinase inhibitors. Analysis of these cDNA libraries yielded at least 3 different isoforms of this peptide in L. vannamei (crustin Lv1-Lv3) and 3 in L. setiferus (crustin Ls1-Ls3). Further analysis of a second L. vannamei cDNA library revealed the presence of 3 more possible isoforms (crustin Lv4-Lv6), which differed from those seen in the first L. vannamei cDNA library. Genomic Southern blot analysis revealed a complex family of crustin-related sequences. However, full-length crustin appears to be encoded by a much more restricted subset of sequences within this family.

Penaeidins, a unique family of antimicrobial peptides (AMPs) with both proline and cysteine-rich domains, were initially identified in the hemolymph of the Pacific white shrimp, Litopenaeus vannamei. Described here are the results of an investigation of penaeidin diversity in individual shrimp from two species, L. vannamei and L. setiferus (Atlantic white shrimp). We report the discovery of a novel penaeidin class, designated penaeidin 4 present in both L. vannamei and L. setiferus, and that all penaeidin classes were expressed in a single individual. In addition, nearly all penaeidins, regardless of class, shared an identical leader sequence while differing dramatically in the remainder of the peptide. Several new class 3 isoforms were identified, as well as sequence variants of Lv3a, which differ in the 3' untranslated region. Penaeidin sequence variability (especially of class 3), within and between individuals, is not interpretable as simple allelic polymorphism and may reflect alternate transcriptional mechanisms. Penaeidins are encoded by a small number of genetic loci and are not likely representatives of a large gene family produced by whole gene duplication, but rather may be products of a multi-component locus. Based on phylogenetic analysis, penaeidins fall into three classes where 1 and 2 are combined while classes 3 and 4 remain distinct. Phylogenetic analysis indicates that all classes of penaeidin were likely present in both species prior to speciation.

ABSTRACT:In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 ± 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27°C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD 50 and LD 90 . The LD 50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 × 10 7 (Trial 1) and 1:6.667 × 10 7 (Trial 2). The LD 50 s of 2 different WSSV injection preparations were estimated at 1:4.444 × 10 6 and 1:4.505 × 10 6 . The LD 50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20°C and 1:1272 at 27°C. A second waterborne TSV inoculum challenge at 27°C produced an LD 50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens. KEY WORDS: TSV · WSSV · Bioassay · Shrimp · Litopenaeus vannameiResale or republication not permitted without written consent of the publisher

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