Insights into the antiviral functions of the RNAi machinery in penaeid shrimp.

Over the last decade, RNA interference pathways have emerged in eukaryotes as critical regulators of many diverse biological functions including, among others, transcriptional gene regulation, post-transcriptional gene silencing, heterochromatin remodelling, suppression of transposon activity, and antiviral defences. Although this gene silencing process has been reported to be relatively well conserved in species of different phyla, there are important discrepancies between plants, invertebrates and mammals. In penaeid shrimp, the existence of an intact and functional RNAi machinery is supported by a rapidly growing body of evidence. However, the extent to which this process participates to the host immune responses remains poorly defined in this non-model organism. This review summarizes our current knowledge of RNAi mechanisms in shrimp and focuses on their implication in antiviral activities and shrimp immune defences.

Quantitative methods to characterize morphological properties of cell lines.

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

Health status, infection and disease in California sea lions (Zalophus californianus) studied using a canine microarray platform and machine-learning approaches.

Conservation biologists face many challenges in assessing health, immune status and infectious diseases in protected species. These challenges include unpredictable sample populations, diverse genetic and environmental backgrounds of the animals, as well as the practical, legal and ethical issues involved in experimentation. The use of whole genome scale transcriptomics with animal samples obtained in a minimally invasive manner is an approach that shows promise for health assessment. In this study we assessed the utility of a microarray to identify changes in gene expression predictive of health status by interrogating blood samples from California sea lions (Zalophus californianus) in rehabilitation. A custom microarray was developed from the commercially available dog microarray (Canis familiaris) by selecting probes that demonstrated reliable cross-hybridization with RNA in sea lion blood. This custom microarray was used for the analysis of RNA from 73 sea lion blood samples, from animals with a broad spectrum of health changes. Both traditional classifying techniques and newer artificial neural network approaches correctly classified sea lions with respect to health status, primarily distinguishing between leptospirosis infection and domoic acid exposure. Real time PCR validation for a small set of genes, followed by sequencing, showed good correlation with array results and high identity (96-98%) between the dog and sea lion sequences. This approach to health status classification shows promise for disease identification in a clinical setting, and assessment of health status of wildlife.

The transcriptomic response to viral infection of two strains of shrimp (Litopenaeus vannamei).

The extent to which data-intensive studies of the transcriptome can provide insight into biological responses is not well defined, especially in the case of species (such as shrimp) where much physiological and biochemical knowledge is missing. In this study we took a transcriptomic approach to gain insight into the response to viral infection of two strains of the Pacific whiteleg shrimp (Litopenaeus vannamei) that differ in their resistance to Taura Syndrome Virus (TSV). Changes in gene expression in the hepatopancreas following infection with TSV and Yellow Head Virus (YHV) were assessed using a cDNA microarray containing 2469 putative unigenes. The null hypothesis tested was that significant differences between the transcriptomic responses to viral infection of resistant and sensitive strains would not be detected. This hypothesis was broadly rejected, with the most surprising observation being that the baseline (control, unchallenged) sensitive and resistant strains expressed distinguishable transcriptomic signatures. The resistant line was pre-disposed to lower expression of genes encoding viral (and host) proteins. Many of the genes differentiating resistant and sensitive lines are involved in protein metabolism, cellular trafficking, immune defense and stress response, although it was not possible to clearly identify candidate genes responsible for TSV resistance. In contrast to TSV challenge, YSV either failed to perturb the host transcriptome or created a "confused" response that was difficult to interpret.

Non-specific activation of antiviral immunity and induction of RNA interference may engage the same pathway in the Pacific white leg shrimp Litopenaeus vannamei.

Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length. The findings show that the length of small RNA duplexes plays a critical role in the activation of both RNAi-related and innate antiviral responses. They also suggest that these two mechanisms of antiviral response may activate the same pathway, requiring Lv-Sid 1 and Lv-Ago 2 induction.

Modelling interactions of acid-base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica.

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001-0.400 microM), Zn (0.001-3.059 microM) or Cu (0.002-0.787 microM), either alone or in combination for 1 to 27 days. We measured indicators of acid-base balance (hemolymph pH and total CO(2)), gas exchange (Po(2)), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid-base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid-base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of sub-acute exposure to contaminant mixtures.

Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis.

The reptiles are the last major group of jawed vertebrates in which the organization of the IGH locus and its encoded Ig H chain isotypes have not been well characterized. In this study, we show that the green anole lizard (Anolis carolinensis) expresses three Ig H chain isotypes (IgM, IgD, and IgY) but no IgA. The presence of the delta gene in the lizard demonstrates an evolutionary continuity of IgD from fishes to mammals. Although the germline delta gene contains 11 C(H) exons, only the first 4 are used in the expressed IgD membrane-bound form. The mu chain lacks the cysteine in C(H)1 that forms a disulfide bond between H and L chains, suggesting that (as in IgM of some amphibians) the H and L polypeptide chains are not covalently associated. Although conventional IgM transcripts (four C(H) domains) encoding both secreted and membrane-bound forms were detected, alternatively spliced transcripts encoding a short membrane-bound form were also observed and shown to lack the first two C(H) domains (VDJ-C(H)3-C(H)4-transmembrane region). Similar to duck IgY, lizard IgY H chain (upsilon) transcripts encoding both full-length and truncated (IgYDeltaFc) forms (with two C(H) domains) were observed. The absence of an IgA-encoding gene in the lizard IGH locus suggests a complex evolutionary history for IgA in the saurian lineage leading to modern birds, lizards, and their relatives.

Lack of evidence for Litopenaeus vannamei Toll receptor (lToll) involvement in activation of sequence-independent antiviral immunity in shrimp.

Injection of non-specific dsRNA initiates a broad-spectrum innate antiviral immune response in the Pacific white shrimp, Litopenaeus vannamei, however, the receptor involved in recognition of this by-product of viral infections remains unknown. In vertebrates, dsRNA sensing is mediated by a class of Toll-like receptors (TLRs) and results in activation of the interferon system. Because a TLR (lToll) was recently characterized in L. vannamei, we investigated its potential role in dsRNA recognition. We showed that injection of non-specific RNA duplexes did not modify lToll gene expression. A reverse genetic approach was therefore implemented to study its role in vivo. Silencing of lToll did not impair the ability of non-specific dsRNA to trigger protection from white spot syndrome virus and did not increase the shrimp susceptibility to viral infection, when compared to controls. In contrast, gene-specific dsRNA injected to specifically silence lToll expression activated an antiviral response. These data strongly suggest that shrimp lToll plays no role in dsRNA-induced antiviral immunity.

A transcriptomic analysis of land-use impacts on the oyster, Crassostrea virginica, in the South Atlantic bight.

Increasing utilization and human population density in the coastal zone is widely believed to place increasing stresses on the resident biota, but confirmation of this belief is somewhat lacking. While we have solid evidence that highly disturbed estuarine systems have dramatic changes in the resident biota (black and white if you will), we lack tools that distinguish the shades of grey. In part, this lack of ability to distinguish shades of grey stems from the analytical tools that have been applied to studies of estuarine systems, and perhaps more important, is the insensitivity of the biological end points that we have used to assess these impacts. In this study, we will present data on the phenotypic adjustments as measured by transcriptomic signatures of a resilient organism (oysters) to land-use practices in the surrounding watershed using advanced machine-learning algorithms. We will demonstrate that such an approach can reveal subtle and meaningful shifts in oyster gene expression in response to land use. Further, the data show that gill tissues are far more responsive and provide superior discrimination of land-use classes than hepatopancreas and that transcripts encoding proteins involved in energy production, protein synthesis and basic metabolism are more robust indicators of land use than classic biomarkers such as metallothioneins, GST and cytochrome P-450.

Sampling the skin transcriptome of the North Atlantic right whale.

As an initial step in defining the transcriptome of the North Atlantic right whale (Eubalaena glacialis) and developing functional genomic tools to study right whale health at the molecular physiological level, a cDNA library has been constructed from a skin biopsy. 2496 randomly selected clones (expressed sequence tags, ESTs) have been sequenced, and genes identified as important in the response to stress and immune challenges have been cloned by targeted RT-PCR from skin cDNA. The analysis of the EST collection (archived at www.marinegenomics.org and GenBank) showed a 34.79% redundancy, yielding 1578 unigenes and 27 potential microsatellite markers. 96 genes were cloned by targeted PCR; moreover, 52 of these genes are stress and immune function related. A Gene Ontology analysis of the unigene collection indicates that the skin is a rich source of expressed genes with diverse functions, suggesting an important role in multiple physiological processes including those related to immunity and stress response.

BOB.1 of the channel catfish, Ictalurus punctatus: not a transcriptional coactivator?

Expression of the immunoglobulin heavy chain (IGH) locus of the channel catfish (Ictalurus punctatus) is driven by the Emu3' enhancer, whose core region contains two octamer motifs and a muE5 site. Orthologues of the Oct1 and Oct2 transcription factors have been cloned in the channel catfish and shown to bind to the octamer motifs within the core enhancer. While catfish Oct2 is an activator of transcription, catfish Oct1 failed to drive transcription and may act as a negative regulator of IGH transcription. In mammals, the Oct co-activator BOB.1 (B cell Oct-binding protein1, also known as OCA-B and OBF-1) greatly enhances the transcriptional activity of Oct factors and plays an important role in the development of the immune system. An orthologue of BOB.1 has been cloned in the catfish, and its function characterized. The POU binding domain of the catfish BOB.1 was found to be 95% identical at the amino acid level with the binding domain of human BOB.1, and all the residues directly involved in binding to the Oct-DNA complex were conserved. Despite this conservation, catfish BOB.1 failed to enhance transcriptional activation mediated by endogenous or co-transfected catfish Oct2, and failed to rescue the activity of the inactive catfish Oct1. Electrophoretic mobility shift assays showed that catfish BOB.1 was capable of binding both catfish Oct1 and Oct2 when they formed a complex with the Oct motif. Analysis of recombinant chimeric catfish and human BOB.1 proteins demonstrated that the failure to drive transcription was due to the lack of a functional activation domain within the catfish BOB.1.

Interaction between E-protein and Oct transcription factors in the function of the catfish IGH enhancer.

Transcriptional control of the immunoglobulin heavy chain (IGH) locus in the channel catfish, Ictalurus punctatus, is incompletely understood. It is, however, known that 2 variant octamer motifs and a microE5 motif in the core region of the enhancer (Emicro3') are important in driving transcription, and it has been suggested that interaction between transcription factors (Oct factors and E-proteins) bound to these sites contributes to enhancer function. In this study, the functional relationships between the microE5 motif, the proximal octamer motif, and the factors that bind them have been examined. The results of mutational analysis of these motifs showed that their interaction is important to driving transcription from the enhancer. Furthermore, the catfish Oct transcription factors were capable of a physical interaction with the catfish E-proteins. These results support a role for interaction between transcription factors bound to the octamer and microE5 motifs in the function of the Emicro3' enhancer.

Function of E-protein dimers expressed in catfish lymphocytes.

E-proteins are essential class I bHLH transcription factors that play a role in lymphocyte development. In catfish lymphocytes the predominant E-proteins expressed are CFEB (a homologue of HEB) and E2A1, which both strongly drive transcription. In this study the role of homodimerization versus heterodimerization in the function of these catfish E-proteins was addressed through the use of expression constructs encoding forced dimers. Constructs expressing homo- and heterodimers were transfected into catfish B cells and shown to drive transcription from the catfish IGH enhancer. Expression from an artificial promoter containing a trimer of muE5 motifs clearly demonstrated that the homodimer of E2A1 drove transcription more strongly (by a factor of 10-25) than the CFEB homodimer in catfish B and T cells, while the heterodimer showed intermediate levels of transcriptional activation. Both CFEB1 and E2A1 proteins dimerized in vitro, and the heterodimer CFEB1-E2A1 was shown to bind the canonical muE5 motif.

Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection.

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp (Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences (LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2. A reverse genetic approach was implemented to study the in vivo role of LvALF1 in protecting shrimp from bacterial, fungal and viral infections. Injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of LvALF1 mRNA transcript abundance as determined by qPCR. Following knockdown, shrimp were challenged with low pathogenic doses of Vibrio penaeicida, Fusarium oxysporum or white spot syndrome virus (WSSV) and the resulting mortality curves were compared with controls. A significant increase of mortality in the LvALF1 knockdown shrimp was observed in the V. penaeicida and F. oxysporum infections when compared to controls, showing that this gene has a role in protecting shrimp from both bacterial and fungal infections. In contrast, LvALF1 dsRNA activated the sequence-independent innate anti-viral immune response giving increased protection from WSSV infection.

A cDNA microarray for Crassostrea virginica and C. gigas.

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus).

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.

Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: a negative regulator of immunoglobulin gene transcription?

BACKGROUND: The enhancer (Emu3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding muE5 site. An orthologue to the Oct2 transcription factor has previously been cloned in catfish and is a functionally active transcription factor. This study was undertaken to clone and characterize the Oct1 transcription factor, which has also been shown to be important in driving immunoglobulin gene transcription in mammals. RESULTS: An orthologue of Oct1, a POU family transcription factor, was cloned from a catfish macrophage cDNA library. The inferred amino acid sequence of the catfish Oct1, when aligned with other vertebrate Oct1 sequences, revealed clear conservation of structure, with the POU specific subdomain of catfish Oct1 showing 96% identity to that of mouse Oct1. Expression of Oct1 was observed in clonal T and B cell lines and in all tissues examined. Catfish Oct1, when transfected into both mammalian (mouse) and catfish B cell lines, unexpectedly failed to drive transcription from three different octamer-containing reporter constructs. These contained a trimer of octamer motifs, a fish VH promoter, and the core region of the catfish Emu3' IGH enhancer, respectively. This failure of catfish Oct1 to drive transcription was not rescued by human BOB.1, a co-activator of Oct transcription factors that stimulates transcription driven by catfish Oct2. When co-transfected with catfish Oct2, Oct1 reduced Oct2 driven transcriptional activation. Electrophoretic mobility shift assays showed that catfish Oct1 (native or expressed in vitro) bound both consensus and variant octamer motifs. Putative N- and C-terminal activation domains of Oct1, when fused to a Gal4 DNA binding domain and co-transfected with Gal4-dependent reporter constructs were transcriptionally inactive, which may be due in part to a lack of residues associated with activation domain function. CONCLUSION: An orthologue to mammalian Oct1 has been found in the catfish. It is similar to mammalian Oct1 in structure and expression. However, these results indicate that the physiological functions of catfish Oct1 differ from those of mammalian Oct1 and include negative regulation of transcription.

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues.

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g. the Interferon response) have long been thought to be restricted to vertebrates. Recent work in an underappreciated experimental model, the penaeid shrimp, is challenging these traditional distinctions, by demonstrating the existence of both innate (non sequence-specific) and RNAi-related (sequence-specific) antiviral phenomena in crustacea. Here we discuss the evidence for this bivalent role of dsRNA in the initiation of antiviral responses in shrimp, and present new data that suggest that the antiviral functions of the shrimp RNAi machinery have imposed selective pressures on an evolving viral pathogen. These findings open the door for the discovery of novel mechanisms of innate immunity, and provide a basis for the future development of strategies to control viral diseases in the commercially important penaeid shrimp.