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.

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.

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.

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.

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.

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.

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.

Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge.

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.

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.

Enhancer and promoter activity in the JH to IGHM intron of the Pekin duck, Anas platyrhynchos.

A transcriptional enhancer, Emu, was defined in the IGH locus of the Pekin duck, Anas platyrhynchos. Regions of DNA from the JH to IGHM intron were cloned into reporter constructs containing the SV40 promoter and transiently transfected into chicken B and T lymphocytes. A strong transcriptional activity, of several hundred-fold greater than that of a reporter construct with the promoter alone, was localized to a 281bp region that contains 2 E-box motifs, CAGCTG. This fragment showed enhancer activity in both orientations and was active in chicken B cells but not in T cells. When the activity of the enhancer was tested in constructs without a promoter, it showed high transcriptional activity in the forward orientation, but much less activity (by two orders of magnitude) when tested in the reverse orientation. This suggests that the fragment contains not only enhancer activity but may contain promoter activity analogous to that of the Imu promoter described in mammals. Thus it appears that the location, but not the fine structure, of the Emu enhancer was established before the evolutionary divergence of the avian and mammalian lineages some 300Myr ago.

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.

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.

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.

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.

Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors.

Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.g., Jenny et al., Eur. J. Biochem. 2005; 271:1702-1712) defines an unprecedented diversity of MT primary structure and domain organization in the American oyster, Crassostrea virginica. Two novel molluscan MT families are described. One of these (CvMT-III) is characterized by the presence of two beta-domains and the absence of alpha-domains. This family exhibits constitutive expression during larval development and is the dominant CvMT isoform expressed in larvae. CvMT-III displays low basal levels of expression in adult tissues and only moderate responsiveness to metal challenges in both larvae and adults. A second novel MT isoform (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques following a 4-hour immune challenge with heat-killed bacteria (Vibrio, Bacillus, Micrococcus spp. mixture). Based on conservation of the cysteine motifs, this isoform appears to be a sub-family related to the molluscan alphabeta-domain MTs. A series of amino acid substitutions has resulted in four additional cysteines which give rise to a Cys-Cys motif and three Cys-Cys-Cys motifs. Northern blot analyses demonstrate that CvMT-IV is down-regulated upon sterile wounding and immune challenge, displays moderate expression in larvae and adults and differential gene induction in response to metals exposure.

Regulation of the immunoglobulin heavy chain locus expression at the phylogenetic level of a bony fish: transcription factor interaction with two variant octamer motifs.

Transcriptional control of the IGH locus in teleosts is not fully understood, but evidence from catfish and zebrafish indicates major roles for octamer-binding (Oct) and E-protein transcription factors. A pair of variant octamer motifs in the Emu3' enhancer of the catfish has been shown to be particularly important in driving expression, justifying detailed study of their function. These octamer motifs were examined to determine if they bound Oct2 POU domains in monomeric or dimeric (PORE and MORE) configurations. While catfish Oct2 was shown to be capable of binding PORE and MORE motifs in dimeric conformation, the two octamer motifs in Emu3' bound Oct2 POU domains only in monomeric configuration. Catfish Oct2, when bound in this monomeric conformation, was shown to bend the DNA helix. The relative position of the two octamer motifs in Emu3' affected the activity of the enhancer, and moving the octamer motifs closer together by 5 bp greatly reduced the activity of the enhancer. This effect was not due to steric hindrance preventing the binding of Oct transcription factors to the two motifs, but rather was shown to be due to the disruption of an additional transcription factor binding site lying between the two octamer motifs.

Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck.

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.

Channel catfish immunoglobulins: repertoire and expression.

The channel catfish, Ictalurus punctatus, is widely recognized as an important model for studying immune responses in ectothermic vertebrates. It is one of the few fish species for which defined viable in vitro culture systems have been established and is currently the only fish species from which a variety of functionally distinct clonal leukocyte lines are available. Moreover, there is a large basis of biochemical and molecular information on the structure and function of catfish immunoglobulins (Igs). Catfish, as other teleosts, have a tetrameric homolog of IgM as their predominant serum Ig plus a homolog of IgD. They also have genetic elements basically similar to those of mammals, which encode and regulate their expression. The catfish Ig heavy (H) chain locus is a translocon-type locus with three Igdelta genes linked to an Igmu gene or pseudogene. The catfish IgH locus is estimated to contain approximately 200 variable (V) region genes representing 13 families as well as at least three diversity (D) and 11 joining (JH) genes. The catfish has two light (L) chain isotypes, F and G, both encoded by loci organized in multiple cassettes of VL-JL-CL with the VL in the opposite transcriptional orientation. Hence, all requisite components for encoding antibodies are present in the catfish, albeit with certain variations. In the future, whether or not additional unique features of Ig function and expression will be found remains to be determined.

Evolution of antibody class switching: identification and transcriptional control of an Inu exon in the duck (Anas platyrhynchos).

Immunoglobulin class switching is characteristic to the tetrapod lineage, but the nature of this process has been elucidated only in mammals, where I-exon transcription initiates and directs the recombination in the IgH locus. Here, it is shown that an I-exon occurs 5' of the nu (IgY constant region) gene of the duck (Anas platyrhynchos): it is longer than mammalian I-exons and comprised primarily of tandem repeats. The Inu promoter was identified and shown to be responsive to stimulation with IL-4 but not LPS. It contains Oct, LYF-1, ATF, and C/EBP motifs. Site directed mutagenesis indicates that 2 C/EBP motifs are uniquely necessary for the response of the promoter to IL-4, as tested in the mouse pre-B cell line, 70Z/3. These results support the conclusion that the signal transduction pathways controlling I-exon promoter responses to cytokines have been highly conserved in vertebrate evolution.