Gregory W. Warr

IgY: clues to the origins of modern antibodies

IgY is the functional equivalent of IgG in birds, reptiles and amphibia, but many aspects of its biology are poorly understood. Recent studies have increased awareness of the genetics and functions of this molecule, and have revealed its position as the ancestor of the uniquely mammalian antibodies IgG and IgE. Here, Greg Warr, Kathy Magor and David Higgins review current knowledge of IgY structure, function and expression in the context of the evolutionary role of this primitive immunoglobulin.

Induction of Antiviral Immunity by Double-Stranded RNA in a Marine Invertebrate

ABSTRACT 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 dsRNA-mediated 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.

Double-Stranded RNA Induces Sequence-Specific Antiviral Silencing in Addition to Nonspecific Immunity in a Marine Shrimp: Convergence of RNA Interference and Innate Immunity in the Invertebrate Antiviral Response?

ABSTRACT 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, sequence-dependent 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.

Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus.

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.

Fish immunoglobulins and the genes that encode them

Abstract The nature of fish antibodies (concentrating primarily on the most studied species of bony and cartilaginous fishes) is discussed in terms of their immunoglobulin biochemistry and immunobiology. The major serum immunoglobulin (IgM) is described in detail, and structural variants of IgM are discussed in terms of their distribution in different fish species, and different anatomical sites within a fish (e.g. blood, mucus, bile). Structural variation in IgM includes the size of the constituent heavy and light polypeptide chains, and the extent to which they are covalently associated with one another. The intramolecular heterogeneity of binding sites for antigen on IgM is reviewed and possible mechanisms for the phenomenon are presented. The evidence suggests that some, but not all, species of fish possess a detectable J chain in their IgM. The general nature of the fish immune response is that IgM antibody of moderate affinity is produced and prolonged or repeated immunization: (a) fails to produce a switch to production of a non-IgM class of antibody, and (b) fails to induce substantial increases in the affinity of the specific antibodies. Evidence supports a conclusion that fish lack the typical secondary antibody response seen in mammals, and possess antibodies of limited heterogeneity. Our current understanding of the genetic basis for fish antibodies is presented. Fish appear to utilize the same basic genetic elements as mammals to encode and regulate the expression of their immunoglobulins. The teleost heavy chain (IgH) locus resembles that of mammals and amphibia in its organization. The IgH locus of elasmobranchs is arranged in a unique multicluster organization. The light chain loci of elasmobranchs are organized analogously to the heavy chain locus (in multiclusters). The structure of the light chain locus of teleosts is presently unknown. Teleost fish utilize a unique pattern of RNA processing to generate the secreted and membrane receptor forms of the IgM heavy chain. The genes encoding the unique low molecular weight Ig heavy chain found in skates and rays have been cloned and sequenced, and also display the multicluster pattern of organization. Teleost fish appear to have normal numbers of variable regions: it is hypothesized (but as yet unproven) that the failure of their IgM to increase in affinity is due to a deficiency of somatic hypermutational mechanisms in their Ig gene variable regions during B lymphocyte differentiation.

The immunoglobulin genes of fish

Abstract The current state of knowledge concerning the structure, organization, and functional expression of immunoglobulin genes in chondrichthyan and osteichthyan fish is presented.

The IgH Locus of the Channel Catfish, Ictalurus punctatus, Contains Multiple Constant Region Gene Sequences: Different Genes Encode Heavy Chains of Membrane and Secreted IgD

The δ-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to Cμ1, seven C domain-encoding exons (δ1–δ7), and a transmembrane tail. The presence of cDNA forms showing splicing of δ7 to an exon encoding a secretory tail was interpreted to indicate that membrane (δm) and secreted (δs) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three δ C region genes, each linked to a μ gene or pseudogene. The first (IGHD1) is located 1.6 kb 3′ of the functional Cμ (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo Cμ (IGHM3P), ∼725 kb 5′ of IGHM1. These two δ genes are highly similar in sequence and each contains a tandem duplication of δ2-δ3-δ4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a μ pseudogene indicates that the putative δs product may not be expressed as a chimeric μδ molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish δm transcripts appear to originate from IGHD1, whereas δs transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed δ gene. The third δ (IGHD2) is associated with a pseudo Cμ (IGHM2P); its presence is inferred by Southern blot analyses.

Enhanced Bacterial Disease Resistance of Transgenic Channel Catfish Ictalurus punctatus Possessing Cecropin Genes

The cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus promoter, was transferred to the channel catfish Ictalurus punctatus. Transgenic individuals (P1) were mated to produce individuals (F1) that exhibited enhanced disease resistance and survival when challenged with pathogenic bacteria. During the epizootic of Flavobacterium columnare in an earthen pond, the percentage of transgenic individuals containing preprocecropin B construct that survived (100%) was significantly greater (P <0.005) THAN THAT OF NONTRANSGENIC CONTROLS (27.3%). ALSO, WHEN CHALLENGED IN TANKS WITH EDWARDSIELLA ICTALURI, THE CAUSATIVE AGENT OF ENTERIC SEPTICEMIA OF CATFISH, THE PERCENTAGE OF TRANSGENIC INDIVIDUALS CONTAINING CATFISH IG LEADER CECROPIN B CONSTRUCT THAT SURVIVED (40.7%) WAS SIGNIFICANTLY GREATER (P <0.01) THAN THAT OF NONTRANSGENIC CONTROLS (14.8%). THERE WERE NO PLEIOTROPIC EFFECTS OF THE TRANSGENES, AND GROWTH RATES OF THE TRANSGENIC AND NONTRANSGENIC SIBLINGS WERE NOT DIFFERENT (P > 0.05). Inheritance of the transgene by the F1 generation, 20.2% to 30.7% was typical of that in studies with transgenic channel catfish.

THE CASE FOR SEQUENCING THE PACIFIC OYSTER GENOME

Abstract An international community of biologists presents the Pacific oyster Crassostrea gigas as a candidate for genome sequencing. This oyster has global distribution and for the past several years the highest annual production of any freshwater or marine organism (4.2 million metric tons, worth

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

3.5 billion US). Economic and cultural importance of oysters motivates a great deal of biologic research, which provides a compelling rationale for sequencing an oyster genome. Strong rationales for sequencing the oyster genome also come from contrasts to other genomes: membership in the Lophotrochozoa, an understudied branch of the Eukaryotes and high fecundity, with concomitantly high DNA sequence polymorphism and a population biology that is more like plants than any of the model animals whose genomes have been sequenced to date. Finally, oysters play an important, sentinel role in the estuarine and coastal marine habitats, where most humans live, environmental degradation is substantial, and oysters suffer intense fishing pressures and natural mortalities from disease and stress. Consumption of contaminated oysters can pose risks to human health from infectious diseases. The genome of the Pacific oyster, at 1C = 0.89 pg or ~824 Mb, ranks in the bottom 12% of genome sizes for the Phylum Mollusca. The biologic and genomic resources available for the Pacific oyster are unparalleled by resources for any other bivalve mollusc or marine invertebrate. Inbred lines have been developed for experimental crosses and genetics research. Use of DNA from inbred lines is proposed as a strategy for reducing the high nucleotide polymorphism, which can interfere with shotgun sequencing approaches. We have moderately dense linkage maps and various genomic and expressed DNA libraries. The value of these existing resources for a broad range of evolutionary and environmental sciences will be greatly leveraged by having a draft genome sequence.