Bl Munday

M6P/IGF2R Imprinting Evolution in Mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.

Evolution of interleukin-1β

Abstract All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawless vertebrates posses an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it has been speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little has been done to look at the evolution of cytokine genes, despite well-known biological activities within vertebrates. In recent years, cytokines, which have been well characterised within mammals, have begun to be cloned and sequenced within non-mammalian vertebrates, with the number of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalian homologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are unique to certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a wide variety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1β sequences there are now full cDNA sequences and genomic organisations available from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach. This review considers how the IL-1β gene has changed through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates.

Nodaviruses as pathogens in larval and juvenile marine finfish

Nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish in aquaculture worldwide. The causative agents are non-enveloped, icosahedral, RNA viruses with diameters in the range of 25–34nm. They display considerable serological and molecular homology, although the present evidence suggests that there is more than one agent causing disease in a range of species. The diseases produced by these nodaviruses invariably involve the central nervous system and the retina where they usually produce vacuolation and cell necrosis. Virus particles are numerous within the cytoplasm of affected cells and extracellularly. As a result of the lesions, affected larvae/juveniles exhibit a range of neurological signs usually culminating in high mortality rates (not uncommonly 100%). One virus, that of the European sea bass, has recently been cultured in a fish cell line, but to date techniques such as the fluorescent antibody test, enzyme-linked immunosorbent assay and polymerase chain reaction have relied upon the harvest of purified viral antigen from infected tissues rather than obtaining these reagents from viruses grown in cell cultures. The epidemiology of these diseases is only partly understood. All appear to transmit readily by cohabitation of infected fish with naive larvae or juveniles, but vertical transmission has only been recognized with striped jack nervous necrosis and sea bass nervous necrosis viruses. Consequently, some aspects of disease control are based on first principles, rather than application of a full understanding of epidemiological factors.

PHYLOGENY OF THE MULTIVALVULIDAE (MYXOZOA: MYXOSPOREA) BASED ON COMPARATIVE RIBOSOMAL DNA SEQUENCE ANALYSIS

Fish parasites of the Multivalvulida (Myxozoa, Myxosporea) are widespread and can be associated with mortality or poor flesh quality in their commercially important marine hosts. Traditional classifications divide members of this order into families based on spore valve and polar capsule numbers. Analyses of the small-subunit (SSU) ribosomal DNA (rDNA) sequences from all representative families in the order (Trilosporidae, Kudoidae, Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae) indicate that a revision of the taxonomy and nomenclature is warranted. In our phylogenetic analysis of (SSU and large subunit) rDNA sequences, members of Pentacapsula, Hexacapsula, and Septemcapsula root within a clade of Kudoa species with Unicapsula (Trilosporidae) as an outlier to these genera. Therefore, we propose to synonymize Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae with Kudoidae alter the diagnosis of Kudoidae and Kudoa to accommodate all marine myxozoan parasites having 4 or more shell valves and polar capsules.

Evidence for an early appearance of modern post‐switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE, IgG1 and IgG2 from a monotreme, the duck‐billed platypus, Ornithorhynchus anatinus

To trace the emergence of the modern post‐switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg‐laying mammal, a monotreme, the duck‐billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR‐based screening using degenerate primers. The fragments obtained were used as probes to isolate full‐length cDNA clones of three platypus post‐switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post‐switch isotypes appeared very early in the mammalian lineage, possibly already 310–330 million years ago.

Heavy Chain V Region Diversity in the Duck-Billed Platypus (Ornithorhynchus anatinus): Long and Highly Variable Complementarity-Determining Region 3 Compensates for Limited Germline Diversity

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in ∼36% of the platypus VH sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.

Further studies on acquired resistance to amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L.

Trials were designed to test the efficacy of freshwater treatments for amoebic gill disease (AGD) of Atlantic salmon, Salmo salar L., and the effect they had on the acquisition of resistance to reinfection with AGD. The first trial involved fish being given an industry-simulated freshwater bath of 2-3 h duration which simulated treatments given on farms. These fish did not display appreciable resistance to reinfection. The second trial involved four groups of fish which had been infected with and treated for AGD in a number of different ways. Once again the fish that had been infected for the first time and given a single 2-3 h freshwater bath and then re-exposed did not exhibit appreciable resistance to reinfection. In contrast, those fish that had been given a second 2-3 h freshwater bath and those that had been maintained in freshwater for 4 weeks displayed high levels of resistance. There is preliminary evidence to suggest that this resistance could be related to stimulation of the non-specific immune system.

The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

The relative susceptibility of fish to infections by Flexibacter columnaris and Flexibacter maritimus

Abstract The susceptibility of freshwater and marine fish to infection by Flexibacter columnaris and Flexibacter maritimus was assessed in laboratory based experiments. Flexibactor columnaris produced a more severe disease in barramundi compared to goldfish. Both species suffered greater mortality at 25 °C than at 20 °C. Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) had similar levels of susceptibility to F. maritimus infection in seawater with approximate LC 50 values of 2.3 × 10 5 and 1.6 × 10 6 CFU mL −1 respectively. However, at salinity of 15 %. greenback flounder ( Rhombosolea tapirina ) were more resistant to F. maritimus than Atlantic salmon.

Possible differences in pathogenicity between cane toad-, frog- and platypus-derived isolates of Mucor amphibiorum, and a platypus-derived isolate of Mucor circinelloides

Platypuses (Ornithorhynchus anatinus) in the north of the island state of Tasmania, Australia, suffer from a serious disease called ulcerative mycosis, which is responsible for high morbidity and, presumably, mortality rates in areas where it occurs. The disease is caused by the dimorphic fungus Mucor amphibiorum, which is also found in Queensland, New South Wales and Victoria. However, it does not cause disease in platypuses in those states. It has been previously reported that a closely related fungus, Mucor circinelloides, may also be capable of causing this disease. This paper describes pathogenicity trials involving cane toads (Bufo marinus) as the experimental model. The toads were infected with either Tasmanian, platypus-derived M. amphibiorum, West Australian, frog-derived M. amphibiorum, Queensland cane-toad-derived M. amphibiorum or Tasmanian platypus-derived M. circinelloides. The Tasmanian isolates of M. amphibiorum were more likely to cause a serious, long-term infection than were Queensland or West Australian isolates, and (+) mating types caused a more serious infection than the (-) mating type. The isolate of M. circinelloides was incapable of infecting the toads, lending further weight to the theory that it represents an environmental contaminant. The results suggest that an endemic strain of M. amphibiorum has mutated and become pathogenic to platypuses. Alternatively, a pathogenic strain of M. amphibiorum may have been introduced into Tasmania, where it is infecting a naïve population.