The effects of warming on the ecophysiology of two co-existing kelp species with contrasting distributions

The northeast Atlantic has warmed significantly since the early 1980s, leading to shifts in species distributions and changes in the structure and functioning of communities and ecosystems. This study investigated the effects of increased temperature on two co-existing habitat-forming kelps: Laminaria digitata, a northern boreal species, and Laminaria ochroleuca, a southern Lusitanian species, to shed light on mechanisms underpinning responses of trailing and leading edge populations to warming. Kelp sporophytes collected from southwest United Kingdom were maintained under 3 treatments: ambient temperature (12 °C), +3 °C (15 °C) and +6 °C (18 °C) for 16 days. At higher temperatures, L. digitata showed a decline in growth rates and Fv/Fm, an increase in chemical defence production and a decrease in palatability. In contrast, L. ochroleuca demonstrated superior growth and photosynthesis at temperatures higher than current ambient levels, and was more heavily grazed. Whilst the observed decreased palatability of L. digitata held at higher temperatures could reduce top-down pressure on marginal populations, field observations of grazer densities suggest that this may be unimportant within the study system. Overall, our study suggests that shifts in trailing edge populations will be primarily driven by ecophysiological responses to high temperatures experienced during current and predicted thermal maxima, and although compensatory mechanisms may reduce top-down pressure on marginal populations, this is unlikely to be important within the current biogeographical context. Better understanding of the mechanisms underpinning climate-driven range shifts is important for habitat-forming species like kelps, which provide organic matter, create biogenic structure and alter environmental conditions for associated communities

Detection of Minchinia occulta in samples of pearl oysters Pinctada maxima infected by Haplosporidium hinei

Objective To determine if juvenile pearl oysters (Pinctada maxima) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta. Design Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144-bp and 220-bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin-fixed H. hinei-infected pearl oyster samples was sequenced. A 25-bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH. Results The results of DNA-based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks. Conclusion Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta, which was recently identified in rock oysters within the pearl-producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co-infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease

Spore ornamentation of Haplosporidium hinei n. sp. (Haplosporidia) in pearl oysters Pinctada maxima (Jameson, 1901)

An infection of pearl oysters, Pinctada maxima, attributed to a Haplosporidium sp. by Hine and Thorne (1998) has been detected on 3 occasions and is considered to represent a serious concern to the pearling industry in Australia. The spore ornamentation of the parasite was determined by scanning electron microscopy and transmission electron microscopy. Spores of the parasite were pleomorphic, or elongated 3.5-4 mcm x 2.5-3.0 mcm in size. Two filaments were wound around the spore and originated from 2 'knob-like' posterior thickenings. Both filaments passed up one side of the spore together until just below the operculum whereupon each split and passed obliquely under the lip of the opercula lid. Each filament wrapped around the spore 4 times. The posterior thickenings seem to appear late in the development of the spore and were composed of spore wall material. A second set of branching tubular filaments composed of a different material was observed on the spore body although not on mature spores possessing a 'knob-like' posterior thickening. The ornamentation on the spores of the pearl oyster parasite was unique amongst described haplosporidian species where spore ornamentation is known. The parasite is named in this manuscript as Haplosporidium hinei n. sp

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 mcm-5.0 mcm x 3.5-4.1 mcm in size. The nucleus of the sporoplasm measured 1.5-2.3 mcm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures

Intracellular ciliated protozoal infection in silverlip pearl oysters, Pinctada maxima (Jameson, 1901)

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 μm (range of 2.73–7.47 μm, n = 9) in width and 11.15 μm (range of 9.02–16.2 μm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p < 0.001, C = 0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38–50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection

Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters (Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude -20.4'S longitude 115.53'E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite's rRNA gene. An oligonucleotide probe was constructed from the parasite's SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite's rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123-13], in archived rock oyster tissues from the same coastline

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166 bp polynucleotide probe while the second used a 30 bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220 bp region and detected Minchinia sp. DNA from 50 ng of genomic DNA extracted from the tissues of infected oysters and 10 fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite

Aquatic Animal Health Subprogram: Development of diagnostic tests to assess the impact of Haplosporidium infections in pearl oysters

Haplosporidians are protozoa that are internal parasites of lower vertebrates and invertebrates, and can be amongst the most dangerous of diseases of molluscs. They are able to cause disease particularly where naïve hosts are exposed to the parasite