The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7% of the 'lightly' infected oysters, and 66.7% of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia. KEY WORDS: Bonamia ostreae · PCR · Haplosporidia · Ostrea edulisResale or republication not permitted without written consent of the publisher

Infectious marine diseases can decimate populations and are increasing among some taxa due to global change and our increasing reliance on marine environments. Marine diseases become emergencies when significant ecological, economic or social impacts occur. We can prepare for and manage these emergencies through improved surveillance, and the development and iterative refinement of approaches to mitigate disease and its impacts. Improving surveillance requires fast, accurate diagnoses, forecasting disease risk and real-time monitoring of disease-promoting environmental conditions. Diversifying impact mitigation involves increasing host resilience to disease, reducing pathogen abundance and managing environmental factors that facilitate disease. Disease surveillance and mitigation can be adaptive if informed by research advances and catalysed by communication among observers, researchers and decision-makers using information-sharing platforms. Recent increases in the awareness of the threats posed by marine diseases may lead to policy frameworks that facilitate the responses and management that marine disease emergencies require.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus.Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 lM while a mixture of histones H2B and H4 was active at 3 lM. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.

Examination of the oyster Ostreola equestris as a potential reservoir host for a species of Bonamia discovered in Crassostrea ariakensis in North Carolina (NC), USA, revealed a second novel Bonamia sp. Histopathology, electron microscopy, and molecular phylogenetic analysis support the designation of a new parasite species, Bonamia perspora n. sp., which is the first Bonamia species shown to produce a typical haplosporidian spore with an orifice and hinged operculum. Spores were confirmed to be from B. perspora by fluorescent in situ hybridization. Bonamia perspora was found at Morehead City and Wilmington, NC, with an overall prevalence of 1.4% (31/2,144). Uninucleate, plasmodial, and sporogonic stages occurred almost exclusively in connective tissues; uninucleate stages (2-6 microm) were rarely observed in hemocytes. Spores were 4.3-6.4 microm in length. Ultrastructurally, uninucleate, diplokaryotic, and plasmodial stages resembled those of other spore-forming haplosporidians, but few haplosporosomes were present, and plasmodia were small. Spore ornamentation consisted of spore wall-derived, thin, flat ribbons that emerged haphazardly around the spore, and which terminated in what appeared to be four-pronged caps. Number of ribbons per spore ranged from 15 to 30, and their length ranged from 1.0 to 3.4 microm. Parsimony analysis identified B. perspora as a sister species to Bonamia ostreae.

A "universal non-metazoan" polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of nonmetazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5'-GTGCCAGCAGCCGCG-3') and 18S-EUK1134-R (5'-TTTAAGTTTCAGCCTTGCG-3') with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a nonmetazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000 x more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.