In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages.

Hematodinium is a parasitic dinoflagellate of numerous crustacean species, including the economically important Atlantic snow crab, Chionoecetes opilio. The parasite was cultured in vitro in modified Nephrops medium at 0 °C and a partial characterization of the life stages was accomplished using light and transmission electron microscopy (TEM). In haemolymph from heavily infected snow crabs two life stages were detected; amoeboid trophonts and sporonts. During in vitro cultivation, several Hematodinium sp. life stages were observed: trophonts, clump colonies, sporonts, arachnoid sporonts, sporoblasts and dinospores. Cultures initiated with sporonts progressed to motile dinospores; however, those initiated with amoeboid trophonts proliferated, but did not progress or formed schizont-like stages which were senescent artefacts. Plasmodial stages were associated with both trophonts and sporonts and could be differentiated by the presence of trichocysts on TEM. Macrodinospores were observed but not microdinospores; likely due to the low number of Hematodinium sp. cultures that progressed to the dinospore stage. No early life stages including motile filamentous trophonts or gorgonlocks were observed as previously noted in Hematodinium spp. from other crustacean hosts. All Hematodinium sp. life stages contained autofluorescent, membrane-bound electron dense granules that appeared to degranulate or be expelled from the cell during in vitro cultivation.

Bacterial communities associated with lesions of two forms of shell disease in the American lobster (Homarus americanus, Milne Edwards) from Atlantic Canada.

Shell disease is a major threat to the American lobster (Homarus americanus, Milne Edwards) fishery. Here we describe the composition of microbial communities associated with lesions of 2 forms of shell disease in Atlantic Canada, (i) a trauma shell disease (TSD) characterized by massive lesions and (ii) an enzootic shell disease (EnSD) characterized by irregularly shaped lesions with a distinct orange to yellow color. The microbiology of the lesions was described by polymerase chain reaction and denaturing gradient gel electrophoresis of 16S rDNA amplified from scrapings of the shell lesions and was compared with communities of unaffected carapaces and previously described forms of shell diseases. Both TSD and EnSD lesions were dominated by members of Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria, all commonly detected in other forms of shell disease; however, unique members of Epsilonproteobacteria were also present. Two Vibrio spp. and 2 Pseudoalteromonas spp. were dominant in lesions of TSD and a Tenacibaculum sp. and Tenacibaculum ovolyticum were dominant in lesions of EnSD. The TSD and EnSD in this study contained similar taxa as other shell disease forms; however, their microbiology is mostly different and neither resembles that of epizootic shell disease.

Genomic characterization of Neoparamoeba pemaquidensis (Amoebozoa) and its kinetoplastid endosymbiont.

We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan Neoparamoeba pemaquidensis. The genome of this "Ichthyobodo-related organism" was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp.

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus).

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.

Diseases of American lobsters (Homarus americanus): a review.

The American lobster fishery is a significant economic driver in coastal communities of North America. Increasingly, the impacts of infectious disease are recognized as important components and factors in the population ecology and subsequent management of the lobster fishery. Both environmental and anthropogenic factors impact marine diseases. The review herein highlights aspects of several important bacterial, fungal and protistan diseases, including gaffkemia, shell disease, vibriosis, disease caused by species of Lagenidium, Haliphthoros and Fusarium, paramoebiasis and Bumper Car disease. As the global environment continues to change, these diseases could more severely affect both wild caught and impounded lobsters.

Microheterogeneity and coevolution: an examination of rDNA sequence characteristics in Neoparamoeba pemaquidensis and its prokinetoplastid endosymbiont.

Neoparamoeba pemaquidensis, the etiological agent of amoebic gill disease, has shown surprising sequence variability among different copies of the 18S ribosomal RNA gene within an isolate. This intra-genomic microheterogeneity was confirmed and extended to an analysis of the internal transcribed spacer (ITS) region. High levels of intra-genomic nucleotide diversity (Pi=0.0201-0.0313) were found among sequenced ITS regions from individual host amoeba isolates. In contrast, the ITS region of its endosymbiont revealed significantly lower levels of intra-genomic nucleotide diversity (Pi=0.0028-0.0056) compared with the host N. pemaquidensis. Phylogenetic and ParaFit coevolution analyses involving N. pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The observation of non-shared microheterogeneity and coevolution emphasizes the complexity of the interactions between N. pemaquidensis and its obligate endosymbiont.

Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont.

Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to correctly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA).

Genetic characterization of the lobster pathogen Aerococcus viridans var. homari by 16S rRNA gene sequence and RAPD.

A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.