Abstract

Bodonid flagellates, of the genus Ichthyobodo, have long been recognized as significant ectoparasitic pathogens of freshwater and marine fish in aquaculture, and have been the subject of extensive research. In contrast, there are only intermittent reports of bodonids and "Ichthyobodo-like" flagellates from cephalopods, and their host-parasite interaction and identity have not been reported in detail.

The recent deaths of three giant Pacific octopi, Octopus (Enteroctopus) dofleini, wild caught in Alaska, and held at the National Aquarium in Baltimore, allowed us to study the host-parasite interactions, ultrastructure, and molecular taxonomy of the Ichthyobodo-like flagellates that infected their gills.

Two of the octopi died after 12 months at the facility, following normal gamete release, and 2-4 weeks of inappetence: a 16 kg female ('C') in September 2006 and a 29 kg male ('JB') in December 2007. The third was a female ('L') who had been at the facility for 13 months, and died in November 2006 before gamete release; there was some concern that she had been exposed to a potential red algal toxin after some Palmaria algae were harvested 10 days previously.

At necropsy, tissue samples were preserved in buffered 10% formalin for histopathology. From octopus 'L', some gill tissues were subsequently transferred to a 2% paraformaldehyde-2% glutaraldehyde mixture, and then post-fixed in osmium tetroxide for ultrastructural studies; other gill tissues were frozen, and then processed for genomic DNA extraction, PCR amplification, and cloning and sequencing of the rRNA genes; phylogenetic analyses was conducted on the SSU, D1-D3 and D8-D10 LSU rDNA sequences. Gene sequences were deposited in GenBank.

On histopathology, all three individuals had moderate to heavy density infections of Ichthyobodo-like flagellates on the gills; two of the three ('JB' and 'L') showed diffuse, severe, acute, hemocytic branchitis associated with the protozoa.

Scanning electron microscopy showed that the flagellates were flattened pyriform to trapezoidal forms, approximately 6-10 µm long and 3-6 µm wide, and attached into the host cell cytoplasm by a narrow cytostome. In some regions the infection was so dense that the gill surface was completed covered with the flagellates. At the point of attachment, there was a puncture in the smooth mucus covering of the gill epithelium that formed a crater-like lesion. Transmission electron microscopy showed numerous ultrastructural features, including two unequal-width flagella lying in a U-shaped flagellar pocket, microtubules and striated fibers surrounding the flagellar pocket, and radiating fibers lying in a semi-circle around the cytostome primordial. The spherical nucleus of the flagellate had a large central nucleolus and peripheral chromatin, and the cytoplasm also contained rough endoplasmic reticulum, and mitochondria. There was an attachment plate anchoring the flagellate to the gill epithelial cell, and the cytostome process, reinforced with fibrillar structures, passed though the plate into the cytoplasm of the host cell.

Phylogenetic analysis indicated that the Ichthyobodo species identified from the octopus in this study was most closely related to those isolated from freshwater fish, including a dwarf cichlid (Apistogramma sp.), a hybrid striped bass (Morone saxatilis x Morone chrysops) and a swordtail (Xiphophorus helleri).

This study has shown the Ichthyobodo sp. can be a significant ectoparasitic pathogen of captive cephalopods. The host-parasite interaction and parasite ultrastructure are essentially similar to those of the better known Ichthyobodo spp. affecting teleosts. Molecular phylogeny showed that the Ichthyobodo sp. from the giant Pacific octopi held at the National Aquarium in Baltimore was most closely related to those from several species of freshwater teleosts.

Acknowledgements

The National Aquarium in Baltimore is thanked for financial support of this study. Denise Schultz, Sarah Trembly, and Katie Kelly are supported by the Ruth L Kirschstein National Research Service Award T32 RR007002.