Adult, female, wild-caught Giant Pacific octopus (Octopus (Enteroctopus) dofleini).Three Giant Pacific octopuses, Octopus (Enteroctopus) dofleini, wild-caught in Alaska and held in
captivity, died or were euthanized following periods of decreased appetite and lethargy, despite treatments for
possible septicemia and heavy metal exposure. This particular wild-caught female came in October 2006 and
spawned 20 April 2008. She continued to eat, interact with the environment, and behave normally until found
moribund 4 December 2008, requiring euthanasia. Lesions were similar in all three giant octopus submissions.
Multiple gill biopsies were submitted in formalin and 95% ethanol.
Gills: There are abundant piriform structures lining up along the apical surface of
columnar gill epithelium.Â The parasites extend slender structures connecting them to apical aspects of the
epithelium.Â The submucosa is diffusely and variably expanded by numerous degenerate and viable hemocytes with
abundant granular eosinophilic cytoplasm and reniform-to-bilobed nuclei.Â Within the inflammatory hemocytes, and
often extracellularly, is variably-sized spherical, amphophilic, pink-to-olive brown, refractile material.Â Individual
inflammatory cells are observed transmigrating through the crowded tall columnar pseudostratified epithelial cells
and occasionally coalescing into small aggregates of exudated hemocytes.Â There is some epithelial loss with
parasites adhered to areas of disruption; adjacent degenerate to necrotic epithelial cells are vacuolated or collapsed
with nuclear pyknosis.Â There are multifocal areas of increased clear space between haphazardly arranged bundles
and individual connective tissue fibers of the submucosa (edema).
Gills: Inflammation, subacute, hemocytic, diffuse, severe with epithelial
Histopathological findings showed extensive gill degeneration and necrosis and
hemocytic inflammation associated with a heavy infection of Ichthyobodo-like flagellates.Â The gills had a large
number of the piriform protozoal structures queued up along the apical margin of the gill epithelium, and attended
by variable submucosal hemocytic inflammation.
Samples of gills tissues were processed for genomic DNA extraction, PCR amplification, and cloning and sequencing of the rRNA genes; phylogenetic analysis was conducted on the SSU, D1-D3 and D8-D10 LSU rDNA sequences.Â Gene sequences were deposited in GenBank.Â Phylogenetic analysis indicated that the Ichthyobodo species identified in this study was most closely related to those from freshwater fish.
Ultrastructural features were similarly characteristic of Ichthyobodo species.Â Scanning electron microscopy showed that the flagellates were flattened pyriform to trapezoidal, approximately 6-10 Î¼m long and 3-6 Î¼m wide.Â In some regions the infection was so dense that the gill surfaces were completely covered with the flagellates.Â Flagellates were attached to the host cells cytoplasm by a narrow cytostome via a crateriform puncture through the smooth mucous covering of the gill epithelium.Â Transmission electron microscopy revealed two unequal-width flagella lying in a U-shaped flagellar pocket, microtubules and striated fibers surrounding the flagellar pocket, and radiating fibres lying in a semi-circle around the cytostome primordial.Â The spherical nucleus had a large central nucleolus and peripheral chromatin, and the cytoplasm also contained rough endoplasmic reticulum, and mitochondria.Â There was an attachment plate anchoring the flagellate to the epithelial cell, and the cytostome process, reinforced with fibrillar structures, passed though the plate into the cytoplasm of the host cell.
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.Â The parasite can be found both free-swimming in the water column and attached to epithelial surfaces such as gills and skin.(6,13) The free-swimming form moves in a spiral motion with the aid of flagella.Â Ichthyobodo attaches to epithelial cells via a long, slender organelle that facilitates the ingestion of host cellular contents.(6,13) Ichthyobodiasis can impede osmoregulation and predispose to secondary infection; alternatively, Ichthyobodo infections are often found in association with stressful circumstances in freshwater fish.(6,13) 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.
Invertebrates, including mollusks, rely on innate immunity to combat disease, with no evidence of acquired immunity.(9) Members of the phylum Mollusca, which includes the class Cephalopoda (squids, cuttlefish, octopus and nautilus), have both cellular and humoral mechanisms of defense.Â Hemagluttins are the best studied and understood component of molluscan humoral immunity.Â These soluble factors, named after the agglutination of erythrocytes from other species, is thought to play a role in the recognition and opsonization of foreign material, although cell-free hemolymph is reported to have limited ability to agglutinate bacterial isolates in vitro.(3) The hemocyte is the primary component of cephalopod cellular immunity and develops from stem cells found in the white body, a multilobed leukopoietic organ located in the retrobulbar areas.(4) The role of octopus hemocytes in wound healing is well documented; a progression from skin wound infiltration, transformation of hemocytes to fibroblastic morphology and establishment of a dermal plug with subsequent contraction culminates in wound repair.(2) A multi-function cell with oxygen-carrying and nutrient transport capacities, the octopus hemocyte also performs phagocytosis, producing reactive oxygen species and lysozyme to destroy pathogens.(8,11) Stress and low temperature have been shown to alter the phagocytic function of hemocytes in vivo.(7) In addition to phagocytosis, hemocyte encapsulation serves as defense mechanism against foreign substances.Â Resident hemocytes are described in gill tissues of octopus, complicating the evaluation and characterization of potential gill pathogens.(12)
The reproductive physiology of cephalopods is interesting and enigmatic.Â In cephalopods including Giant Pacific octopus, endocrine secretions from the paired optic glands direct the development of senescence, characterized by loss of appetite, change in feeding behavior, loss of condition and behavioral changes that culminate in death. Hence these species of octopus have a brief lifespan of only about 3 years and inevitably die after spawning.(1) In the captive octopus of this report, it is unclear whether there is any association of senescence and enhanced susceptibility to the Ichthyobodo infections.Â Removal of the optic glands is reported to reduce senescenceassociated behavior and greatly extends the lifespan of cephalopods.(14)
These observations demonstrate that Ichthyobodo sp.Â can be a significant ectoparasitic pathogen of captive cephalopods.Â The host-parasite interaction and parasite ultrastructure are essentially similar to those of the better known Ichthyobodo sp.Â affecting teleosts.Â Molecular phylogeny showed that the Ichthyobodo sp.Â from these wildcaught captive Giant Pacific octopuses was most closely related to flagellates from several species of freshwater teleosts.
Gill: Branchitis, hemocytic, multifocal, moderate, with many surface epithelial-associated
The contributor provides an excellent synopsis of the entity in this mysterious species.
Conference participants discussed the challenge of estimating a lesions chronicity based solely on microscopic
examination in invertebrate species.Â In contrast to most vertebrates, in which the composition of inflammatory
infiltrates correlates fairly reliably with chronicity, to the best of our knowledge, the composition of the
inflammatory infiltrate has not been reported relative to lesion chronicity in the octopus, in which the hemocyte is
the primary inflammatory cell.Â Accordingly, participants debated the merits of including the modifier hemocytic
in the morphologic diagnosis, with some favoring its omission on the basis of redundancy, though most agreed with
the contributor that its inclusion is an appropriate enhancement to the morphologic diagnosis, most precisely
reflecting the pathologic processes involved.
Because Ichthyobodo infection is commonly associated with stress in freshwater fish, participants speculated that senescence-associated immunosuppression may have rendered this octopus particularly vulnerable to parasitism by Ichthyobodo-like flagellates.Â Curiously, some species of Ichthyobodo are capable of survival both in saltwater and freshwater; for instance, I.Â necator, which survives and causes disease over a wide range of temperatures, can survive in saltwater and cause mortality in marine-adapted salmonids.Â Other Ichthyobodo-like flagellates exclusively infect marine fish.(10) Ichthyobodo-like flagellate infection was reported in a laboratory cultured population of California mud-flat octopus (Octopus bimaculoides) and two related species (O.Â maya and O.Â digueti), in which the organism was initially found on the gill, then spread over the course of the disease to involve the internal surface of the mantle cavity and the body surface, leading the authors to conclude that the gill was the initial site of infection.Â Gross lesions included small white foci on the dorsal surfaces of the arms and mantle; however, these may have been partly attributed to co-infection with an ancistrocomid ciliate.(5)
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3.Â Fisher WS, DiNuzzo AR: Agglutination of bacteria and erythrocytes by serum from six species of marine molluscs.Â J Invertebr Pathol 57:380-94, 1991
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12.Â Schipp R, Mollenhauer S, von Boletzky S: Electron microscopical and histochemical studies of differentiation and function of the cephalopod gill (Sepia officinalis L.) Zoomorphology 93:193-207, 1979
13.Â Thune RL: Parasites of catfishes.Â In: Fish Medicine, ed.Â Stoskopf MK, pp.Â 528-529.Â W.B.Â Saunders, Philadelphia, PA, 1993
14.Â Wodinsky J: Hormonal inhibition of feeding and death in octopus: control by optic gland secretion.Â Science 198:948-951, 1977