Since 1976 we have worked extensively on the capture, maintenance and culture of cephalopod species with potential or demonstrated value in biomedical research (Forsythe & Hanlon, 1980; Hanlon et al., 1983; Hanlon & Forsythe, 1985). We have maintained, reared or cultured 14 octopus species, eight squid species and two species of cuttlefish. Animal densities and system crowding have increased as our research emphasis has shifted from capture to culture, resulting in an increased occurrence of disease. Some form of pathologic condition has been observed in all the species ranging from mild and insignificant to severe or fatal. In this paper the range of conditions will be described briefly with some comments on antibiotic therapies.

Skin Pathology

Cephalopod skin consists of a thin epidermis and a thicker dermis that covers the muscle layers beneath. The epidermis is microvillous and composed of a monolayer of simple cuboidal cells interspersed with mucous-secreting cells. The underlying dermis is thicker and contains connective tissue, blood vessels, nerves, amoebocytes, and chromatophore organs and iridescent cells used for color change. Subjacent to the dermis is a uniform band of longitudinal muscles and a thicker band of transverse muscles. All species of cephalopods have been found to be susceptible to bacterial infections of the skin. Evidence suggests these are secondary infections by opportunistic pathogens.

The major cause of initial skin damage in octopuses has been inter-animal contact, since skin lesions are seen rarely in individually cultured octopuses. Apparently when one octopus touches another with its suckers, sufficient damage to the epidermis can occur in some cases to allow secondary infections. The resultant lesions usually appear on the dorsal mantle. Lesions on the arms are sometimes the result of injury incurred during the capture of food organisms, principally crabs and shrimps. Some octopuses develop single, large chronic lesions on the distal tip of the mantle. It is unclear how these begin, but they appear to be related to stress that causes the animals to swim more than normal and hit their mantle tips on tank walls.

It has been well documented that squids readily sustain mantle tip damage and fin abrasion from capture trauma and confinement (Leibovitz et al., 1977; Hulet et al., 1979). During full-life-cycle culture experiments with squids of the genus Loligo, secondary infections of fin and mantle tip damage are the principal cause of mortality before sexual maturity. As these infections progress, behavior becomes erratic and animals swim along the tank bottom causing further fin abrasion and ventral mantle lesions that actually penetrate through the mantle. All field-collected squids sustain skin damage in collection.

Microbiological examinations or cephalopod skin lesions have found bacteria of the genus Vibrio , Psendomonas and Aeromonas present, usually simultaneously. Vibrios appear to be the principal pathogens because they are always present and have been shown experimentally to cause lesions in octopuses (Hanlon et al., 1984). We have cultured at least twelve species of Vibrio from skin lesions in octopuses and squids (Table 1). The skin lesions described above have ranged from acute to chronic in duration, possibly reflecting the bacterial species involved. Acute lesioning in Octopus has caused death in as little as four days from onset of infection, while large chronic lesions have persisted for months. In cultured squids, mantle tip infections are normally chronic and cause death after weeks or months.

Table 1. Bacteria isolated from infection sites of octopuses and squids at this laboratory

The only non-bacterial skin pathology we have encountered has been caused by an ectoparasitic protozoan very similar to the Bodonid parasite Ichtyobodo necator. These organisms ape small, 4-20µm, and can infest any surface of the epidermis exposed to sea water, including the internal surfaces of the mantle cavity and gills. The parasite feeds by attaching to the host cell and passing a cytostome or feeding tube into it, digesting cytoplasmal components. When the host cell dies, a new one is parasitized, Recent reports in the literature have described infections of this mainly freshwater parasite on fishes in marine habitats (Morrison & Cone, 1986); however, to our knowledge, this is the first case of an invertebrate host for this genus of parasites. Infestations were first manifest on the dorsal arms and mantle surfaces of Octopus bimaculoides as white spots (visible when the animals were dark in coloration). Lesions eventually formed at the centers of the spots and as the conditioned progressed, the interspersed lesions eventually connected. It appears that once the parasite causes damage to the epidermis, secondary bacterial infections are responsible for the lesioning. The parasite also heavily infests the gills, impairing respiratory function. It is noteworthy that this parasite often assumes a giant morph (15-20µm) when on the gills versus the smaller size (4-8µm) seen externally on the epidermis. This parasite proved devastating to two culture populations of O. bimaculoids. The disease first appeared in tow-month-old octopuses (0.5g). Animals seem to become refractory to the disease beyond a size of 25g, although small numbers of parasites may be present on the gills. This parasite also attacked young O. maya, but occurred only on the gills, while young O. digueti in the same tank as the O. maya were not parasitized. This form of Ichtyobodo tolerated culture temperatures from 15-30°C and salinities of 20-45 ppt.

Internal Pathologies

Vibrio carchariae has recently been shown to cause internal infections in O. bimaculoids. This disease was characterized by sudden death of octopuses. No behavioral symptoms were apparent in the 12 to 18 hour period preceding death and in some cases animals even fed normally within hours of death. Dead animals typically had no external damage or only a few (<5) very small (<3mm diameter) skin lesions that would not be sufficient to cause death. Approximately half the dead animals had signs of autophagy of one or more arms very near the arm bases. On all moribund animals without signs of autophagy, it was possible to find short sections of certain arms with no muscle firmness. Histology of the eaten and soft areas of the arms showed the tissue to be heavily infected with rod-shaped bacteria, and smears of tissue on TCBS agar plates produced almost entirely V. carchariae. Further histological and microbiological investigation revealed the same bacteria in the branchial hearts and kidney. At this point, we are working on the assumption that the bacteria entered the gut via food and were carried in the blood to other parts of' the body.

Skin lesions sustained when squids contact tank walls often result in internal infections as well as the usual external secondary bacterial infections found in the lesion. These internal infections are chronic, usually requiring several weeks or months to kill the animal. The most common infection is a localized necrosis (limited to less than 1 cm 2) of mantle tissue surrounding the lesion. Recovery and tissue repair can occur in otherwise healthy squids, hut often it evolves into the following condition. The infection can spread from the apex (posterior) of the mantle toward the rear mantle appearing as an opaque dagger-shaped abscess in the lateral mantle tissue. No squid has been observed to recover from either of' these infections and the exact cause or death has not been identified.

Octopuses and squids have pathologies of the eye. In octopuses, the eyeball occasionally swells and ruptures. This condition is very rare, but always fatal within two days. No causative organism has been implicated. Squids and octopuses have developed white opaque lenses (cataracts?), but the condition typically is not fatal.

The development of an infected and swollen eye is less common in the squid than are mantle infections, but occasionally these are observed. The swelling is due to a buildup of humor in the retinal cavity and results in the eye becoming noticeably larger than normal (0.5x - 1x). Planococcus sp. and Micrococcus sp. have been isolated from the humor, with the former being in much higher concentration. The lens was also found to be encrusted with colonies of' Planoroccus sp. and had turned opaque as had the corneal covering or the eye. The same bacteria were found in the hemolymph and on the skin. This is one of the few times that we have observed a systemic bacterial infection in Loliginidae squids. Planococcus sp. seems to be quite specific to these eye infections since it was not isolated from squids in the same tank that had severe tail damage and mantle infections.

We have evaluated a broad range or antibiotics, particularly on octopuses (Table 2). Octopuses have been treated with bath applications, intramuscular injections and via food. Bath applications of nitrofurazone (1.6 - 3.2 ppm for 6 days) and nifurpirinol (1 - 2 ppm for 10 min., 2x/day for 10 - 30 days) have been most effective for treating skin lesions of octopuses. Intramuscular injections of' chloramphenicol at 10 mg/1/kg octopus have yielded equivocal results, but this method appears generally ineffective. The recent outbreak or internal infestations of Vibrio carchariae was halted by feeding small portions or frozen shrimp injected with chloramphenicol at 100 mg/l/kg of octopus once a day for six days.

Table 2. Treatments used against disease. These agents were applied to octopuses in various dosages, durations and frequencies

Squids appear to be more sensitive to antibiotic treatments than octopuses. Exposure to nifurpirinol (0.1 ppm bath) and nitrofurazone (2 ppm bath) resulted in 37% mortality for squids Lolliguncula brevis) within a 18 hour period (Ford et al., 1986). Higher concentrations resulted in 1001 mortality within 24 hours. My one attempt to medicate injected squids has been made. The squids (12 Loligo plei) were exposed to the slow introduction (over a 4 hour period) or nifurpirinol to a final concentration of 0.1 ppm. The antibiotic was removed by activated charcoal filtration after a 16-hour exposure. No improvement in tail damage could be detected and all 12 animals were dead within 50 hours A later experiment using a concentration of 0.05 ppm nifurpirinol resulted in good survival, but no improvement was seen in squid condition nor were bacterial populations ions in the tank or on the skin of the squid reduced significantly. Because of this sensitivity to antibiotics, our main defense against the bacterial infection associated with tail damage has been to lower the water temperature of our systems (from 15 - 17° C to 12 - 13 °C) The lower water temperature apparently slows the growth of' bacteria and reduces the severity or mantle damage through lower squid activity levels.

No effective treatment has been found (see Table 2) for the octopus parasite Ichtyobodo, which is resistant and adaptable. Known cures for the disease are lethal to octopuses. However, by no longer bringing in brood stock females from California and by prefiltering water to 1 µm Followed by U/V sterilization, we have been able to eliminate the parasite from cultures.

Acknowledgements

We acknowledge grant support from the Division of Research Resources of the NIH on grants RRO1024 and RR01279. Fine technical assistance was supplied by Randal DeRusha and Karen Gilligan in the application and evaluation of antibiotics. Kay Cooper prepared and viewed SEM & TEM material. Anthony DiNuzzo performed bacterial identifications and Drs. Dean Folse and John Christie of the Department of Pathology (UTMB) were most helpful in evaluating histological sections. Dr. M. Stoskopf supplied useful information on antibiotic applications while Drs. Gene Burreson and Ed Noga helped in identification of Ichthyoboda.

References

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