NOTES

Special Considerations for Keeping in Laboratory Facilities

DANIELJ. OESTMANN, DVM, PHD, JOSEPH M. SCIMECA, DVM, PHD, JOHN FORSYTHE, MS, ROGER HANLON, MS, PHD, AND PHILLIP LEE, MS, PHD

Abstract I Cephalopods have been used for a wide variety of biomedical and basic science research projects and their use has been growing. Advances in culture techniques pioneered at the National Resource Center for Cephalopods (NRCC) have enabled the NRCC to culture cephalopods year-round, rather than relying seasonally on wild-caught cephalopods. These cultured cephalo- pods are then provided to visiting investigators or shipped to investigators in remote areas. This article describes how an investigator in a remote area can contravene shipping stress and, in turn, maintain small colonies of healthy cephalopods for long periods of time. The NRCC has established protocols for health monitoring involving behavior and water chemistry analyses. Disease preven- tion is accomplished through rigorous environmental control, water treatment and adequate feeding. Treatment is usually a less-effective option, involving dips and injections of antibiotics. The list of effective antibiotics is short (i.e., chloramphenicol, gentamicin, and nitrofurazone). The NRCC also air-freights cephalopods routinely via overnight delivery service to remote or inland institutions for inunediate use on arrival. As a result, these cephalopods often become stressed during shipment. The NRCC's goals are for investigators in remote areas to avoid potential problems in their research results due to stress and to extend the time frame during which cephalopods can be maintained at these remote institutions.

The use of aquatic models continues to be an important tory, which means that they grow rapidly to sexual maturity, component of laboratory research world-wide. In Canada, for in- once, and die. Life span and growth rate in laboratories are tem- stance, fishes represent the largest group of laboratory perature dependent, but rarely exceed a year, and often are only used in government laboratories (1). The use of cephalopods as 5 to 6 months for tropical . Animals brought into the labo- laboratory animals is also well established, as they have long been ratory as juveniles or sub-adults may only have a few months to models used in neurophysiology and basic physiology studies (2, live. Hatchlings display true exponential growth for the first third 3). Aquatic species, especially invertebrates (e.g., crayfish, sea ur- of the life cycle, growing at rates of6 to 12% of wet body weight chins, horseshoe crabs, and ) can provide researchers with a per day. Asjuveniles reach maturity, these rates decrease to 4 to viable alternative to traditional terrestrial vertebrates. 5% per day (7). To fuel such growth, and Ten percent of the known living species have been consume a virtually pure protein diet from their prey of fishes, maintained, reared, or cultured in laboratories (4). Culturing shrimps, and crabs, converting 30 to 50% of the diet into growth. of cephalopods through multiple generations has been achieved. This high-protein diet results in production oflarge amounts of Seven generations of European ( officinalis) and nitrogenous waste in the form of ammonia. Cephalopods ex- 6 generations of Pacific long-finned squid (Sepioteuthis lessoniana) crete 2 to 3 times the amount of ammonia per kg of body weight, have been cultured at the National Resource Center for Cepha- compared with fishes (8). lopods (NRCC) of the Marine Biomedical Institute (MBI) in Squids and cuttlefishes are active, mobile predators in nature. Galveston Texas (5, 6). Advances in laboratory care and hus- Since they compete with fishes in the sea, they have evolved a bandry have allowed the development of a year-round source of sophisticated sensory neurophysiologic mechanism (3) .They have these research animals from the NRCC. The most important superb vision, although apparently they detect only black and white, advances have been through improvements in tank design, feed- relying on vision for orientation and prey selection. They have ing methods, handling and transport methods, and water complex behavior, particularly in the areas of reproduction and filtration techniques. predator avoidance (camouflage). Both of these are reflected in Advances in design of sea water filtration equipment elimi- the complex neurally controlled system in the skin nated the requirements for a coastal location; thus, numerous that allows them to change their coloration and body patterns in a inland laboratories are now keeping cephalopods routinely. fraction of a sec. As a defensive ploy, they can excrete copious Long-term success in maintaining laboratory populations of amounts of ink to confuse potential predators. The ink is an im- cephalopods requires rigorous water-quality management com- portant issue to be dealt with in their captive maintenance, because bined with an aggressive health monitoring program. It is our it increases water turbidity and can foul gills. purpose to provide investigators and animal care professionals Basic Tank and Sea Water System Requirements: We have with the basic information needed to ensure colony health for described elsewhere, in detail, designs of closed sea water sys- small cephalopod populations « 20 cephalopods/ colony). tems suitable for culturing of cephalopods (5,6,9-15). Readers are urged to consult these publications for details on system Cephalopod Biology and Life History design. Although some of these articles deal with culturing of To appreciate the health maintenance requirements of cepha- octopuses, the criteria for water quality and filtration design apply lopods, it is necessary to understand their biology and life history. equally well to squids and cuttlefishes. The actual tanks used Foremost, squids and cuttlefishes have a semelparous life his- and precise layout of filtration apparatus is highly adaptable to constraints of space and number of animals to be housed. It is NationalResource Centerfor Cephalopods, University of Texas Medical Branch, essential that water filtration is processed in the following order: Marine Biomedical Institute, 301 University Boulevard, Galveston, Texas first, water leaves the animal holding tanks and then passes 77555-1163 through a foam fractionator (protein skimmer), which strips

Volume 36, No. 21 March 1997 CONTEMPORARY TOPICS © 1997 by the American Association for Laboratory Animal Science 89 dissolved organic compounds including ink. The water then Receiving and Post-Shipment Handling: A bacterial filter bed passes through a mechanical filter, removing particles down to (nitrifying biofilter) must be conditioned prior to the arrival of 100 /lm. It then passes through high-grade activated carbon, animals if it has not been supporting any animals during the through a biologic filter where ammonia is broken down to less- preceding 2 weeks. This can be done by maintaining fishes or toxic forms by nitrifying bacteria (we generally use down-flow other invertebrates in the tanks or by adding increasing amounts sub-gravel filters that have crushed oyster shell as a media), and of ammonium chloride (18). One or 2 days prior to arrival of lastly through an ultraviolet (UV) sterilizer before returning to new animals, steps should be taken to match temperature, salin- the animal holding tank. System design should produce flow ity, and pH of the water as closely as possible to those of the rates that allow the entire water volume of the culture system to provider institution (i.e., NRCC) or the natural environment of pass through the filtration loop a minimum of 2 times per h. field-collected animals. Natural and artificial sea water have been used successfully to On arrival, shipping containers should be opened in dim light- maintain and culture cephalopods. When natural sea water is used, ing so that the animals, which have acclimated to darkness during particulate and carbon filtration prior to use in closed, recirculat- transport, will not be startled. The high metabolic rate of cepha- ing sea water systems is recommended, especially when the water lopods results in high ammonia concentration during transport is to be stored prior to use. When an artificial sea water is used, that should be corrected as soon as possible during acclimation. the fresh water must be filtered through activated carbon or must This is accomplished by slowly removing transport water from be dechlorinated with sodium thiosulfate to remove chemical ad- the shipping container and replacing it with tank water. The ditives commonly used by municipal water authorities (e.g., time required for acclimation will depend on the difference chlorine and chloramines). De-ionized water also is safe to use. between water conditions in the shipping container and the We have successfully used Instant Ocean™, HW Marine MixTM, housing tank. As a rule, you should not acclimate most cephalo- and Fritz Super Salt™ artificial sea salts. Artificial sea water should pod species faster than 10 C/h or 2-3 ppt salinity Ih. be mixed and aged at least 48 h before it is added to a tank con- When animals are removed from transport containers or taining cephalopods. When converting existing fish tank systems, moved from one tank to another, they should be slowly maneu- it is essential to determine whether copper treatments have ever vered into a submerged bucket, beaker, or bowl that is then used been used to combat fish parasite problems. Copper is highly toxic to transfer the animal to a new tank. Netting should be avoided to many marine invertebrates, especially to cephalopods. If cop- to prevent epithelial trauma. per has been used in that system in the past, it may be necessary to Lifting squids and cuttlefishes completely out of the water build a new system for cephalopods, because it is extremely diffi- causes unnecessary stress resulting in inking and jetting upon cult to eliminate residual copper from a system. return to the water. Newly received animals should be left alone Squids are pelagic (dwelling in the water column) and require for 1 to 2 h so that they can acclimate to their new surroundings. tanks that are at least I-m deep, whereas the benthic (bottom dwell- When possible, an initial feeding of live fishes should be pro- ing) nature of cuttlefishes requires tanks that maximize horizontal vided after 2 h. A strong initial feeding response indicates that surface area. Round tanks eliminate areas of stagnant circulation the animals have recovered from transport and the new envi~ and corner crowding (16), but often are not space efficient. Rect- ronment is satisfactory. Ammonia concentrations in the receiving angular tanks with strong water-flow patterns are acceptable. Tanks tank should be monitored carefully thereafter to make sure that should have tops, because cuttlefishes and especially squids can biologic filtration is adequate. leap from tanks when they jet in response to sudden motion or Feeding: One of the major maintenance constraints of cuttle- light changes. This behavior also results in damage to the epider- fishes and squids is the lack of a defined processed diet. This has mis of the fins and on collision with tank walls (17). To been mitigated in cuttlefishes by training them to eat a frozen avoid injury to the cephalopods, fiberglass or polyethylene tanks shrimp or fish diet. The NRCC is continuing research to develop with small observation windows should be used, because animals a processed diet (19, 20). Cuttlefishes and squids assimilate pro- will not be startled by activity in the facility. Glass aquarium tanks tein almost exclusively with a high degree of efficiency. should be avoided for housing squids and cuttlefishes because of Hatchlings undergo exponential growth up to the juvenile stage the sensitivity of the animals to human activity. Holding tanks (one fourth to one half of the life cycle), consuming 80 to 100% should be in low traffic areas with dim lighting. of their body weight per day. The growth phase from juvenile to The number of animals that can be successfully maintained adult is logarithmic with food consumption decreasing to 10% varies according to their species, age, and size. Younger animals (for cuttlefishes) and 30% (for squids) of body weight per day are smaller, but space must be allotted to compensate for their (6, 8, 13). Frozen-fresh shrimp or fresh non-oily fish from gro- exponential growth. Male squids and cuttlefishes display aggres- cery stores may be used to maintain cuttlefishes. Squid may be sive sexual behavior as they reach maturity. General guidelines fed live guppies or goldfish obtained from pet store wholesalers. have been developed for population densities on the basis of However, feeder fish have the potential to introduce diseases. species, age, and size (Table 1). Plans must be made so that adequate food supplies are

Table 1. Density and water quality parameters

N0 -Nb Growth Mantle weight density NH4-N" 2 N03-N' stage length (g) (#/m') (ppm) (ppm) (ppm) (cm)

Cuttlefish (Sepia o(ficinalis) hatchling 2-3 1.4-4.6 250-300 <0.5 < 0.5 < 80 juvenile 5-15 139-441 20 <0.5 < 0.5 < 80 adult 15-22 1000-2000 2 < 0.5 < 0.5 < 80 Squid (Sepioteuthis lessoniana) hatchling 0.7-1.5 0.4-1.0 100-200 < 0.1 < 0.05 < 50 juvenile 5-15 50-500 50 < 0.1 < 0.05 < 50 adult 15-30 2500 3-4 < 0.1 < 0.05 <50

aAmmonia-nitrogen, bNitrite-nitrogen, "Nitrate-nitrogen

90 CONTEMPORARY TOPICS © 1997 by the American Association for Laboratory Animal Science Volume 36, No.2 / March 1997 readily available prior to arrival of the animals. irritation. The animals are thought to be "scratching" the irritated Water Quality: General water quality parameters are more area, similar to fishes "flashing" in response to a skin irritation. stringent for the maintenance of cuttlefishes and squids than Without antibiotic treatment, death will result 7 to 10 days after for most marine fishes (Table 1). Cephalopods are sensitive to the appearance of the scratching behavior. Mortalities have been rapid changes in pH, salinity, low-dissolved oxygen concentra- minimized by injecting chloramphenicol into the food for 7 days tions, and nitrogenous wastes. These inherent sensitivities may after an ammonia spike. be due to the highly absorptive surface area of their microvil- Crowding may initiate aggressive behavior, resulting in tank lous epidermis and high metabolic rates (8). In addition, the wall collisions and caudal mantle damage or cuttlebone fractures rapid growth rate, high feeding rate, and dependence on pro- (see Table 1 for population density recommendations). The tein metabolism for energy result in a nitrogen load much greater former may resolve without treatment or can result in erosions (2 to 3 times on a per-mass basis) than that of fishes. We cannot in the muscle and dermal tissues, requiring antibiotic treatment. overemphasize the importance for monitoring of nitrogen con- Cuttlebone fractures may cause remodeling, and the animal usu- centration in the health management of captive cephalopods. ally will survive, but sporadically the stomach and cecum will

The low tolerance of cephalopods to ammonia (NH3) and herniate or the dorsal mantle will detach from the cuttlebone, ) nitrite (N02 dictate the use of efficient and high-capacity nitri- resulting in death. When the cuttlebone heals, the cuttlefishes fYingfiltration systems. Ammonia-nitrogen and nitrite-nitrogen do not grow from that point. Chronic low-impact trauma that concentrations should be monitored daily for a week after ar- results when cuttlefishes infrequently hit the tank walls may re- rival and twice weekly thereafter. Sea water kits are sult in a prolapse and proliferation of the caudal cuttlebone commercially available and appropriate for this purpose. Water membranes and dermis that appears as a tumor-like outgrowth. changes must be performed to maintain ammonia and nitrite Edema, hemocyte infiltration, and necrosis of mantle muscle concentrations within recommended parameters until the nitri- and the dermis with accompanying epithelial loss have been fying filter can compensate for the added nitrogen load. observed by the authors as a result of the application of identifi- Cuttlefishes will survive nitrate concentrations of up to 150 ppm, cation tags. Attachment of a numbered identification tag is but animals will become more aggressive toward each other and performed by passing a needle and thread through the left staff, displaying short, jerky swimming behavior rather than re- craniodorsal mantle and then loosely knotting the thread. Swell- maining placidly on the tank bottom. Cuttlefishes constantly ing around the thread sometimes impinges the nerves of the release ink when nitrate concentrations exceed 80 ppm. Squids stellate ganglia and giant axon which results in unilateral paraly- are even more sensitive to nitrate, requiring concentrations to sis of the mantle and fin muscles and a hemispheric asynchronous be kept below 50 ppm. The ink is not toxic chemically, but when chromatophore display. secreted in large quantities, it can adsorb to the gills, mechani- A rare condition in cuttlefishes has been described in which a cally blocking oxygen uptake. The ink can be removed by foam raised ridge on the dorsal mantle appears. The condition fractionation (protein skimmers) and activated carbon. How- progresses, and swelling involving the greater part of the dorsal ) ever, denitrification (N03 converted to N02 is a better technique mantle is evident within 18 h, and death often occurs within 24 when housing a large number of cephalopods (15,21). Nitrate h. The condition was attributed to a bacterial infection, because concentration should be measured every 2 weeks. the lesion resolved with aggressive gentamicin antibiotic treat- ment (29). Dangling tentacles and arms that become white have Common Health Problems been observed. Because bacterial septicemia was confirmed in The pathogen defense system of cephalopods is primitive when these animals, the tentacle and arms may have died as a result of compared with that of mammals or even fishes. Their thin, mi- a hemocyte/bacterial embolism in the central artery. crovillar epidermis is easily traumatized during confinement or handling; minor skin lesions and abrasions can lead to opportu- Health Monitoring and Treatment nistic bacterial infections and death (17, 22). Cellular immunity Frequent observation of animal behavior is the single most im- consists of one type of circulating hemocyte that clumps and iso- portant management strategy in a quality animal health program. lates, but does not phagocytize, foreign material (23,24). Humoral Much of the behavior of diseased cephalopods appears to be analo- immunity consists of a hemagglutinin protein that may function gous to similar behaviors in diseased captive fishes. The general similar to lectins in other invertebrates (25-27). The relative sim- behavioral state of cephalopods should be noted frequently (2 to plicity of cephalopod immunity predisposes the animals to higher 4 time per day). Because cephalopods eat relatively large amounts risk of morbidity and mortality from diseases. Injured or sick ani- of food, altered feeding behavior is usually the first and most ap- mals should be isolated and aggressively treated. parent change in behavior. Animals should be especially alert and Specific animals may have discrete external lesions; however, feed aggressively in the morning. Agitated behavior, swimming the underlying dermal and iridocytes can make into the tank sides, or reaching the tentacles over the back may be injured skin appear normal. Ulcers on the distal tip of the mantle signs of deteriorating water ..quality, and all indices should be from handling or collision with tank walls may erode through checked. Passive floating is a sign of suspended feeding and may the epidermis and dermis, exposing the mantle muscle. Ulcers on be observed in subordinate animals that are having trouble com- the arms and tentacles may be the result of bites from live prey. peting for food or in an entire population through which an Epithelial loss readily progresses to secondary bacterial infections, infection is spreading. Behavioral changes also include subtle because the surface bacterial population of captive cephalopods changes in chromatophore display patterns or changes in orienta- can be up to 100 times greater than that of wild cephalopods (28). tion to other animals in the tank. These behavioral changes are For this reason, IN treatment of the sea water is essential to re- often the only clinical signs of disease prior to animal deaths. duce the bacterial concentrations in the water (5, 12). Another Antibiotics have been used subjectively at the NRCC to pre- skin condition develops in cuttlefishes when they begin reaching vent or treat diseases (Table 2). A prophylactic antibiotic regimen over the dorsal mantle with their arms, as if they were scratching; should be initiated when diseases or stresses are suspected be- the behavior begins 3 to 7 days after spike in ammonia concentra- cause of the limited ability of cephalopods to fight infection. tion (> 0.3 ppm). As in fishes, it is possible that a deterioration in Intramuscular injection should be given at the base of the arms water quality causes a loss of the secreted mucous layer, resulting to confined animals (30); intramuscular injections should be in bacterial colonization of the skin's surface that leads to skin given only when animals are not feeding. Once feeding resumes,

Volume 36, No.2 / March 1997 CONl}.'MPORARY TOPICS© 1997 by the American Association fOf Laboratory Animal Science 91 antibiotics can be provided via the food. When antibiotic treat- Acknowledgments ment was necessary at the NRCC, chloramphenicol and The authors express their appreciation to the Nationallnsti- gentamicin have been used with the best success (30). tutes of Health National Center of Research Resources (grant DHHS 5P40RR01024-20), the Texas Institute of Oceanography, Postmortem Evaluation and the Marine Medicine account of the Marine Biomedical Cephalopod tissues contain high activity for protease enzymes, Institute for support of this research. causing rapid tissue autolysis. Necropsies should be performed as soon as it is determined that an animal will not survive, be- cause autolysis will begin before death in some organs. Animals References should be euthanatized by rapid cooling in iced sea water or 1. Canadian Council On Animal Care. 1994. CCAC survey administration of a 10% solution of magnesium chloride in sea shows changing animal use trends, p. i-iii. In M. Silverstone water (31) or a 10% solution of ethanol in sea water. In our (ed.), Resource. Vo1.l8:1-2 experience, issues from animals found dead in a tank have lim- 2. Gilbert, D. J., W. J. Adehnan, and J. M. Arnold. 1990. In ited diagnostic value. For maximum recovery, hemolymph should Squid as experimental animals, Plenum Press, NY. be collected immediately after immobilization, but before death. 3. Abbott, N. J., R. Williamson, and L. Maddock. 1995. In Squid and cuttlefish hemolymph can be withdrawn from the Cephalopod neurobiology neuroscience studies in squid, muscular portion of the cephalic vein by inserting a syringe with octopus, and cuttlefish, Oxford University Press. Oxford, a 22 gauge needle directed caudodorsally at the ventral midline United Kingdom. caudal to the eye and cranial to the funnel. A bacterial septice- 4. Hanlon, R. T. 1987. Mariculture, p. 291-305. InP. R. Boyle mia is suspected when hemocytes have aggregated into visible (ed.), Cephalopod life cycles, Vol. 11.Comparative Reviews clumps. Systemic bacterial infections are confirmed by bacterial Academic Press, Inc., London. culture of the hemolymph. 5. Forsythe,J. W., R. T. Hanlon, and R. DeRusha. 1991. Pilot Most standardized methods for finfish postmortem examina- large-scale culture of Sepia in biomedical research, p. 313- tion can be applied to cephalopods. External lesions are blotted 323. In E. Boucaud-Camou (ed.), The cuttlefish: Acta I. to remove excess mucous, then, aseptically, specimens are ob- International symposium. Cuttlefish Sepia. Centre de Pub- tained by use of swabs and submitted for bacterial culture. Smears lications de I Universite de Caen, Caen, France obtained from the swab or skin scrapings are air dried and stained 6. Lee, P. G., P. E. Turk, W. T. Yang, andR. T. Hanlon. 1994. for bacterial or fungal observation. The mantle cavity is opened Biological characteristics and biomedical applications of by a ventral midline incision, the viscera are examined visually, the squid Sepioteuthis lessoniana cultured through multiple and samples are obtained and processed. Tissue sections are fixed generations. Bio. Bull. 186:328-341. in neutral-buffered 10% formalin, and standard histologic tech- 7. Forsythe,J. W. and W. F. Van Heukelem. 1987. Growth, p. niques are used for microscopic evaluation. Particular attention 135-155. InP. R. Boyle (ed.), Cephalopod life cycles, Vol. should be paid to the digestive gland and the digestive gland 11.Comparative reviews. Academic Press, London. duct diverticula, because these are the largest organs and the 8. Lee, P. G. 1994. Nutrition of cephalopods: fueling the sys- center of energy processing. tem. Mar. Fresh. Behav. Physiol. 25:35-51. 9. Forsythe, J. W. and R. T. Hanlon. 1980. A closed marine Summary culture system for rearing Octopus joubini and other large- egged benthic octopods. Lab. Anim. 14:137-142. Rapid growth rate, short life span, and a well-developed ner- 10. Hanlon, R. T., R. F. Hixon, and W. H. Hulet. 1983. Sur- vous system make cuttlefishes and squids useful laboratory vival, growth, and behavior of the loliginid squids Loligo animals. Advances in mariculture techniques have enabled the Plei, Loligo pealei, and brevis : NRCC to provide large numbers of high-quality cephalopods for Cephalopoda in closed sea water systems. BioI. Bull. biomedical researchers on a consistent year-round basis. Tradi- 165:637-685. tionally, cephalopods have been used for experimentation 11. Hanlon, R. T. andJ. W. Forsythe. 1985. Advances in the immediately following collection at only a few laboratories that laboratory culture of octopuses for biomedical research. are located near fishing grounds (e.g., Woods Hole, Mass.; Lab. Anim. Sci. 35:33-40. Monterey, Calif.); however, holding tanks with off-the-shelf fil- 12. Yang, W. T., R. T. Hanlon, P. G. Lee, andP. E. Turk. 1989. tration systems can be built at a moderate cost, and animal care Design and function of closed sea water systems for cultur- staff can be trained to recognize animal behavior particular to ing loliginid squids. Aquacul. Eng. 8:47-65. these species. Careful observation of these behaviors can alert 13. Forsythe, J. W., R. H. DeRusha, and R. T. Hanlon. 1994. the animal care staff of impending system problems or disease Growth, reproduction and life span of Sepia officinalis outbreaks. When water quality parameters are maintained and (Cephalopoda: Mollusca) cultured through seven consecu- nutritional requirements are met with available fresh, fresh-fro- tive generations.]. Zool. (Lond.). 233: 175-192. zen, or live food, small populations of these animals can be 14. Hanlon, R. T., P. E. Turk, and P. G. Lee. 1991. Squid and maintained for weeks to months. When preventative and pro- cuttlefish mariculture: an updated perspective.]. Ceph. BioI. phylactic approaches to water quality and health maintenance 2:31-40. are adopted, this unique animal group can become a routine 15. Lee, P. G. 1995. A review of automated control systems for component of virtually any animal care facility. aquaculture and design criteria for their implementation. Aquacul. Eng. 14:205-227. Table 2. Antibiotic use and dosage 16. Boletzky, S. V. and R. T. Hanlon. 1983. A review of the Antibiotic Dosage Administration laboratory maintenance, rearing and culture of cephalo- Chloramphenicol 40 mg/kg Injected into food for 7 days pod molluscs. Mem. Natl. Mus. Victoria 44:147-187. 40 mg/kg Intramuscular injection once a day 17. Hulet, W. H., M. R. Villoch, R. F. Hixon, andR. T. Hanlon. for I week, or as needed 1979. Fin damage in captured and reared squids. Lab. Anim. Gentamicin 20 mg/kg Intramuscular injection once a day Sci. 29:528-533. for 1 week, or as needed 18. Emmens, C. W. 1985. In The marine aquarium in theory Nitrofurazone 2mg/1 Use as bath for 72h and practice. T. F. H. Publications, Neptune City, NJ.

92 CONTEMPORARY TOPICS© 1997 by the American Association for Laboratory Animal Science Volume 36, No.2 / March 1997 19. Castro, B. G., F. P. Dimarco, R. H. DeRusha, and P. G. 26. Rogener, W., L. Renwrantz, and G. UWenbruck. 1985. Iso- Lee. 1993. The effects of surimi and pelle ted diets on the lation and characterization of a lectin from the hemolymph laboratory survival, growth, and feeding rate on the cuttle- of the cephalopod Octopus vulgaris (Lam.) inhibited by a fish SePia officinalis L.]. Exp. Mar. BioI. Ecol. 170:241-252. D-Lactose and N-acetyl-Iactosamine. Dev. Compo Immunol. 20. Castro, B. G. and P. G. Lee. 1994. The effects of semi-puri- 9:605-616. fied diets on growth and condition of Sepia officinalis L. 27. Olafsen,J.A.1988. Role oflectins in invertebrate humoral (Mollusca: Cephalopoda). Compo Biochem. Physiol. defense. Am. Fish. Soc., Spec. Publ. Ser. 18:189-205. 109A:I007-1016. 28. Ford, L. A., S. K. Alexander, K. M. Cooper, and R. T. 21. Whitson,J., P. G. Lee, andP. E. Turk. 1983. Biological deni- Hanlon. 1986. Bacterial populations of normal and ulcer- trification in a closed, recirculating marine culture system, ated mantle tissue of the squid, Lolliguncula brevis.]. Invert. p. 458-466. In]. K. Wang (ed.), Techniques for modern Path. 48:13-26. aquaculture, American Society of Agricultural Engineers, 29. Hanlon, R. T. andJ. W. Forsythe. 1990. 1. Diseases of Mol- St.joseph, Mo. lusca: Cephalopoda. 1.1. Diseases caused by microorganisms, 22. Hanlon, R. T. 1990. Maintenance, rearing and culture of p. 23-46. InO. Kinne (ed.), Diseases of marine animals, Vol. teuthoid and sepioid squids, p. 35-62. In D. L. Gilbert, W. III. Biologische Anstalt Helgoland, Hamburg, Germany. ]. Adelman,jr., and]. M. Arnold (eds.), Squid as experi- 30. Forsythe, J. W., R. T. Hanlon, and P. G. Lee. 1990. A for- mental animals, Plenum Press, New York. mulary for treating cephalopod mollusc diseases, p. 51-63. 23. Cowden, R. R. andS. K. Curtis. 1981. Cephalopods, p. 301- In F. O. Perkins and T. C. Cheng (eds.), Pathology in ma- 323. InN. A. Ratcliffe and A. F. Rowley (eds.), Invertebrate rine science, Academic Press Inc., San Diego, Calif. blood cells. Vol. 1. Academic Press, London. 31. Messenger,J. B., M. Nixon, and K. P. Ryan. 1985. Magne- 24. Claes, M. F. 1995. Functional morphology of the white bod- sium chloride as an anaesthetic for cephalopods. Compo ies of the cephalopod mollusc, Sepia officinalis. Acta Zool. Biochem. Physiol. 82C:203-205. (In Press). 25. Russo, G., and G. Tringali. 1983. Hemagglutinating and antibacterial activity in hemolymph of Octopus vulgaris. Rev. Int. Oceanogr. Med. 70/71:49-54.

Volume 36, No.2 / March 1997 CONTEMPORARY TOPICS© 1997 by the American Association for Laboratory Animal Science 93