NOTES Special Considerations for Keeping Cephalopods 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 animal models continues to be an important tory, which means that they grow rapidly to sexual maturity, spawn 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 animals 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 species. 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 squid) 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, squids and cuttlefishes Ten percent of the known living cephalopod 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 cuttlefish (Sepia 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 chromatophore 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