The California sea hare (Aplysia californica) is recognized as a key model for studies of memory and learning. Despite the use of this model system by hundreds of investigators over the past several decades, little research has been done into baseline values or stress induced changes of hemolymph chemistry and cytology for this species (or any closely related gastropod mollusk). We hypothesized that commonly used clinical pathology methods should be applicable to Aplysia hemolymph and that stressors associated with husbandry and shipping should alter values of hemolymph constituents. The ability to identify shifts in hemolymph values that predict health status would be a valuable tool for both culture facilities and researchers alike who need to assess the health status of their study animals.

A three phase pilot project was conducted to evaluate changes in hemolymph variables induced by a variety of stressors, including air exposure, anesthesia, hypo- and hyperthermia. These stressors were chosen to mimic conditions likely to be encountered in culture facilities or research laboratories as well as in the wild (with the exception of anesthesia). Values including total hemocyte count (THC), osmolality, electrolytes (K⁺, Ca²⁺, Mg²⁺, Na⁺, and Cl^-), total protein (TP), and glucose were monitored pre- and post-exposure Additionally, protein electrophoresis was performed, and techniques for differential cell counts and lactate and cortisol assays were explored. Baseline values were established for a set of twenty animals. All animals were housed at the National Resource for Aplysia in Miami, Florida. Study animals were age and weight matched. Individuals within a given treatment group were uniquely identified and from a single hatchery batch.

For controls, TP increased initially while both K⁺ and Ca²⁺ decreased initially, then returned to baseline, and THC decreased and did not return to baseline. Following heat exposure, TP decreased at 24 h, glucose increased and then decreased, THC increased and then decreased, K+ increased and remained elevated, and Ca+ increased 24 h later. Following air exposure, glucose increased at 24 h, THC decreased and remained low. Cold exposure resulted in a sustained increase in THC. Electrophoresis showed increased ~70 kDa band intensity immediately after air exposure, increased then decreased 70 kDa band intensity after heat exposure, and decreased 30 kDa band intensity immediately after cold exposure.

THC, osmolality, K⁺, Ca²⁺, glucose by hexokinase method, TP by Bradford method, and protein electrophoresis all provided literature supported or biologically sensible results. Mean value for glucose, TP and osmolality were similar to those reported in the literature for other mollusks.

Changes from baseline values in THC, ions, TP, glucose, and electrophoretic band patterns indicate that the stressors examined impact hemolymph composition. With further work including establishing baseline values for Aplysia that account for variables like age and season, a baseline picture of a healthy Aplysia could be established. These results constitute an initial step in identifying a minimum database of values useful for assessing Aplysia health.

Acknowledgements

This work was supported by a Frontiers of Veterinary Medicine Fellowship from the Geraldine R. Dodge Foundation. All animals used in the studies and additional supplies and equipment were provided by the National Resource for Aplysia supported by PHS grant RR01094. Equipment and supplies were also loaned from and donated by North Carolina State University, College of Veterinary Medicine.