To our knowledge, no other study of invertebrates examines whether environmental factors directly influence learning and task completion. Furthermore, no other study directly examines learning in cave crayfish. Materials and Methods Animals Procambarus clarkii (sighted crayfish; 5.08- to 6.35-cm body length) were obtained commercially

from Atchafalaya Biological Supply Co. (Raceland, LA). Orconectes australis packardi (blind crayfish; Rhoades; 4.5- to 6.35-cm body length) were obtained from Sloan’s Valley Cave System (Somerset, KY; collecting permits obtained). These studies were conducted in Lexington, KY, between 2006 and 2009. A total of 24 sighted Inhibitors,research,lifescience,medical and 24 blind crayfish were used. Both sexes were used, but learning differences between the sexes were not analyzed. Animals Inhibitors,research,lifescience,medical were housed individually in rectangular plastic containers and cared for in the same manner in an aquatic facility within our temperature-regulated laboratory (17–20°C). All animals were on a 12-h light–dark cycle, but O. a. packardi were covered with black plastic to omit light. They were fed dried fish pellets weekly until 2 weeks prior

to experimentation. During experimentation, food was restricted to 30% of normal amounts. Because the crayfish were kept in a small container, Inhibitors,research,lifescience,medical their energetic needs were likely reduced. They were fed 1.2 g of “shrimp and plankton sticks: sinking mini sticks” (Aquadine, AquaDine Nutritional System, Healdsburg, CA). Crayfish handling was conducted using a glass beaker to transfer crayfish between containers. Because containers were cleaned weekly, the crayfish Inhibitors,research,lifescience,medical were handled often. This limited handling during experimentation is assumed to have little to no effect on the internal status of the crayfish. Only crayfish in the intermolt stage, possessing all Inhibitors,research,lifescience,medical walking legs and both chelipeds, were used. Chamber design Four rectangular experimental chambers were constructed from Plexiglas (18 × 8 × 8 cm) with an 8-cm Plexiglas divider dividing one third of the container from the rest (Fig. 1). Sand was permanently glued

to the bottom mTOR inhibitor surface for traction. The crayfish were placed in the larger chamber. A vertical platform was placed in the smaller chamber, approximately 1 cm from the divider. The platform was a square plastic object (5.5 cm2) with mesh material on the surface. The access point was a half-oval shaped opening in the Plexiglas divider. This allowed only a single cheliped to enter into the smaller PDK4 portion of the chamber (the hole was adjusted in size based on the species to be only slighter larger than a single cheliped). The food reward was five thawed bloodworms (mosquito larvae, PetCo, Lexington, KY) attached through the mesh material and placed into the chamber before the crayfish were added. The worms were centered 3 cm above the access point, which required the animal to reach in and up to obtain the food source.