Documentation

The Crayfish Y-Maze is a flow-through two-choice maze used in the evaluation of choice behaviors in crayfish. The apparatus consists of a large tank that is divided at one end into the start area and the other end into two choice arms by a partition. The back walls of each of the choice arms are connected to an inlet through which odor stimuli are introduced as the waters flow into the arms. The design of the apparatus takes into consideration the role the olfactory system plays in crayfish behaviors such as foraging, mating, and predator recognition (Horner, Schmidt, Edwards, & Derby, 2007; Jurcak & Moore, 2014).

Studies such as those by Belanger, Evans, Abraham, and Barawi  (2017), and Lahman, Trent, and Moore (2015) have highlighted the growing impact of pollution on natural behaviors of crayfish. The Crayfish Y-Maze allows the opportunity to observe the impacts of different pollutants and other pharmacological manipulations on chemoreception in the crayfish. Understanding the modification or deficits in behaviors can be used as a potential indicator of aquatic pollution. The maze can also be extended to evaluate learning and memory behaviors of crayfish using olfactory cue-based protocols. Further, observation of preference behaviors and social behaviors can also be performed in the Crayfish Y-Maze using chemosensory cues. The simple design of the maze makes it easy to modify for different investigatory needs, and modifications such as live stimuli holders or reward holders may allow mimicking of natural encounters.

Other Y-Mazes used to observe foraging behaviors in other animals include the Rodent Y-Maze, the Zebrafish Y-Maze, the Pig Y-Maze, and the Bat Y-Maze.

The Crayfish Y-Maze is composed of an opaque flow-through tank and reservoirs. The tank measures 76 × 40 × 30 cm and is divided into two sections; start area and choice arm area. The choice arms are created by using a partition to create two 50 × 20 × 30 cm arms. The start area serves as the neutral zone and is equipped with a gated shelter that can be used to acclimate the subjects. Each arm is connected to a 4.45 L elevated reservoir tank through an inlet at the back walls. The flow-rate of each reservoir tank can be controlled using the in-line flow meters. Water exits the tank through 5 outflow pipes present at the start area wall at the height of 5 cm above the maze floor.

Based on the experiment requirements, clean the apparatus and change the water to remove any lingering cues. It is advised to rinse the maze using hot and distilled water in alternation for 10 minutes to remove any lingering odors after every trial. Appropriately illuminate the apparatus. A tracking and recording system such as the Noldus Ethovision XT can be used to assist with observations.

Following is a sample protocol for Crayfish Y-Maze using appetitive rewards and a constant flow rate. The subjects may be required to be food-restricted prior trials to increase the motivation to perform the task.

Habituation and Pretraining

Place the subject in the gated shelter for 10 minutes and initialize the flow. Remove the shelter once the 10 minutes have elapsed, and allow the subject to explore the maze 15 minutes at the set constant flow rate. Place the subjects back in the shelter at the end of the 15 minutes.

Crayfish Y-Maze Task

Begin the Y-Maze Task immediately after the pretraining session. Select at random the choice arm to be rewarded and place the food reward 3 cm in front of the corresponding inlet nozzle with the subject still in the shelter. Remove the shelter and allow the subject to explore and consume the reward for a full 15 minutes trial. At the end of the 15 minutes, place the subject back in the shelter to repeat trials, if required.

Investigation of the effects of toxicant exposure via turbulent dispersion on foraging behavior

Ludington and Moore (2016) investigated the effects of exposure to sublethal concentrations of carbaryl under different turbulent and spatial conditions on foraging behaviors of female crayfish (Orconectes virilis). Crayfish were starved for 7 days prior to and during the 2 days of the experiment to maintain their motivation. Exposure treatments were based on a 2 × 2 × 2 fully factorial experimental design that involved the following factors: toxicant or no toxicant (control), upstream flow or downstream flow, and obstruction or no obstruction. Thus, a total of 8 exposure conditions were formed. The treatments were applied over the course of 48 hours using flow-through trough corresponding to each condition group. Following the treatment, the crayfish were evaluated for their foraging behavior in the Crayfish Y-Maze using fish-flavored gelatin as the odor source and the reward in one of the arms. In comparison to the control groups, carbaryl treated groups displayed significant behavioral impairments in the Crayfish Y-Maze task. Among the downstream carbaryl groups, the group that had an obstruction during the exposure was observed to spend significantly more time in the wrong arm in comparison to the group without any obstruction. Further, the group was also observed to spend less time on the reward in comparison to the latter group. No significant difference in the time spent in either arm or on the reward could be observed in the upstream carbaryl groups. However, in comparison to the carbaryl downstream group with no obstruction, the corresponding upstream group was observed spending significantly less time on the reward.

Evaluation of the effects of acute atrazine exposure on the chemosensory responses of crayfish

Belanger et al. (2015) evaluated whether chemosensory responses of male and female crayfish (Orconectes virilis) recovered following acute exposure to atrazine (ATR) in a Crayfish Y-Maze task. Two weeks prior to experimentations, the crayfishes were maintained on a diet of rabbit pellets and fish-flavored gelatin three times per week. The Crayfishes were starved for 1 week and divided into atrazine exposed and controls by placing them individually in 1500 L with the corresponding treatment solution. The treatment period lasted for 96 hours, during which the ATR group was placed in an environmentally relevant concentration of 80 ppb (lg/L) of ATR. Following the treatment period, all subjects were placed in 500 mL of clean, dechlorinated water for 3 days as part of recovery treatment. During the treatment and recovery, water was changed every morning with fresh solutions. The subjects were then tested in the Y-Maze for their responses to food odor with distilled water flowing through the maze. The performances were evaluated at 0, 24, 48, and 72 hours post-treatment for 4 days using fish-flavored gelatin for the food odor and plain gelatin as the control placed at the back wall of the choice arms. The subjects were individually placed in the start area, and behaviors were recorded. The ATR exposed group was observed to spend significantly less time in the food arm and near the odor source (10 cm area zone near the source) of the maze as trials progressed in comparison to the controls. By the 48- and 72-hour mark, the number of ATR exposed crayfish finding and handling the food odor source had decreased relative to controls (3 vs. 9 at 48 h and 4 vs. 10 at 72 h). Additionally, it was observed that in comparison to the ATR group, the controls consumed significantly more fish-flavored gelatin following 72 hours. Based on the observations, it was concluded that atrazine results in chemosensory deficits in the crayfish.

Evaluation of the effects of herbicides on the feeding behaviors of crayfish

Browne and Moore (2014) evaluated the effect of sub-lethal levels of 2,4-diclorophenocyacetic acid (2,4-D) on the feeding behaviors of crayfish (Orconectes rusticus). Male and female crayfish with intact appendages were fed fish gelatin for 2 weeks and then starved for 1 week prior to the trials. The subjects were evaluated for 3 concentrations of 2,4-D: 32.69 mg/L (high), 14.07 mg/L (medium), and 7.65 mg/L (low). The subjects were placed in groups of 3 in the corresponding exposure tanks over the course of 96 hours. The exposure tank solutions were replaced for each exposure cycle. Following the exposure treatment, the subjects were tested on the Crayfish Y-Maze using the same 2,4-D concentrations in the tank that they had been exposed to. Foraging behaviors were observed using fish gelatin in the randomly selected rewarded arm of the maze. A significant effect of 2,4-D concentrations on the foraging behavior of crayfish could be observed. In comparison to the controls (256.6 ± 49.8 s) and the medium concentration (315.2 ± 67.6 s) group, the high concentration group was observed to take longer to find the food (559.6 ± 87.9 s). In comparison to the controls, all exposure groups spent significantly less time in the correct arm. In terms of speed, exposure groups had higher walking speed than the controls. However, the overall speed of the controls and high concentration were similar, though medium and low concentration groups displayed faster overall speeds than the former two groups. Additionally, all exposure groups displayed a decrease in reward consumption. The high concentration group consumed significantly lower food in comparison to the controls as well as the two other exposure groups.

Investigation of responses of male crayfish to molt-related chemical stimuli

Adams and Moore (2003) investigated differential responses of male crayfish (Orconectes rusticus) to male molt odor signals in a Crayfish Y-Maze. In order to obtain molt cues, a separate group of crayfish was placed in isolated pots (no external flow) within which molting was induced. Water from the isolation tanks of recently molted male crayfish and intermolt male crayfish were collected and used as molt and intermolt chemical cues, respectively. For control cue, aged tank water was used while food cue was obtained from a blended and strained mixture of haddock and aged tank water. The subjects were starved 2 days prior to testing and were assessed under either pair-wise combination of the 4 stimuli or with identical stimuli in both arms. A significant initial preference for the molt arm was observed in the combinations with control stimuli. Further, subjects were observed spending more time in the molt arm as well as the food arm in the combinations with control stimuli. In molt vs. intermolt choice, crayfish had a significant initial preference for the molt arm. Further, in molt vs. food combination, crayfish were observed spending significant time in the food arm; however, such an observation was not made in intermolt vs. food combination. The preferences observed were also reflected in the time spent near the nozzle. When presented with identical cues in the maze, the subjects were observed to walk faster in the molt cue than in the intermolt cue test. In comparison to the intermolt cue and control cue tests, the time spent moving was significantly lower in the molt condition.

The following can be observed on the Crayfish Y-Maze:

• Time taken to leave the shelter
• Time spent in the start area
• Initial arm choice
• Latency to choose
• Time spent in each choice arm
• Time spent consuming/exploring the reward
• Total distance covered
• Walking speed
• Number of correct choices
• Number of incorrect choices
• Time taken to find the reward
• Amount of food consumed

Strengths

The Crayfish Y-Maze is designed as a flow-through maze to mimic certain parameters of the natural environment of crayfish. The flow in the maze can be controlled using in-line flow meters connected to each of the elevated reservoir tanks. Since each arm is connected with its own elevated reservoir tank, the tanks can be filled with different solutions, and responses to a different olfactory stimulus can be evaluated within the two-choice set-up. The two-choice set-up can be easily adapted to evaluate behavioral responses to cues such as predator cue, prey cue, and conspecific cue to observe the social behaviors of crayfish. Additionally, the maze can be used to evaluate learning and memory behaviors following pre-trial treatments such as pharmacological manipulation and stress. The maze also comes equipped with a shelter that can be used for maze acclimation and to introduce delays.

Limitations

Improper handling and maintenance of the subject can impact task performances. The presence of unintended cues can result in an erroneous result. AN improper flow rate in the choice arms may affect the rate at which cues are dispersed. For experiments using food rewards, changes in quality and quantity of the rewards may affect the subject’s motivation to perform the task. Factors such as age, gender, and strain of the subjects may affect task performance.

• The Crayfish Y-Maze is used in determining choice behaviors and learning and memory behaviors of crayfish.
• The apparatus is a flow-through maze composed of a maze tank and solution holding reservoirs.
• Each choice arm of the maze is connected to one elevated reservoir tank, thus, allowing 2 different solutions to be simultaneously used.
• The inflow can be controlled by in-line flowmeters, while the five outflow pipes at the end of the maze ensure the drainage of the water.
• The apparatus comes equipped with a gated shelter that can be used for acclimation and task delays.

1. Adams, J. A., & Moore, P. A. (2003). Discrimination of conspecific male molt odor signals by male crayfish, Orconectes rusticus. Journal of Crustacean Biology, 23(1), 7-14. doi:10.1163/20021975-99990309
2. Belanger, R. M., Evans, K. R., Abraham, N. K., & Barawi, K. M. (2017). Diminished Conspecific Odor Recognition in the Rusty Crayfish (Orconectes rusticus) Following a 96-h Exposure to Atrazine. Bulletin of Environmental Contamination and Toxicology, 99(5), 555–560.doi:10.1007/s00128-017-2178-3
3. Belanger, R. M., Mooney, L. N., Nguyen, H. M., Abraham, N. K., Peters, T. J., Kana, M. A., & May, L. A. (2016). Acute atrazine exposure has lasting effects on chemosensory responses to food odors in crayfish (Orconectes virilis). Archives of environmental contamination and toxicology, 70(2), 289-300. doi: 10.1007/s00244-015-0234-8.
4. Browne, A. M., & Moore, P. A. (2014). The effects of sublethal levels of 2, 4-dichlorophenoxyacetic acid herbicide (2, 4-D) on feeding behaviors of the crayfish O. rusticus. Archives of environmental contamination and toxicology, 67(2), 234-244. doi: 1007/s00244-014-0032-8
5. Horner, A. J., Schmidt, M., Edwards, D. H., & Derby, C. D. (2007). Role of the olfactory pathway in agonistic behavior of crayfish, Procambarus clarkii. Invertebrate Neuroscience, 8(1), 11–18. doi:10.1007/s10158-007-0063-1
6. Jurcak, A. M., & Moore, P. A. (2014). Behavioral decisions in sensory landscapes: crayfish use chemical signals to make habitat use choices. Journal of Crustacean Biology, 34(5), 559–564.doi:10.1163/1937240x-00002266
7. Lahman, S. E., Trent, K. R., & Moore, P. A. (2015). Sublethal copper toxicity impairs chemical orientation in the crayfish, Orconectes rusticus. Ecotoxicology and Environmental Safety, 113, 369–377. doi:10.1016/j.ecoenv.2014.12.022
8. Ludington, T. S., & Moore, P. A. (2016). The Degree of Impairment of Foraging in Crayfish (Orconectes virilis) due to Insecticide Exposure is Dependent upon Turbulence Dispersion. Archives of Environmental Contamination and Toxicology, 72(2), 281–293. doi:10.1007/s00244-016-0341-1