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Do Not Pet the Crawfish: Starting an Invertebrate Behavioral Lab.

Historic sources e.g. (A.P.A., 2007; Norcross, Sayette, Stratigis, & Zimmerman, 2014) extol the virtues of undergraduate behavioral research with animals in a laboratory (e.g. such training is favored by graduate school selection committees). In past decades, faculty and students in our psychology department at a south Louisiana university maintained a "rodent lab" where students learned about the care and handling of lab animals, participated in faculty research, and, in some instances, designed, conducted and published their own studies.

The department has been without a lab for several years due to retirements and funding cutbacks. Restarting a viable rodent lab requires considerable financial resources and Herculean effort. Given these considerations our lab has transitioned away from small mammals to invertebrate research.

In an editorial, Brembs (2013) provided a rationale for using invertebrates for experimental study. He cited examples of using invertebrate responses as an analog for more complex behavioral systems (reductionism) (Brembs, 2013). Finding simpler ways of explaining phenomena enables us to understand complexities and provides opportunities to study parallel mechanisms. After considerable deliberation and a brief foray into a "Ladybug Lab," we had a flash of insight: "Crawfish!"

This article describes our experiences over the past two years in founding our undergraduate crawfish lab, a practical, cost-effective alternative to behavioral research with vertebrates. Now that the lab is well-established we can offer interested colleagues insights that might prevent a "Crawpocalypse" in future labs. Mistakes that we made, as well as useful tips on how to prepare tanks, populate and maintain a healthy colony will be discussed. (1)

Early Lessons.

Rough start. After acquiring a donated 20 gallon tank, we bought a power filter, gravel, heater, dip net, water conditioner/dechlorinator, a few (1"x 5") PVC pipes, a 1/4 inch galvanized wire mesh as an aquarium hood, sinking spirulina food pellets for freshwater invertebrates (Aquatic Arts, USA), a siphon to clean the gravel (TERA PUMP[C] Genuine Aquarium Cleaner with Long Nozzle) and a timer for the lights (Model 63-864, Radio Shack, USA).

"Slough" crawfish (P. fallax), common to Louisiana and nearby states, are widely available by the sack. As the first tank was a pilot, a sack crawfish was put in; it scurried into a pipe and hid. For about a week, students fed it and observed its behavior. Crawfish are nocturnal, but especially when fed, do venture into the light. Crawfish are highly mobile and agile creatures; ours climbed up the heater cord and out of the tank. Lesson learned, crawfish are escape artists and lids are a must!

Our next lesson followed shortly thereafter when we added a second crawfish to the tank. It scurried into another pipe seemingly safe and sound. Flush with success, well on our way towards the inevitable self-sustaining Craw-colony, we left.

The next day we observed a horrific Gotterdamme-craw. Claws, carapaces, and antennae scattered in a watery killing-field! "Craw 1" had killed "Craw 2"; sack crawfish are highly territorial and aggressive!

Choosing the right crawfish and several cauCrawtions.

The solution was the discovery of P. virginalis (a.k.a. Marmorkrebs" or "Marbled Crawfish") which are remarkably docile. "Crawtion#1": MARBLED CRAWFISH ARE AN INVASIVE SPECIES AND MUST NOT BE RELEASED INTO THE WILD. IF YOU CANNOT SAFEGUARD AGAINST THIS, FIND ANOTHER INVERTEBRATE. THEY ARE ALSO ILLEGAL IN PLACES LIKE MISSOURI, TENNESEE AND THE EUROPEAN UNION. Marmorkrebs are relatives of "slough" crawfish, Aquatic Arts, (2019) notes that Marmorkrebs are "an all-female mutation that continually produces its own fertilized [sic] eggs, which develop into exact clones of the mother"; of course it is parthenogenesis that takes place in the female crawfish. The eggs are not actually fertilized. According to the Invasive Species Compendium (CABI), Marbled Crawfish are typically 10 cm but can be up to 13 cm long (CABI, 2019). Marmorkrebs are sold on the internet. Purchasing led to several "real-world" learning experiences, as in: "there's a head-sucker (2) born every minute."

At first, we bought dud-Marmorkrebs--crawfish of another species (some of them males) sold to us as Marmorkrebs. "Crawtion 2": Caveat Emp-craw! Buyers beware! Later, we bought and placed "berried" (3) (pregnant) mothers" in the aquariums (by this time we had four tanks) and waited for the joyful clatter of tiny craw feet; only the berries vanished, stress led the faux-Marmorkreb to infanticide. Finally, an honest marketeer mailed an authentic Marmorkreb; like new parents everywhere, our craw-babies (4) changed our lives forever!

Some owners grow Marmorkrebs in an empty bucket. Aside from the ethical/safety issues, the current authors view this as cruel.

A final note. The path to Hell is paved with good intentions. This article's title reflects a lab worker's bonding with her Marmorkreb so much that she'd pet it. She was inconsolable when it died, not because her grade might suffer but because, unaware, she was stressing her Marmorkreb; she had innocently "petted her craw to death."

In our experience, tropical fish in aquariums are exquisitely sensitive; however, crawfish are much less so. None the less, our lab is over-engineered with backups. It has one extra of everything and the equipment was designed for much larger tanks than the ones we used.

CRAWFISH HUSBANDRY

Materials

Marmorkrebs. We bought five berried craws; and after establishing vender reliability, bought an additional five (total $120.00).

Aquariums. In addition to a donated 20 gallon tank, one 40 gallon and four 10 gallon tanks came from a local PetSmart[c]. PetSmart[c] has a yearly sale where tanks of 40 gallons or less cost about $1.50 per gallon. Total cost was approximately $120.00. We recommend that you buy locallyfor easy return if something is wrong, and suggest that you test the tanks immediately. Juvenile/adult Craws live in a 10 gallon tank and hatchlings are raised in the 10 gallon tanks; all aquariums are kept in optimal condition.

Gravel, power filters and other necessities. White gravel (to ease Craw visibility) was purchased locally and six AquaClear[c] Power Filters (total ~$240.00) were internet purchases. The AquaClears[c] (rated for 20 to 50 gallon tanks) were used on the 10 and 20 gallon aquarium(s), larger filters ensured cleaner water. Filter material (AquaClear[c] Foam Inserts) are a continuing expense at $10.00 total, filters are changed every four months and can be rinsed out in between).

The six Aqueon[c] Pro Submersible 100W heaters (one in reserve) totaled $156.00; like the pumps, the heaters were rated for larger tanks. An HDE Digital pH Meter and LCD Thermometer[C] (~$28.00), a bottle of Tetra SafeStart Plus[c]dechlorinator ($11.00) and a box of Aquatic Arts[c] Sinking Pellets ($15.00) (another continuing expense) were ordered online. The 40 gallon tank, filled with clean treated water is used to replenish the other tanks.

At first, live aquatic plants were used, but the Craws ate them, so replica plants, coral, and in the hatchling tank, plastic grass was used. Experimenters who start a Marmorkreb lab must check that anything in the tank with the Craws does not decompose and is not made of, and does not leach, toxic chemicals. Before anything inside the tank is disposed of, any Craws or eggs on it must be euthanized.

Food. Crawfish were fed sinking spirulina pellets with an occasional piece of raw chicken or vegetables. As an aside, the current first author wanted to supplement the Craw's diet and developed an elaborate procedure to keep cucumber slices on the tank's floor. Little glass vials were filed with small rocks and water and cubes of cucumber were jammed into the vials; this made the greenery stay on the gravel.

Months later, the second author took some Marmorkrebs home. Fascinated, his children fed their new friends a bit of fresh cucumber. They dropped small pieces of raw cucumber into the tank; the Craws swam to the top of the water and happily chomped away.

If asked, however, the triumphant first author will wax poetically about his method of steering food pellets to a specific craw with a drinking straw (5).The method involves keeping one end of the straw above the water; the other end is lowered into the tank and placed just in front of the craw. A pellet is placed in the top of the straw and (if the inside of the straw remains dry) the pellet will fall just in front of the intended recipient.

Procedure

The Lab's Craws are housed approximately 10 adults to each 10 gallon tank. Water is maintained at ~22[degrees]C, filtered continuously (1/3 was replaced every 2 weeks) and gravel is cleaned often. Area lighting is on a 12 hour cycle (12 on/12 off). Tanks contained plastic aquatic plants and coral and 3" sections of 1 1/2 PVC pipe (at least one for each mature resident craw). Each mature Craw was fed 2-3 spirulina pellets (0.05g) about every 2-3 days.

Any water removed from aquariums (e.g. siphoned water from the fortnightly gravel cleaning) was filtered through a fine wire strainer into buckets with 20% dilution of chlorine/water to ensure no live crawfish or eggs were introduced to the local sewer system.

CABI, (2019) reports Marmorkrebs mature fast, "berry" early, and have a long breeding period. Growth rate varies with water temperature, fastest growth is at 30[degrees]C, but more survive at a temperature 10[degrees] lower. Under optimal conditions, they "berry" in 141-255 days after birth. Depending upon the environment, Marmorkrebs give birth from March through December.

CRAWFISH EXPERIMENTS

Materials

The plus-maze. A transparent, watertight Plexiglas[R]plus-maze with four 30cm x 15cm arms branching from a 15cm x 15cm central start area was built in-house. For each experiment the maze rested in the middle of a flat tabletop surface.

Video camera. A Logitech[R] HD C270 Webcam, (Office Depot, USA) was mounted 2 meters above the center of the maze on a custom mount. It was controlled by a laptop computer running the device drivers that came with the webcam. The camera was placed directly above the maze to record crawfish movements.

Analysis of behavior. AnyMaze Software[R]for Windows[R]v.4.82 (Stoelting, USA) was used to analyze the videos. This software allows automatic measurement of distance traveled, speed, and time spent immobilized. It is optional but handy.

Procedure

Habituation. Prior to each of the protocols below, crawfish were allowed to freely explore the maze for two 10 minute trials. No food or escape was available during habituation. If crawfish preferred a certain arm, the goal arm was assigned to the non-preferred arm. Random assignment was used in the case of no preference.

Three different learning paradigms were tried in the lab. They were, from least to most effective, a Warm-water Escape paradigm (Tierney & Lee, 2011), a food-reward paradigm, and a light escape paradigm.

Warm-WaterEscape. The warm-water escape paradigm was developed by Tierney and Lee (2011). The maze was flooded to a depth of 5cm. On the right wall at either end of the T-maze was a 10cm wide opening, 5cm above the maze floor that led to a separate holding tank. A small set of steps allowed crawfish to climb through the opening. Openings are blocked or unblocked by a removable Plexiglas barrier.

Maze Preparation. The maze was filled with 29-31[degrees]C water for each of the 5 consecutive days of testing. Water at this temperature induces heat-stress (Tierney& Lee, 2011). The holding tank at the end of the goal arm was filled with room temperature (22[degrees]C) water.

Testing. Crawfish were placed into the central start area and latency to exit the maze through the opening in the goal arm was measured. We had difficulty maintaining the water temperature differences and this could account for the difference between the current efforts (Soignier, Rosenthal & Barr, 2018; Tierney & Lee, 2011).

Food-reward paradigm. The classic T-maze paradigm was employed (Tolman, 1948), except the maze was constructed of waterproof PVC plastic and the dimensions were smaller to accommodate our subjects. Briefly, in this paradigm animals were placed in a central start area and allowed to explore the maze until all food had been recovered or 20 minutes elapsed, at which point they were returned to their home tank.

Maze Preparation. The same T-Maze was used except the openings were obstructed and small food cups were placed at the end of each arm.

Testing. Following the habituation procedure, the number of incorrect arm entries and latency of the craw to retrieve food (0.005g spirulina pellet) from the food cup was measured. To maintain the appetitiveness of food reward, trials occurred every 2-3 days and animals were only fed their remaining allotment of food after completion of a trial.

Light-Escape Paradigm. This novel paradigm was developed after we noted the crawfish's strong affinity for dark spaces like the interior of the PVC pipe.

Maze preparation. The same T-Maze was used except the illumination of the maze was increased and the end of each arm contained a 3" section of 1.5" opaque PVC pipe as an escape chamber. The entrance to the PVC was blocked by Plexiglas in each of the non-goal arms.

Testing. The number of incorrect arm entries and latency to enter the PVC pipe in the goal arm was recorded for 5 consecutive trials.

Suggested experiments. Data from the above learning paradigms could be outcome measures in a variety of experiments. Examples of suggested hypotheses garnered from previous studies of other invertebrates follow:

1) Do crawfish respond to specific conditioned stimuli X, Y, or Z? This type of activity would be designed to introduce students to controlled experimentation. Students could be asked to choose stimuli, such as a red LED, to serve as a discriminant stimulus for the availability (S+) or absence (S-) of reinforcement (e.g. Sherman & Sherman, 1976). If crawfish can learn the discrimination, then they must be able to discern the discriminant stimulus.

2) Does LED wavelength have an effect on the circadian entrainment of crawfish? Observations of a "cleaner shrimp" Stenopushispidus in field and lab studies indicate that they exhibit several measurable behaviors when active, e.g. they clean parasites and other debris. When kept in aquaria and exposed to 12 hr light and 12-hr darkness they were active during darkness and inactive during light, however, the wavelength of light mattered: where blue or green LED were used, a day-inactive and night-active rhythmicity was observed. This was not observed when red LED was used. This design and results of this project provide a basis for studying the effects of LED on the behavior of crawfish (Yuriko, Yoshioka, Takruchi, Hur & Takemura, 2016).

3) What is the effect of short wavelength versus UV light range radiation on the phototaxis of crawfish? This suggested experiment is an extension of the LED wavelength on phototaxis study. Short-wavelength (blue through UV) light is "toxic" to some organisms, especially to insects. Research indicates that among insects, the response to blue light and other shorter UV light wavelengths appears to be species specific (Hori, Shibuya, Sato, & Saito, 2014). Like insects, crawfish are arthropods. The impact of various short wavelength light sources on the activity of crawfish may be observed using the same methods as used on other arthropods.

4) Do Crawfish have a substrate preference? A substrate is the surface substance on which an organism lives and may include living, nonliving, natural or artificial substances. Karadal and Turkmen, (2014) investigated the effects of different substrates on the growth, survival and substrate preference in juvenile blue tiger crawfish (Cheraxalbertisii). Pebbles was the most preferred when compared to the other substrates.

DISCUSSION

Marmorkrebs as lab animals

Interested researchers can communicate with Zen Faulkes to procure what he calls the "Crayfish"; his NSF funded lab's webpage is: Marmorkreb.org (2019). It has over 50 Marmorkreb research references.

Marmorkrebs offer the benefits of docility. Not a single act of "Marmorkreb on Marmorkreb violence" occurred in our lab, or in the cloning population. Another benefit is that all Marmorkrebs are genetically identical (perhaps why they don't kill tank mates).

But, there are drawbacks to a Marmorkreb lab; researchers must exercise utmost caution that Marmorkrebs do not escape into the wild. All waste (e.g. water/gravel) from our aquariums is placed in a 20%/80% bleach/water solution for at least 10 minutes; please do not forget to treat the filter material (foam inserts) in the pumps. There are other protocols that must be observed as well.

An essential entreaty

We started this presentation with the humorous neologism "Crawpocalypse"; in all seriousness, if you experiment with Marmorkrebs, please use the rigorous safeguards you need to exercise with any invasive species.

REFERENCES

A.P.A. (2007). Getting In: A step-by-step plan for gaining admission to graduate school in psychology, (2nded.) Washington, D.C.: American Psychological Association.

Aquatic Arts (2019). Self-Cloning Marmorkreb Crayfish (Procambarus fallax cf. virginalis)-Tank-Bred! Retrieved from https://aquaticarts.com/products/selfcloning-marmorkreb-crayfish-juveniles

Brembs, B. (2013, November). Invertebrate behavior-actions or responses? Frontiers in Neuroscience, 7, 1-2. Doi: 10.3389/fnins.2013.00221

CABI (2019).Procambarus fallax f. virginalis (Marmorkrebs). [original text by Christoph Chucholl]. In: Invasive species compendium. Wallingford, UK: CAB International. https://www.cabi.org/isc/datasheet/110477

Hori, M., Shibuya, K., Sato, M. & Saito, Y. (2014).Lethal effects of short-wavelength visible light on insects. Scientific Reports, 12/12/2014, p1-6. DOI: 10.1038/srep07383

Karadal, O. & Turkmen, G. (2014). Effects of substrate preference on growth and survival of blue tiger crayfish (Cheraxalbertisii).Su UrunleriDergisi. 31(1): 1 4. DOI 10.12714/egejfas.2014.31.1.01

Marmorkreb.org (2019). Advancing marbled crayfish research. [original text by Zen Faulkes].https://faculty.utrgv.edu/zen.faulkes/marmorkrebs/

Norcross, J.C., Sayette, M.A., Stratigis, K..Y., & Zimmerman, B.E. (2014). Of course: Prerequisite courses for admission into APA-accredited clinical and counseling psychology programs. Teaching of Psychology, 41(4), 360-364.

Sherman, I.W., & Sherman, V.G. (1976). The invertebrates: Function and form: A laboratory guide. New York: Macmillan.

Soignier, R. D., Rosenthal, G. T., & Barr, J. E. (2018). Classic learning paradigms in Marmokrebs. Unpublished raw data.

Tierney, A.J., & Lee, J. (2011).Spatial learning in a T-maze by the crayfish Orconectesrusticus. Journal of Comparative Psychology, 125(1), 31-39.

Tolman, E.C. (1948). Cognitive maps in rats and men. Psychological Review, 55(4), 189-208.

Yuriko, E., Y., Yoshioka, E., Takruchi, Y., Hur, S., & Takemura, A. (2016). The effect of light Intensity and wavelength on diurnal activity of the banded coral shrimp Stenopushispidus (Decapoda, Stenopodidae): A possible adaptation for a cleaner shrimp in reef environments. Pacific Science, 70(2), 191-200.

Footnotes:

(1) The authors have no financial interest in any of the products discussed in this article.

(2) True sack crawfish aficionados will get this pun!

(3) "berried"-crawfish eggs appear as little "berries" underneath the mother's abdomen where she continually fans oxygenated water past them.

(4) Yes, that makes us Crawdaddies.

(5) Science lurches on.

Gary T. Rosenthal, R. Denis Soignier, James E. Barr

Nicholls State University

Barlow Soper

Ruston, Louisiana

Author info: Correspondence should be sent to: Dr. Gary Rosenthal, Ph.D., Department of Psychology. Nicholls State University, Box 2075, Thibiodaux, LA 70310 Email: gary.rosenthal@nicholls.edu
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Author:Rosenthal, Gary T.; Soignier, R. Denis; Barr, James E.; Soper, Barlow
Publication:North American Journal of Psychology
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Date:Dec 1, 2019
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