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Research Organism

Thirty form II (non-reproductive form) male crayfish of F. rusticus and F. virilis were utilized in this experiment. Individuals were collected from two bodies of water in northern Michigan, Carp River (45.680000 °N, -84.814797 °W) and Burt Lake (45.404175 °N, -84.628630 °W). Animals were collected via Gee’s© Minnow Traps (Carp River) baited with sardines (Beach Cliff© sardines in water) and hand collection with nets (Maple Bay, Burt Lake) based on the collection requirements of the waterbody. All individuals captured were transported back to the Experimental Stream Research Facility at the University of Michigan Biological Station (UMBS). All crayfish were housed in a flow-through mesocosm, individually contained within plastic containers (16 x 14 x 6 cm, l x w x h) with opaque sides and an ID number. Thus, crayfish were separated from each other physically and visually. Post-orbital carapace length (cm), and left propodus (cm) measurements were collected for each crayfish along with species, and any alterations to normal morphology that were of note. Crayfish with notable differences between chelae or loss of antenna were removed from the study. Pairings of crayfish were made after the boldness/shyness value was determined. A carapace length difference of 10% or less was used to match F. rusticus and F. virilis together for the duration of the experiment.

The holding mesocosm (203.2 x 81.3 x 40.6 cm, l x w x h) was a flow-through system created with cinder blocks and lined with 6-mil black plastic sheeting. Untreated river water entered the system from the East Branch of the Maple River, which runs adjacent to the facility and contains both species of crayfish. Individuals were housed between June and July of 2024, in ambient temperatures, as well as light and dark cycles for that time. All individuals were marked with Wite-Out© (BIC© Wite-Out© 2-in-1 Correction Fluid) as a unique ID before the start of the experimental portion of the study.

Mesocosm Design

In order to determine the impact of the different treatment types and focal boldness/shyness on shelter use, foraging, and consumption behaviors, flow-through mesocosms were built. The mesocosms consisted of a smaller upstream section (termed the cue donor section: 40.6 x 61 x 40.6 cm, l x w x h), to hold the cue donor crayfish and a larger downstream behavioral section (termed the assay section: 80 x 61 x 40.6 cm, l x w x h) for the focal crayfish were created. A total of eight mesocosms were built for the behavioral assays constructed from cinderblocks and 6 mil plastic sheeting. Between the two sections, a single cinderblock was placed such that the cinderblock openings were horizontal and allowed water to flow from the cue donor section into the focal crayfish section. This cinderblock was covered with a mesh window screening that prevented the crayfish from moving between the two sections but still allows for movement of water and odors. Four mesocosms were fed by a single 208 L head tank which fed unfiltered water from the Maple River into each mesocosm through two garden hoses (inner diameter = 1.9 cm) with a flow-through water rate of 0.1 ± 0.05 L/s per garden hose. A total of two head tanks were used, one for each set of four mesocosms, and were filled from a 7.6 cm PVC pipe with a filter made from nylon stockings to prevent detritus and macroinvertebrates from the river entering the mesocosms.

The focal section also contained approximately 2 cm of sand covering the bottom, a half-cut PVC pipe (9.9 x 7.5 x 4.5 cm, l x w x h) with a single opening that served as a shelter and a food gelatin cap (described below). A wooden structure was constructed above the lower portion of the mesocosm to attach a security camera approximately 1.7 meters above the mesocosm, with video footage being recorded onto a DVR (Zosi, H.265+ 8-Channel 5MP-LITE DVR 1TB Hard Drive and 1080P Wired Bullet Cameras). Red lights were placed by the mesocosm for better observation of the animal’s behavior at night.

Food gelatin for the foraging and consumption assays was created using 13 g of sardines (Beach Cliff© sardines in water), 7 g of unflavored gelatin (Knox©), and 175 mL of water to make approximately 40 food cups (10 ml), with approximately 2.3 g of emulsified sardine gelatin in each.

Assay Protocol

To account for the interaction between crayfish personality and sensory cues on resource use, crayfish with known personality values were run in one of four conditions. Two of the conditions were termed unpaired (because a single animal was run) and considered control treatments: F. rusticus alone with the resources, F. virilis alone with the resources. Two other conditions were termed paired (because size-matched pairs were run) and considered experimental treatments: F. rusticus in the assay section with F. virilis in the cue donor section, and a F. virilis in the assay section with a F. rusticus cue donor section. All crayfish were run through a personality test first (see below) and then size-matched (within 10% of carapace length). Crayfish pairs were run through the unpaired (control) assays first and then run through the paired assays (one assay as the cue and one as the focal) with F. rusticus being placed upstream first, followed by the trials with F. virilis being placed upstream. All behavioral assays in mesocosms were run between 1400 and 0900 with video recordings of the behaviors occurring between 0000 and 0400. If the trial was an experimental treatment, a cue donor crayfish was placed upstream in a weighted deli container (18.5 x 18.5 x 8.5 cm, l x w x h) within the 10 minutes before the focal crayfish placement. The deli container had four windows with screening that allowed water and odor to flow out of the container. Organisms were removed from their mesocosms at 0900. Change in food weight (g) was determined at this time, after the food was dabbed dry with a microfiber cloth to remove excess water.

Personality Test

Crayfish were run through a behavioral assay for placement on a boldness/shyness spectrum. All crayfish were run three times with thirty minutes of rest between any single behavioral test. To determine the placement of a crayfish on a bold/shyness spectrum, the latency to exit a shelter test was used. The test took place within a 33.3 x 17 x 24.4 cm (l x w x h) arena covered in black plastic with a single shelter (13.5 x 6.2 x 7.3 cm, l x w x h) with a removable door on the front of the shelter. Since four behavioral tests could be run simultaneously, the black plastic served to visually isolate the trials from each other. The top of the shelter had a hole connected to a 0.6 cm PVC tube connected to a funnel at the end opposite the exit door which was used to deliver 25 ml of predatory bass (Largemouth bass, Micropterus salmoides) odor. Each arena was filled with 5000 mL of Maple River water. A test subject was placed inside the closed shelter and allowed 15 minutes to acclimate. After this time, the bass odor water was poured into the shelter while the door was lifted simultaneously. Crayfish were timed on the latency to emerge from the shelter. A crayfish was considered out of the shelter once the entire carapace of the crayfish was outside of the shelter and the crayfish stayed outside of the shelter. Trials were run for a maximum time of 15 minutes. If the crayfish did not exit the shelter within 15 minutes, the trial was ended, and 900 s was noted as the latency time.

Ethical Statement

Largemouth bass were maintained and utilized based on established care and use protocols, with the utilization of vertebrate animals approved by the IACUC at University of Michigan (protocol PRO00010612) and by IACUC at Bowling Green State University (protocol 1861153-3).

Data Collection and Video Analysis

All videos were analyzed by an observer blind to the treatment as the cue donor section of the mesocosm was not visible on the video display. Total time spent foraging (s) and total time spent sheltering (s) were determined. Time spent foraging was defined as the amount of time the crayfish spent over the fish gelatin cap, and the time spent sheltering is the time the crayfish stayed completely or partially in the shelter during the behavioral assay.

A boldness/shyness value was created by averaging the three latencies to exit assays per individual. Since a greater latency to exit the shelter is an indication of shyness, a boldness value was created by subtracting a crayfish’s latency to exit from 900 (maximum time to exit). The new values reflect the condition where the boldest crayfish has the highest value. Finally, a spectrum of boldness for the population was created by dividing all the boldness values by the individual with the greatest boldness value. Thus, the crayfish that exited the fastest had a value of one and all other crayfish fell between this value and zero.