The development of the first Y-maze was supported by cooperative agreements (15-7412-1155-CA, 16-7412-1269-CA, and 17-7412-1318-CA) between James Madison University (JMU) and the USDA Animal and Plant Health Inspection Service. The development of the Y-maze in Everglades National Park was funded by a cooperative agreement (P18AC00760) between JMU and the National Park Service. We thank T. Dean and B. Falk for their facilitation of this project in Everglades NP and assistance with permitting and funding. We thank W. Kellow for assistance in construction of the USGS Y-maze. C. Romagosa, L. Bonewell, and R. Reed provided administrative and logistical support. We thank the two anonymous reviewers who offered helpful feedback. Funding for the Everglades work and in-kind support was provided by U.S. Geological Survey (USGS) Greater Everglades Priority Ecosystem Science Program, National Park Service (P18PG00352), and USGS Invasive Species Program. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The findings and conclusions in this publication have not been formally disseminated by the U.S. Department of Agriculture and should not be construed to represent USDA determination or policy.

<ol>
	<li>Fine, J. M., Vrieze, L. A., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+petromyzontid+lampreys+employ+a+common+migratory+pheromone+that+is+partially+comprised+of+bile+acids.">Evidence that petromyzontid lampreys employ a common migratory pheromone that is partially comprised of bile acids.</a> Journal of Chemical Ecology. 30 (11), 2091-2110 (2004).</li><li>Hesse, S., Bakker, T. C., Baldauf, S. A., Th&#252;nken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kin+recognition+by+phenotype+matching+is+family-rather+than+self-referential+in+juvenile+cichlid+fish.">Kin recognition by phenotype matching is family-rather than self-referential in juvenile cichlid fish.</a> Animal Behaviour. 84 (2), 451-457 (2012).</li><li>Forester, D. 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While Y-mazes are very powerful tools to investigate chemical ecology in reptiles, their limited design can preclude other avenues of inquiry. However, a diversity of other options is available11,12,20,21,22. For example, tongue-flick assays are simpler to execute and allow simultaneous assessment of behaviors exhibited to an array of chemical stimuli relative to control odors23,24,25,26. Open-field tests are another option where a focal animal freely explores an enclosure until it encounters a source of chemical cues, and its behavioral reactions are subsequently scored27,28. Combinations of these approaches can assess discriminatory capacities of reptiles in varying contexts such as presenting a mix of artificial and natural odors along with refugia29. Y-mazes can also be modified to expose animals to airborne chemical cues alone or in combination with substrate-borne cues16,30, and&#160;post hoc&#160;inference can be used to redesign data collection if archived video data are available31. Bioassays should be designed to simplify data collection and minimize conflicting stimuli, especially when a specific source of cues is being assessed (e.g., chemical cues21).
Researchers in animal behavior often observe and quantify focal animal responses in novel, artificial environments (e.g., an enclosed maze with a featureless landscape), and care should be taken to assess whether a given animal is exhibiting natural, exploratory behavior versus avoidance, agitation, or similar distressed behavior. Distressed animal behavior in experimental apparatuses is primarily attributed to neophobia: fear of novelty32. An example is escape behavior, where the focal animal pushes against the joints or the edges of the apparatus to achieve egress. Another example is shyness, where the focal animal demonstrates reluctance to enter the maze, the degree of which can be quantified by latency of maze entry. Apparatus (re)design can facilitate engagement of the focal animal to avoid these confounding effects of distress. The most common approach is repeated introduction of the focal animal to the apparatus to remove the novelty of the environment before testing begins, and contemporary statistical models (e.g., generalized linear mixed models) allow for test animals to be used in multiple trials. An important aside relevant to ecological considerations in behavioral testing is that reduced neophobia is associated with the success of invasive species33. Thus, depending on&#160;a priori&#160;knowledge of the species in question, neophobia may have variable importance as an experimental design consideration.
Acquisition of behavioral data from videos imposes multiple constraints that become major bottlenecks in experimental timelines. For example, the length of a given trial can exponentially increase data extraction time. One workaround is to analyze behavior only until a threshold is met (e.g., total time active). The threshold can be based on the longest video available for a given trial. Alternatively, machine-based observation (e.g., artificial intelligence) can be developed, although this is time- and resource-consuming with considerable effort required for quality control. Another issue is data management: videos must be of sufficient quality to enable behavioral scoring and assessment, resulting in data storage constraints. While cloud storage is now accessible, upload/download rates are often problematic, especially when data acquisition occurs in remote field locations. Additional challenges manifest in the limitations of recording tools that affect the integrity of behavioral observation. Clear viewing of focal animal behavior is always necessary, but visibility is often impeded by uncontrollable factors (e.g., moisture, insects, wind movement). Further, when recordings come from a single perspective (e.g., bird&#39;s eye view), behaviors occurring in the vertical plane (e.g., head raises14) are difficult to assess. A solution is to provide multiple camera angles per trial. Lastly, the time of day significantly affects behavioral recording. Nighttime behavioral analysis requires a camera with a nighttime mode and minimal light projection to avoid obstructive glare on the Y-maze surface or attraction of insects that can interrupt the camera feed. Considering the above, foreknowledge of the study site or species biology can inform which constraints are likely to occur with what frequency and thus inform desirable sample sizes.
Behavior is tightly coupled with physiology, and the utility of Y-mazes for evaluation of behavioral endocrinology in a variety of species has been demonstrated. However, this paper emphasizes some variation in the execution of these experiments depending on the target species, research question, and resources available. Therefore, the selection of materials and dimensions of each testing setup should be carefully considered for potential subsequent research expansion. Section 2 describes modifications made to materials outlined in section 1, which were incorporated to accommodate future, more complex behavioral trials with tegus. The increased vertical depth of the Everglades mazes will allow new questions about chemical ecology in wild-caught tegus to be answered without unduly protracting project design and setup, further demonstrating the translatability of this experimental apparatus.
When employing the above-described techniques in a relatively remote setting (see section 2), there are several limiting factors that must be considered, and project planning is paramount. Depending on the statistical power needed for the prescribed treatment experiment and biological timing of the target species (e.g., seasonality), the resources and labor required will be affected. Further, if single or repeated use of focal animals are desired, careful attention to reducing potential stressors is necessary. Each of these factors will either extend the project timeline or require increased labor, space, and materials. For example, section 2 presents the use of wild-caught male pythons as focal animals trailing another group of wild-caught and hormonally manipulated males, all of which require approximately 24 h of quiet acclimation time in holding boxes to minimize stress effects. Although these acclimation periods extended trial times to over two days, stress due to captivity and handling affect wild animal behavior and must be minimized to generate clean datasets34,35.
In summary, Y-mazes are powerful, adaptable tools that can be used to investigate the chemical ecology of diverse wildlife under widely variable conditions, provided there is vigilant&#160;a priori&#160;planning. Careful consideration must be taken to choose appropriate questions and to properly design the experimental setup for given taxa and conditions. Researchers and managers can significantly benefit from using Y-mazes to better understand animal chemosensory biology as these tools enable flexible experimental designs that provide large volumes of fine-scale behavioral data, especially when combined with remote monitoring tools.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61858">Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles</a>. An author name was updated.
The name of the tenth author was updated from:
Amy Y. Yackel Adams
to
Amy A. Yackel Adams

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61858">hUsing Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles</a>. An author name was updated.

Erratum: Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Y-mazes are common, simple tools in studies of animal behavior that allow for a variety of questions to be addressed. In addition to being widely used in laboratory studies, Y-mazes are also functionally compatible with various field environments to study wild animals in relatively remote settings. Researchers have examined the behaviors of wild vertebrates using Y-mazes in a wide variety of taxa across similarly diverse field applications (e.g., lampreys1; cichlid fish2; poison frogs3; lacertid lizards4; garter snakes5).
Many researchers are focused on how and to what degree chemical cues drive animal behaviors in reproductive, spatial, and foraging ecology6. A variety of chemical stimuli can be tested in Y-mazes and at fine scales, such as two chemical trails that only differ slightly in concentration7, or detection ability based on the reproductive status of the target species8. Chemical trails&#8212;the principal stimulus used in Y-maze tests&#8212;can be naturally created by conspecifics or specifically placed in the environment by a researcher using a defined chemical source1,5. Stimuli can also be tested in unique combinations to determine multimodal influence of cues such as changing contexts of cue presentation (airborne vs. substrate trails9; visual plus chemical cues10). Although there are many other methods for assessing chemosensory responses in reptiles (see discussion section), Y-mazes allow for searching behavior(s) to be assessed and at multiple temporal and spatial scales, which can lead to greater levels of behavioral inference.
Reptiles have been broadly tested for their reliance on chemical cues in reproductive and foraging ecology, and researchers often employ Y-mazes in these studies11,12. The chemical ecology of reptiles continues to be deciphered by studies employing Y-mazes to address a variety of evolutionary and behavioral questions that are valuable to wildlife managers. For example, recent tests with invasive snake and lizard species have revealed that chemical cues alone can influence choice and time allocation within the novel environment of a Y-maze13,14,15.
The use of large Y-mazes for moderately sized focal animals (e.g., large-bodied reptiles) is generally restricted to laboratory settings where the focal animals can be housed easily over the long term, experimental factors (e.g., climate, light, external stimuli) can be controlled, and access to infrastructure (e.g., power, running water) is unlimited. Studies on wild animals, however, are often restricted to specific locations for various reasons (e.g., logistics, permitting). As a result, challenges arise that must be addressed through creative problem solving and methodological adjustments to maintain consistent and comparable results.
Here, two experimental setups have been described using Y-mazes and remote monitoring tools to assess reproductive chemical ecology of invasive squamate reptiles (i.e., snakes and lizards) in different field scenarios: wild-caught, captive Argentine black and white tegu lizards (Salvator merianae) in Gainesville, FL, and wild-caught Burmese pythons (Python bivittatus) in Everglades National Park, FL. As implied by its name, the Y-maze apparatus creates an experimental environment in which an animal enters a main passageway (the base of the Y; &#34;base&#34;) which then leads to two divergent passageways (the arms of the Y; &#34;arms&#34;). In these experiments, two types of animals are used for a single trial: scent-laying animals (provide the stimulus scent in a restricted area of the maze) and focal animals (data are collected on this animal as it explores the scent trail).
As an experimental apparatus in chemoecological studies, any Y-maze must be constructed in a way that allows easy removal of the animal within and can be dissembled for thorough cleaning and reset. Also discussed are the constraints inherent to these different testing environments (e.g., diurnal vs. nocturnal animals, infrastructure differences) that prompted methodological adjustments. Although the focus was on tegu lizards and Burmese pythons, these designs can be applied to a wide range of reptile species. In this research on invasive reptiles, Y-mazes benefit the rate and scale of inference because they enable rapid collection of data to inform management goals that shift in-step with the invasion threat posed by a given species. In particular, studying chemoecology of invasive species is critical for the development of effective chemical control tools.
Discrimination is the key observation from empirical tests using Y-mazes where a focal animal chooses between two stimuli and that decision-making process is assessed. A swath of behaviors can also be scored in Y-maze trials during the trial itself (live) or after the trial (video) to expand inferential power. The complexity of the&#160;a priori&#160;objectives of a given study dictate whether live observation or archived recordings best suit the design. Here, Y-maze methods have been described in detail for addressing chemoecological questions to inform future studies by researchers interested in similar questions on reptile behavior, especially in chemical ecology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#34; Steel zinc-plated corner brace</td>
			<td>Everbilt, The Home Depot</td>
			<td>13619</td>
			<td>See Supplemental File 1, Step 2.1 &#34;90 degree 2.5 cm steel corner brace&#34;</td>
		</tr>
		<tr>
			<td>121.92cm W x 304.8cm&#160; L x 1.27cm H white polypropylene Extended Range High-Heat UHMW Sheet</td>
			<td>TIVAR</td>
			<td>UHMNV SH</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1. &#34;white polpropylene&#34;)</td>
		</tr>
		<tr>
			<td>182.88 cm L x 81.28 cm W x 0.64 cm Thick Clear Acrylic Sheet</td>
			<td>Plexiglass</td>
			<td>32032550912090</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1.6. &#34;Acrylic pieces&#34;)</td>
		</tr>
		<tr>
			<td>2.54 cm W x 2.54 cm H x 243.84 cm L Mill-Finished Aluminum Solid Angle</td>
			<td>Steelworks</td>
			<td>11354</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1.1. &#34;aluminum angle bracket&#34;)</td>
		</tr>
		<tr>
			<td>4.5 kg spool of 5 mm Round Polypropylene Welding Rods</td>
			<td>HotAirTools</td>
			<td>AS-PP5N10</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1. &#34;heat weld&#34;)</td>
		</tr>
		<tr>
			<td>5 mm Plain Aluminum Rivets</td>
			<td>Arrow</td>
			<td>RLA3/16IP</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1.1. &#34;rivet&#34;)</td>
		</tr>
		<tr>
			<td>Aluminum angle, 1.9 cm</td>
			<td>Everbilt, The Home Depot</td>
			<td>802527</td>
			<td>See Supplemental File 1, Step 1.2 &#34;aluminum angle (1.9 cm x 1.9 cm x 0.16 cm thick)&#34;</td>
		</tr>
		<tr>
			<td>Aluminum angle, 2.5 cm</td>
			<td>Everbilt, The Home Depot</td>
			<td>800057</td>
			<td>See Supplemental File 1, Steps 1.2 and 2.2.2 &#34;aluminum angle (2.5 cm x 2.5 cm x 0.16 cm thick)&#34;</td>
		</tr>
		<tr>
			<td>Aluminum angle, 3.2 cm</td>
			<td>Everbilt, The Home Depot</td>
			<td>800037</td>
			<td>See Supplemental File 1, Step 1.2 &#34;aluminum angle (3.2 cm x 3.2 cm x 0.16 cm thick)&#34;</td>
		</tr>
		<tr>
			<td>Aluminum flat bar 1&#34; x 1/8&#34; thick</td>
			<td>Everbilt, The Home Depot</td>
			<td>801927</td>
			<td>See Supplemental File 1, Step 3.2.1 &#34;aluminum strap&#34;</td>
		</tr>
		<tr>
			<td>Avigilon 2.0 MP camera</td>
			<td>Avigilon, a Motorola Solutions Company</td>
			<td>2.0C-H4SL-BO1-IR</td>
			<td>See &#34;1.5 Camera set-up and video acquisition&#34; (step 1.5.1 &#34;Avigilon 2.0 MP&#34;)</td>
		</tr>
		<tr>
			<td>Avigilon NVR</td>
			<td>Avigilon, a Motorola Solutions Company</td>
			<td>HD-NVR3-VAL-6TB-NA</td>
			<td>See &#34;1.5 Camera set-up and video acquisition&#34; (step 1.5.3 &#34;NVR&#34;)</td>
		</tr>
		<tr>
			<td>Clear acrylic sheet (5.6 mm thick)</td>
			<td>United States Plastic Corp.</td>
			<td>44363</td>
			<td>See Supplemental File 1, Step 1.3 &#34;clear acrylic sheet&#34; and step 3.2.1 &#34;clear acrylic door&#34;</td>
		</tr>
		<tr>
			<td>Fillet Weld Nozzle 3/16&#34; x 15/32&#34; / 4.5 x 12 mm</td>
			<td>TRIAC</td>
			<td>107.139</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1. &#34;heat weld&#34;)</td>
		</tr>
		<tr>
			<td>Hanging File Folder Box</td>
			<td>Sterilite</td>
			<td>18689004</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.2.1. &#34;Boxes&#34;)</td>
		</tr>
		<tr>
			<td>HardiePanel HZ10</td>
			<td>James Hardie Building Products</td>
			<td>9000525</td>
			<td>See Supplemental File 1, Step 1.1 &#34;fiber cement siding&#34;</td>
		</tr>
		<tr>
			<td>Heat Welding Gun</td>
			<td>TRIAC</td>
			<td>141.227</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.1. &#34;heat weld&#34;)</td>
		</tr>
		<tr>
			<td>Kraft Butcher Paper Roll, 24&#34;</td>
			<td>Bryco Goods</td>
			<td>24 inch x 175 FT</td>
			<td>See &#34;1.2 Protocol for running scent-laying tegus&#34; (step 1.2.1.2 &#34;butcher paper&#34;)</td>
		</tr>
		<tr>
			<td>Kraft Butcher Paper Roll, 46 cm wide</td>
			<td>Bryco Goods</td>
			<td>BGKW2100</td>
			<td>See &#34;2.3. Protocol for running scent-laying pythons&#34; (step 2.3.4. &#34;scenting paper&#34;)</td>
		</tr>
		<tr>
			<td>Micro-90 Concentrated Cleaning Solution&#160;</td>
			<td>International Products Corporation</td>
			<td>M-9050-12</td>
			<td>See &#34;1.4 Breakdown and clean-up&#34; (step 1.4.4 &#34;laboratory-grade soap&#34;)</td>
		</tr>
		<tr>
			<td>MKV ToolNix - Matroska tools for linux/Unix and Windows</td>
			<td>Moritz Bunkus</td>
			<td>v.48.0.0</td>
			<td>See &#34;2.2. Camera setup and video acquisition&#34; (step 2.2.4.2. &#34;movie processing software&#34;)</td>
		</tr>
		<tr>
			<td>Network Camera</td>
			<td>Axis Communications</td>
			<td>M3104-LVE</td>
			<td>See &#34;2.2. Camera setup and video acquisition&#34; (step 2.2.1. &#34;Project camera&#34;)</td>
		</tr>
		<tr>
			<td>Palight ProjectPVC 1/4&#34;</td>
			<td>Palram</td>
			<td>159841</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.2.3. &#34;faceplate&#34;)</td>
		</tr>
		<tr>
			<td>Palight ProjectPVC 1/8&#34;</td>
			<td>Palram</td>
			<td>156249</td>
			<td>See &#34;2.1. Y-maze components and rationale for changes to USDA design &#34; (step 2.1.2.1. &#34;door&#34;)</td>
		</tr>
		<tr>
			<td>Privacy windscreen (green)</td>
			<td>MacGregor</td>
			<td>Size to fit</td>
			<td>See Supplemental File 1, Step 4.2 &#34;green heavy duty shade cloth&#34;</td>
		</tr>
		<tr>
			<td>Protective Glove, Full-Finger</td>
			<td>ArmOR Hand</td>
			<td>HS1010-RGXL</td>
			<td>See &#34;2.3. Protocol for running scent-laying pythons&#34; (step 2.3.11.2. NOTE: &#34;puncture-resistant glove&#34;)</td>
		</tr>
		<tr>
			<td>REScue Disinfectant</td>
			<td>Virox Animal Health</td>
			<td>44176</td>
			<td>See &#34;1.5. Breakdown and clean-up.&#34; (step 1.5.4. NOTE &#34;sanitation solution&#34;)</td>
		</tr>
		<tr>
			<td>Reversable PVC trim, 1/2&#34; x 24&#34;</td>
			<td>UFP Industries, Veranda products</td>
			<td>H120XWS17</td>
			<td>See Supplemental File 1, Step 2.1 &#34;PVC board partition&#34;, and step 3.2.1 &#34;thinner PVC trim boards&#34;</td>
		</tr>
		<tr>
			<td>S4S / Veranda HP TRIM</td>
			<td>UFP Industries, Veranda products</td>
			<td>H190OWS4</td>
			<td>See Supplemental File 1, Steps 1.2, 2.2.2, and 2.2.3 &#34;PVC board&#34;</td>
		</tr>
		<tr>
			<td>S4S / Veranda HP TRIM (1&#34; x 8&#34; Nominal)</td>
			<td>UFP Industries, Veranda products</td>
			<td>827000005</td>
			<td>See Supplemental File 1, Steps 3.2.1 &#34;PVC trim board&#34;</td>
		</tr>
		<tr>
			<td>ScotchBlue 24 in. Pre-taped Painter&#8217;s Plastic</td>
			<td>3M</td>
			<td>PTD2093EL-24-S</td>
			<td>See &#34;1.2 Protocol for running scent-laying tegus&#34; (step 1.2.1.3 &#34;plastic sheeting&#34;)</td>
		</tr>
		<tr>
			<td>Sterilite 114 L tote box</td>
			<td>Sterilite Company</td>
			<td>1919, Steel</td>
			<td>See Supplemental File 1, Step 3.2 &#34;arm box&#34;</td>
		</tr>
		<tr>
			<td>Sterilite 189 L tote box</td>
			<td>Sterilite Company</td>
			<td>1849, Titanium</td>
			<td>See Supplemental File 1, Step 3.2 &#34;Base box&#34;</td>
		</tr>
		<tr>
			<td>Super Max Canopy</td>
			<td>ShelterLogic</td>
			<td>25773</td>
			<td>See Supplemental File 1, Step 4.3 &#34;white canopy&#34;</td>
		</tr>
		<tr>
			<td>VLC Media Player&#160;</td>
			<td>VideoLAN</td>
			<td>v.3.0.11</td>
			<td>See &#34;2.2. Camera setup and video acquisition&#34; (step 2.2.4.3. &#34;media file reviewing program&#34;)</td>
		</tr>
		<tr>
			<td>White Pavilion Tent</td>
			<td>King Canopy</td>
			<td>BJ2PC</td>
			<td>See Supplimental File 2 &#34;3. Enclosure materials and consideratons&#34; (step 3. &#34;pavilion tent&#34;)</td>
		</tr>
	</tbody>
</table>

using enclosed y-mazes to assess chemosensory behavior in reptiles

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. Decrypting the scent-trailing behavior of vertebrates, particularly invasive species, enables the discovery of cues that induce exploratory behavior and can aid in the development of valuable basic and applied biological tools. However, pinpointing behaviors dominantly driven by chemical cues versus other competing environmental cues can be challenging. Y-mazes are common tools used in animal behavior research that allow quantification of vertebrate chemosensory behavior across a range of taxa. By reducing external stimuli, Y-mazes remove confounding factors and present focal animals with a binary choice. In our Y-maze studies, a scenting animal is restricted to one arm of the maze to leave a scent trail and is removed once scent-laying parameters have been met. Then, depending on the trial type, either the focal animal is allowed into the maze, or a competing scent trail is created. The result is a record of the focal animal&#39;s choice and behavior while discriminating between the chemical cues presented. Here, two Y-maze apparatuses tailored to different invasive reptile species: Argentine black and white tegu lizards (Salvator merianae) and Burmese pythons (Python bivittatus) are described, outlining the operation and cleaning of these Y-mazes. Further, the variety of data produced, experimental drawbacks and solutions, and suggested data analysis frameworks have been summarized.

A multitude of variables can be recorded and/or scored from Y-maze trials. The design of the study should be primarily driven by the desired outcomes/deliverables. Further, if the study is relying on repeated measures (e.g., repeated use of the same focal animals), proper testing and analysis structures are required. For example, as the USDA trials relied on repeated testing of focal tegus, the planning of experimental trials was fully randomized.
Choice data: The majority of studies using Y-mazes report simple binary choice data and analyze the results with parametric statistics such as a binomial test. The chief limitation here is sample size, which directly affects the power of any statistical analysis. In&#160;Figure 2, a series of statistical thresholds per study sample size are depicted that demonstrate how many &#34;successes&#34; would need to occur for a given binomial test to yield statistically significant results. These are mathematically derived and therefore generalizable to any Y-maze test. Binomial statistics are easy to generate using online freeware. For calculating probabilities, one-tail distributions are used if an&#160;a priori&#160;rationale is given; otherwise, the two-tail distribution should be used.
Choice of an arm is often determined by the distance the focal animal moves in a given arm. The simplest way to set this threshold is by establishing a landmark within the maze. For most Y-maze studies, the landmark is the entrance of the arm box. Because reptiles conduct all chemosensory assessment with the chemical-sensing organs in the anterior region of the head, the head is the focal point during a trial. For example, because Burmese pythons are often longer than the entire maze itself, choice is best and most efficiently determined by the movement of the head past a landmark. Other options for determining choice are time spent in an arm and complete movement of the focal animal into a box. Failure is determined by a focal animal not making a choice within a specific period.
More fine resolution analyses can be derived from choice data in the Y-maze. For example, researchers can generate a choice penalty score16.&#160;Here, researchers must track the degree to which the focal animal explored the non-target arm of the maze. Non-target can be defined as the arm the researchers determine&#160;a priori&#160;that the focal animal will not choose based on the alternative hypothesis tested. The simplest example of a non-target arm would be the unscented arm when only one arm contains a target scent. More complex examples would be the choice between two scents from the same source, but presented at different concentrations7. When the experimental design is multi-level and/or the data go from binary to incremental, as with choice penalty, an appropriate statistical approach should be used such as repeated measures analysis of variance (ANOVA) or other methods used with continuous or proportional datasets.
Behaviors: Throughout the duration of an experiment in which focal animals are observed, a variety of individual behaviors can be quantified. This number of variables can either be determined&#160;a priori&#160;depending on what is known16&#160;or&#160;post hoc&#160;following preliminary observations on a subset of data14,15. The study objectives and their degree of resolution determine what behavioral assessments should be made within the maze, if any (i.e., in many studies, only choice data are quantified17). Behaviors can be assessed throughout the maze, in sections, or during specific time periods; for instance, behaviors seen only in the base or at the junction of the arms may be prioritized8. Video recordings facilitate behavioral scoring, although the resolution of the video and its length&#8212;factors that impose data storage constraints&#8212;should be considered before experimentation begins.
Temporal variables: As with behavioral variables, many temporal aspects of animal performance can be quantified during Y-maze trials. For example, researchers can time latency periods (e.g., latency to emerge from the box8). Most temporal variables are associated with exploration of the maze such as total trailing time or time spent in each arm. These variables are usually analyzed in a multi-factor analysis such as multi-way ANOVA.
Observer bias: With any studies involving animal behavior, observer bias significantly influences data collection18. Therefore, observers should be blind to the treatment being tested. The simplest way to do this is to code the video files numerically and then randomly sort them (e.g., random number generator) prior to assigning them to observers. Controlling for observer bias is difficult-to-impossible when live data collection is the only option. In a field setting, this would require two cooperators: an observer blind to the treatment and a coordinator who sets up the trial. Extensive reviews summarize the effects of experimenter bias on data collection and interpretation in behavioral and ecological studies18,19.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61858/61858fig02.jpg" /> 
Figure 2. Sample sizes and P-values for binomial tests from Y-maze results. Each given sample size represents a set number of trials where a scent is tested in one arm of the Y (target arm) while the other could be a control (non-target). Top number above each bar is the one-tail P-value for that number of target arm choices, bottom is two-tail. Numbers within the top bar represent the maximum number of non-target choices that are still traditionally statistically significant (P &#60; 0.05).&#160;<a href="https://www.jove.com/files/ftp_upload/61858/61858fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1.&#160;<a href="https://www.jove.com/files/ftp_upload/61858/ParkerCurrylowetal-JoVE-R1-supp1.docx" style="font-size: 14px;" target="_blank">Please click here to download this file.</a>
Supplemental File 2.&#160;<a href="https://www.jove.com/files/ftp_upload/61858/ParkerCurrylowetal-JoVE-R1-supp2.docx" style="font-size: 14px;" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles at JoVE.com

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Y-mazes enable researchers to determine the relevance of specific stimuli that drive animal behavior, especially isolated chemical cues from a variety of sources. Careful design and planning can yield robust data (e.g., discrimination, degree of exploration, numerous behaviors). This experimental apparatus can provide powerful insight into behavioral and ecological questions.

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. ...

using-enclosed-y-mazes-to-assess-chemosensory-behavior-in-reptiles

Research

JoVE Journal

Behavior

5.4K Views.  James Madison University. Y-mazes enable researchers to determine the relevance of specific stimuli that drive animal behavior, especially isolated chemical cues from a variety of sources. Careful design and planning can yield robust data (e.g., discrimination, degree of exploration, numerous behaviors). This experimental apparatus can provide powerful insight into behavioral and ecological questions.

Video: Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., &#8220;startle probes&#8221;). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

Eye-Tracking Control to Assess Cognitive Functions in Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to progressive loss of motor and speech abilities. In addition, cognitive functions are impaired in a subset of patients. To evaluate these potential deficits in severely physically impaired ALS patients, eye-tracking is a promising means to conduct cognitive tests. The present article focuses on how eye movements, an indirect means of communication for physically disabled patients, can be utilized to allow for detailed neuropsychological assessment. The requirements, in terms of oculomotor parameters that have to be met for sufficient eye-tracking in ALS patients are presented. The properties of stimuli, including type of neuropsychological tests and style of presentation, best suited to successfully assess cognitive functioning, are also described. Furthermore, recommendations regarding procedural requirements are provided. Overall, this methodology provides a reliable, easy to administer and fast approach for assessing cognitive deficits in patients who are unable to speak or write such as patients with severe ALS. The only confounding factor might be deficits in voluntary eye movement control in a subset of ALS patients.

Cognitive deficits are common in about one third of patients with amyotrophic lateral sclerosis, a neurological condition leading to progressive impairments in speech and movement abilities. To conduct cognitive tests in patients unable to speak or write a reliable and easy to administer eye-tracking paradigm was developed.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to ...

Using Gold-standard Gait Analysis Methods to Assess Experience Effects on Lower-limb Mechanics During Moderate High-heeled Jogging and Running

A limited number of studies have explored lower-limb biomechanics during high-heeled jogging and running, and most studies have failed to clarify the wearing experience of subjects. This protocol describes the differences in lower-limb kinematics and ground reaction force (GRF) between experienced wearers (EW) and inexperienced wearers (IEW) during moderate high-heeled jogging and running. A three-dimensional (3D) motion analysis system with a configured force platform was used to synchronously capture lower-limb joint movements and GRF. 36 young females volunteered to participate in this study and were asked about high-heeled shoe-wearing experience, including frequency, duration, heel types, and heel heights. Eleven who had the experience of 3 to 6 cm heels for a minimum of three days per week (6 h per day) for at least two years and eleven who wore high heels less than twice per month participated. Subjects performed jogging and running at comfortable low and high speeds, respectively, with the right foot completely stepping onto a force platform when passing by along a 10 m walkway. EW and IEW adopted different biomechanical adaptations while jogging and running. IEW exhibited a generally larger range of joint movement, while EW showed a dramatically larger loading rate of GRF during running. Hence, further studies on the lower-limb biomechanics of high-heeled gait should strictly control the wearing experience of the subjects.

This study investigated lower-limb kinematics and ground reaction force (GRF) during moderate high-heeled jogging and running. Subjects were divided into groups of experienced wearers and inexperienced wearers. A three-dimensional motion analysis system with a configured force platform captured lower-limb joint movements and GRF.

A limited number of studies have explored lower-limb biomechanics during high-heeled jogging and running, and most studies have failed to clarify the ...

A Community-based Stress Management Program: Using Wearable Devices to Assess Whole Body Physiological Responses in Non-laboratory Settings

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches for emotional regulation and optimizing compensatory reserve mechanisms. This protocol is designed to not only teach mind-body awareness but also to provide feedback utilizing physiological data and survey results. The primary findings of this study showed that heart coherence and alpha variables were significantly correlated after four weeks of the breath-based meditation stress protocol. Meditation and rhythmic breathing produced significant increases in alpha brain activity. These brain physiological responses conformed to the Pleth Variability Index (PVI) changes, suggesting the ability of the human body to enter into a meditative state and effectively manage stress. When assessed after four weeks of daily practicing the techniques employed in the stress management protocol, based on the Five Facet Mindfulness Questionnaire, subjects improved in applying mindfulness skills. The overall mindfulness score, Pleth variability index (PVI), and perfusion index (PI) increased after the 4-week intervention period. Results from electroencephalography (brain waves) were consistent with a meditative state during the post-study follow-up session. This provides evidence that wearable devices are feasible for data collection during a breath-based stress management intervention. This protocol can be easily and efficiently implemented into any study design in which physiological data are desired in a non-laboratory-based setting.

Stress is an unavoidable and persistent component of life and holistic approaches for its management are being considered. A standardized methodology was created to demonstrate the feasibility of a breath-based stress management protocol that can be used with commercially available portable technology.

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Using a Classroom-Based Deese Roediger McDermott Paradigm to Assess the Effects of Imagery on False Memories

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the accuracy of memories. The purpose of the method presented here was to induce false memories using lists of either semantically or phonologically related words and to assess the effects of imagery instructions on the recall and recognition of those false memories. To do this, we used a modified version of the Deese Roediger McDermott (DRM) paradigm. We adapted word lists from previous DRM studies to suit imagery procedures and created an automated presentation to present the word lists in classroom settings. We then recruited undergraduate classes and instructed some of the classes to create mental images of the list words as they were being presented, while instructing the other classes to simply remember the words. The automated presentation presented word lists to participants, one word at a time, alternating between phonologically and semantically related lists. Participants used paper-pencil recall packets to immediately recall list items, complete a distractor activity, and take a subsequent final recognition test. Often, participants immediately recalled and later recognized words that were related to the list items but were not actually presented; these are known as critical lures and indicate a false memory. The protocol detailed here describes a four-step procedure - list presentation, immediate recall, distractor phase, and final recognition - that can assess the effects of list type and imagery instruction within the DRM paradigm on memory. The automated nature of the list presentation provides the ability to systematically vary variables of interest, and the paper and pencil method of data collection affords an easily accessible method for collecting data in classroom settings. The protocol also offers options to modify the procedure to a more traditional DRM paradigm without imagery and/or list type manipulations. The use of this protocol can provide results relevant to both classroom learning and cognitive science principles.

The method presented here induced false memories using lists of related words and also assessed the effects of imagery instructions on the recall and recognition of those false memories. This protocol details a modified version of the Deese Roediger McDermott (DRM) paradigm.

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the ...

Biomechanical Analysis Methods to Assess Professional Badminton Players' Lunge Performance

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge movements of eight professional badminton players and eight amateur players. The purpose of this protocol is to study the differences in kinematics and joint moment of the right knee and ankle. A motion capture system and force plate were used to capture data of the joint movements of the lower extremity and the vertical ground reaction force (vGRF). Sixteen young men who did not have any sports injuries in the past 6 months took part in the study. The subjects performed a maximal right lunge from the start position with their right foot, stepping on and fully contacting with the force plate, hit the shuttlecock with an underhand stroke to the designated position in the backcourt, and then returned to the start/end position. All subjects wore the same badminton shoes to avoid a difference in impact from different badminton shoes. The amateur players showed a greater range of ankle movement and reverse joint moment on the frontal plane, and a larger internal joint rotation moment on the horizontal plane. The professional badminton players exhibited greater knee moment on the sagittal and frontal planes. Therefore, these factors should be considered in the development of the training program to reduce the risk of sports injuries in knee and ankle joints. This study simulates the real badminton court and calibrates the range of activities of each movement of the subjects so that the subjects complete the experimental action in a natural state with high quality. A limitation of this study is that it does not combine joint load and muscle activity. Another limitation is that the sample size is small and should be expanded in future studies. This research method can be applied to the lower limb biomechanical research of other footwork in the badminton project.

Here, we present a protocol to evaluate the differences in injury mechanisms between professional and amateur players when performing a badminton maximal right lunge movement by analyzing lower limb kinematics.

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge ...

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. Touch that optimally activates CT fibers is consistently rated as hedonically pleasant. Patient groups with impaired social-emotional functioning also show disordered affective touch ratings. However, relying on self-reported ratings of touch has many limitations, including recall bias and communication barriers. Here, we describe a methodological approach to study affective responses to touch via facial electromyography (EMG) that circumvents the reliance on self-report ratings. Facial EMG is an objective, quantitative, and non-invasive method to measure facial muscle activity indicative of affective responses. Responses can be assessed across healthy and patient populations without the need for verbal communication. Here, we provide two separate datasets demonstrating that CT-optimal and non-optimal touch elicit distinct facial muscle reactions. Moreover, facial EMG responses are consistent across stimulus modalities, e.g. tactile (experienced touch) and visual (observed touch). Finally, the temporal resolution of facial EMG can detect responses on timescales that supersede that of verbal reporting. Together, our data suggest that facial EMG is a suitable methodology for use in affective tactile research that can be used to supplement, or in some cases, supplant, existing measures.

We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography.

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...